AU749561B2 - Nucleic acid molecules having endonuclease and/or catalytic activity - Google Patents

Description

NUCLEIC ACID MOLECULES HAVING ENDONUCLEASE AND/OR CATALYTIC ACTIVITY Background of the Invention The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.

The Raf family of serine/threonine kinases function as cytoplasmic signaling proteins that transduce mitogenic signals in response to activation of various growth factor receptors (for reviews, see Daum, 1994 Trends in Biochem. Sci. 19, 474; Katz, 1997, Curr. Opin. Genet. Devel. 7, 75; Marais, 1996, Cancer Surveys 27; Naumann, 1997, Cancer Res. 143, 237). c-Raf is the cellular homolog of v-Raf, the transforming element of the murine sarcoma virus 3611. The Raf family consists of three highly conserved isozymes in vertebrates: c-Raf-1, which is constitutively expressed in all 15 tissues, A-Raf, which is expressed in urogenital tissue and B-Raf which is expressed in 0 and cerebrum and testes (Storm, 1990, Oncogene 5, 345). Inappropriate expression of o these key genes involved in cell growth and differentiation can result in uncontrolled cell proliferation and/or propagation of damaged DNA, leading to hyperproliferative disorders such as cancer, restenosis, psoriasis and rheumatoid arthritis.

20 Raf is one of the major downstream effectors of Ras, a member of the class of small GDP/GTP-binding proteins involved in cellular signal transduction pathways (figure 35; Marshall, 1995, Molec. Reprod. Devel., 42, 493). Appropriate mitogenic signals cause an increase in levels of the GTP-bound Ras. In its GTP-bound active state, Ras binds Raf and localizes it to the plasma membrane. This results in activation of the Raf kinase activity. Activated Raf in turn phosphorylates MEK, thereby activating the [R:\LIBZ]02943.doc:lam WO 98/50530 PCT/US98/09249 -2constitutively active MEK is sufficient for oncogenic transformation of fibroblasts (Cowley, 1994, Cell 77, 81; Mansour, 1994, Science 265, 966; Kolch, 1991, Nature 349, 426). In normal cells, the expression level of Raf is limiting in cellular transformation (Cuadrado, 1993, Oncogene 8, 2443). The pivotal position that the Ras and Raf family of proteins occupy in cellular signal transduction pathways emphasizes their importance in the control of normal cellular growth.

Activation of Raf in mammalian cells is triggered by a variety of growth factors and cytokines. Raf activation has been observed in cardiac myocyte cultures stimulated by fibroblast growth factor (FGF), endothelin or phorbol ester (Bogoyevitch, 1995, J. Biol.

Chem. 270, Activation has also been seen in Swiss 3T3 cells treated with bombesin and platelet derived growth factor (Mitchell, 1995, J. Biol. Chem. 270, 8623) or with colony stimulating factor or lipopolysacchride (Reimann, 1994, J. Immun. 153, 398), in L6 myoblasts stimulated with insulin-like growth factor (Cross, 1994, Biochem J 303, 21), as well as in B cells stimulated via the immunoglobulin receptor (Kumar, 1995, Biochem J.

307,215).

There is growing evidence from a number of laboratories that suggests that the Ras/Raf pathway may also be involved in cell motility (Bar-Sagi and Feramisco, 1986 Science 233, 1061; Partin et al., 1988 Cancer Res. 48-6050; Fox et al., 1994 Oncogene 9, 3519). These studies show that cell lines transfected with activated Ras show an increase in ruffling, pseudopod extension and chemotactic response, all of which are cell-motilityrelated processes. Uncontrolled cell motility has been implicated in several pathological processes such as restenosis, angiogenesis and wound healing.

Raf activation leads to induction of several immediate early transcription factors including NF-kB and AP-1 (Bruder, 1992, Genes Devel. 6, 545; Finco, 1993, J. Biol.

Chem. 268, 17676). AP-1 regulates expression of a variety of proteases (Sato, 1994 Oncogene 8, 395; Gaire, 1994, J Biol Chem 269, 2032; Lauricell-Lefebvre, 1993, Invasion Metastasis 13, 289; Troen, 1991, Cell Growth Differ 2, 23). A cascade of MMP and serine proteinase expression is implicated in the acquisition of an invasive phenotype as well as in angiogenesis in tumors (MacDougall, 1995, Cancer and Metastasis Reviews 14, SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 351). Thus, Raf signaling is expected to contribute to increased invasiveness in tumor cells, leading to metastasis.

Coexpression studies of Raf-1 and Bcl-2 have shown that these proteins bind and interact to synergistically suppress apoptosis (Wang, 1994, Oncogene 9, 2751). Thus, overexpression of Raf-1 in tumor cells is likely to contribute to malignant transformation and increased resistance to chemotherapeutic agents. Overexpression of c-Raf-1 is observed in squamous cell carcinomas of the head and neck taken from patients resistant to radiation therapy (Riva, 1995, Oral Oncol., Eur. J. Cancer 31B, 384) and in lung carcinomas (Rapp, 1988, The Oncogene Handbook, 213). Activated (truncated) Raf has been detected in a number of human cancers including small-cell lung, stomach, renal, breast and laryngeal cancer (Rapp, 1988, The Oncogene Handbook, 213).

Therapeutic intervention in down-regulating Raf expression have focused on antisense oligonucleotide approaches: Antisense oligonucleotides targeting c-Raf-1 were used to demonstrate that IL-2 stimulated growth of T cells requires c-raf (Riedel, 1993, Eur. J. Immunol. 23, 3146).

Antisense oligonucleotides targeting c-Raf-1 in SQ-20B cells showed reduced Raf expression and increased radiation sensitivity (Soldatenkov, 1997, The Cancer J from Scientific American 3, 13). Rapp et al. have disclosed a method for inhibiting c-Raf-1 gene expression using a vector expressing the gene in the antisense orientation (International PCT Publication No. WO 93/04170). Antisense oligonucleotides targeting c-Raf-1 in SQ-20B cells showed reduced DNA synthesis in response to insulin stimulation in rat hepatoma cells (Tornkvist, 1994, J. Biol. Chem. 269, 13919). Monia et al. have disclosed a method for inhibiting Raf expression using antisense oligonucleotides (U.S.

Patent No. 5,563,255) and shown that antisense oligonucleotides targeting c-Raf-1 can inhibit Raf mRNA expression in cell culture, and inhibit growth of a variety of tumor types in human tumor xenograft models (Monia et al., 1996, Proc. Natl. Acad. Sci.93, 15481; Monia et al., 1996, Nature Med. 2, 668). No toxicity was observed in these studies following systemic administration of c-Raf antisense oligonucleotides, suggesting that at least partial down regulation of Raf in normal tissues is not overtly toxic.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -4- It has been proposed that synthetic ribozymes can be delivered to target cells exogenously in the presence or absence of lipid delivery vehicles (Thompson et al., International PCT Publication No. WO 93/23057; Sullivan et al., International PCT Publication No. WO 94/02595).

Recently Sandberg et al., 1996, Abstract, IBC USA Conferences on Angiogenesis Inhibitors and other novel therapeutics for Ocular Diseases of Neovascularization, reported pharmacokinetics of a chemically modifies hammerhead ribozyme targeted against a vascular endothelial growth factor (VEGF) receptor RNA in normal and tumor bearing mice after daily bolus or continuous infusion.

Desjardins et al., 1996, J. Pharmacol. Exptl. Therapeutic, 27, 8, 1419, reported pharmacokinetics of a synthetic, chemically modified hammerhead ribozyme against the rat cytochrome P-450 3A2 mRNA after single intravenous injection.

The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the use of ribozymes to cleave Raf RNA.

Furthermore, Applicant believes that the references do not disclose and/or enable the use of ribozymes to down regulate normal Raf gene expression in mammalian cells and/or whole animal.

Summary Of The Invention This invention relates to identification, synthesis and use of nucleic acid catalysts to cleave RNA species that are required for cellular growth responses. In particular, applicant describes the selection and function of ribozymes capable of cleaving RNA encoded by c-rafgene. Such ribozymes may be used to inhibit the hyper-proliferation of tumor cells in one or more cancers, restenosis, psoriasis, fibrosis and rheumatoid arthritis.

In the present invention, ribozymes that cleave c-rafRNA are described. Moreover, applicant shows that these ribozymes are able to inhibit gene expression and cell proliferation in vitro and in vivo, and that the catalytic activity of the ribozymes is required for their inhibitory effect. From those of ordinary skill in the art, it is clear from the SUBSTITUTE SHEET (RULE 26) encoded by c-raf gene. Such ribozymes may be used to inhibit the hyper-proliferation of tumor cells in one or more cancers, restenosis, psoriasis, fibrosis and rheumatoid arthritis.

Thus, in a first embodiment, the invention provides a nucleic acid molecule with an endonuclease activity having the formula III: LA- (N)o-C-G-A-A-A-M-3' wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded tand/or double-stranded region; represents a chemical linkage; and A, C, U and G represent adenosine, cytidine, uridine and guanosine nucleotides, respectively.

S. In a second embodiment the invention provides a nucleic acid molecule with 15 catalytic activity having the formula IV: S.3' m 5 A Z3 A Z4 S: GA

G

A A G GZ 7

A

C G (N)o (N)n

L

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is 2'-C-allyl uridine; Z7 is 6-methyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively.

[R:\LIBZ]02943.doc:lam In a third embodiment the invention provides a nucleic acid molecule with catalytic activity having the formula V: 3' M Q 5 A Z3 A Z4

G

A

A

G GZ 7

A

COG

(N)o (N)n

\L

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when 10 present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is 2 '-methylthiomethyl cytidine; Z7 is 6-methyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively.

S".i In a fourth embodiment the invention provides a nucleic acid molecule with 15 catalytic activity having the formula VI: 3' M AZ3 A Z3 A ZG

G

A

A

Z7 G G7

A

C G (N)o (N)n

\L/

wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, Swherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by C0 1A hydrogen bond interaction; L is a linker which may be present or absent, but [R:\LIBZ]02943.doc:Iam when present, is a nucleotide and/or a non-nucleotide linker, which may be a singlestranded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is 2'-methylthiomethyl cytidine; Z7 is 2'-C-allyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively.

In a fifth embodiment the invention provides a nucleic acid molecule with catalytic activity having the formula VII: A Z3 A Z4

G

A

A

G GZ 7

A

C G (N)o (N)n

L

*wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably 1 0 interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is S 15 2'-methylthiomethyl cytidine; Z7 is pyridine-4-one; and represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively.

S In the present invention, ribozymes that cleave c-rafRNA are described. Moreover, applicant shows that these ribozymes are able to inhibit gene expression and cell proliferation in vitro and in vivo, and that the catalytic activity of the ribozymes is required for their inhibitory effect. From those of ordinary skill in the art, it is clear from the examples described herein, that other ribozymes that cleave target RNAs required for cell proliferation may be readily designed and are within the invention.

By "inhibit" is meant that the activity of c-raf or level of RNAs encoded by c-raf is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes is preferably below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that

RNA.

[R:\LIBz]02943.doc:aak By "nucleic acid catalyst" is meant a nucleic acid molecule capable of catalyzing (altering the velocity and/or rate of) a variety of reactions including the ability to repeatedly cleave other separate nucleic acid molecules (endonuclease activity) in a nucleotide base sequence-specific maimer. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the 0o enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur.

100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic i5 oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, 9 o o oooo *oooo •coo* [RALIBz]02943.doc:aak WO 98/50530 PCT/US98/09249 -6- By "substrate binding arm" or "substrate binding domain" is meant that portion/region of a ribozyme which is complementary to able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in Figure 1 and 3. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions. The ribozyme of the invention may have binding arms that are contiguous or non-contiguous and may be of varying lengths. The length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical each of the binding arms is of the same length; five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical the binding arms are of different length; six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).

In one of the preferred embodiments of the inventions herein, the nucleic acid catalyst is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis d virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; of the hepatitis d virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II SUBSTITUTE SHEET (RULE 26) are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; of the hepatitis d virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No. WO 96/22689; and of the Group I intron by Cech et al., U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is S. is5 important in a nucleic acid catalyst of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

By "equivalent" RNA to c-rafis meant to include those naturally occurring RNA 20 molecules associated with cancer in various animals, including human, rodent, primate, rabbit and pig. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5'-untranslated region, 3'-untranslated region, introns, intron-exon junction and the like.

By "complementarity" is meant a nucleic acid that can form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.

In one aspect, the present disclosure provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a [R:\LIBz]02943.doc:aak WO 98/50530 PCT/US98/09249 -8- By "related" is meant that the inhibition of c-raf RNAs and thus reduction in the level of respective protein activity will relieve to some extent the symptoms of the disease or condition.

In preferred embodiments, the ribozymes have binding arms which are complementary to the target sequences in TablesXII-XIX. Examples of such ribozymes are also shown in Tables XII-XIX. Examples of such ribozymes consist essentially of sequences defined in these Tables.

By "consists essentially of" is meant that the active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

Thus, in a first aspect, the invention features ribozymes that inhibit gene expression and/or cell proliferation. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs.

The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation.

In the absence of the expression of the target gene, cell proliferation is inhibited.

In a preferred embodiment, ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In another preferred embodiment, the ribozyme is administered to the site of c-raf expression tumor cells) in an appropriate liposomal vehicle.

In another aspect of the invention, ribozymes that cleave target molecules and inhibit c-raf activity are expressed from transcription units inserted into DNA or RNA vectors.

The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -9expressing viral vectors could be constructed based on, but not limited to, adenoassociated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510). In another aspect of the invention, ribozymes that cleave target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or viral vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells. However. other mammalian cell vectors that direct the expression of RNA may be used for this purpose.

By "patient" is meant an organism which is a donor or recipient of explanted cells or the cells themselves. "Patient" also refers to an organism to which nucleic acid catalysts can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.

By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

These ribozymes, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with c-raf levels, the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art.

In a further embodiment, the described ribozymes can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described ribozymes could be used in combination with one or more known therapeutic agents to treat cancer.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the tables, shown as Seq. I.D. Nos. 1-501, 1078-1152, 1461-1768, 1841-1912, 2354-2794 and 2846-2956. Examples of such ribozymes are shown as Seq. I.D. Nos. 502-1002, 1003-1077, 1153-1460, 1769-1840, 1913-2353 and 2795-2845. Other sequences may be present which do not interfere with such cleavage.

Ribozymes that cleave the specified sites in Raf mRNAs represent a novel therapeutic approach to treat tumor angiogenesis, ocular diseases, rhuematoid arthritis, psoriasis and others. Applicant indicates that ribozymes are able to inhibit the activity of Raf and that the catalytic activity of the ribozymes is required for their inhibitory effect.

Those of ordinary skill in the art will find that it is clear from the examples described that other ribozymes that cleave Raf mRNAs may be readily designed and are within the invention.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description Of The Preferred Embodiments The drawings will first briefly be described.

Drawings: Figure 1 shows the secondary structure model for seven different classes of nucleic acid catalysts. Arrow indicates the site of cleavage. indicate the target sequence.

Lines interspersed with dots are meant to indicate tertiary interactions. is meant to indicate base-paired interaction. Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al., 1994, Nature Struc. Bio., 1, 273). RNase P (M1RNA): EGS represents external guide sequence (Forster et al., 1990, Science, 249, 783; Pace et a., 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5' splice site; 3'SS means 3'-splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al., 1994, Biochemistry, 33, 2716). VS RNA: I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -11 PCT Publication No. WO 96/19577). HDV Ribozyme: I-IV are meant to indicate four stem-loop structures (Been et al.. US Patent No. 5,625,047). Hammerhead Ribozyme: I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al., 1996, Curr. Op. Struct. Bio., 1, 527).

Hairpin Ribozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is between 3 and 8 base-pairs long; Y is a pyrimidine; Helix 2 (H2) is provided with a least 4 base pairs n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 20 bases, m is from 1 20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases r is 1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs 4 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential basepairing interaction. These nucleotides may be modified at the sugar, base or phosphate.

Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size o and p is each independently from 0 to any number, 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. is 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. refers to a covalent bond. (Burke et al., 1996, Nucleic Acids Mol. Biol., 10, 129; Chowrira et al., US Patent No. 5,631,359).

Figure 2 shows a general approach to accessible site and target discovery using nucleic acid catalysts.

Figure 3 is a diagram of a hammerhead ribozyme. The consensus hammerhead cleavage site in a target RNA is a followed by (anything but The hammerhead ribozyme cleaves after the This simple di-nucleotide sequence occurs, on average, every 5 nt in a target RNA. Thus, there are approximately 400 potential SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 12hammerhead cleavage sites in a 2-Kb mRNA. Stems I and III are formed by hybridization of the hammerhead binding arms with the complementary sequence in target RNA; it is these binding arms that confer specificity to the hammerhead ribozyme for its target. The binding arms of the hammerhead are interrupted by the catalytic domain that forms part of the structure responsible for cleavage.

Figure 4 shows a scheme for the design and synthesis of a Defined Library: simultaneous screen of 400 different ICAM-targeted ribozymes is used as an example.

DNA oligonucleotides encoding each ICAM-targeted ribozyme are synthesized individually pooled then cloned and converted to retroviral vectors as a pool.

The resulting retroviral vector particles are used to transduce a target cell line that expresses ICAM Cells expressing ribozymes that inhibit ICAM expression (ICAMlow) are sorted from cells expressing ineffective ribozymes by FACS sorting effective ribozymes enriched in the ICAM-low population of cells are identified by filter hybridization Figure 5 A) shows randomization of the binding arms of a hammerhead ribozyme to produce a Random Library. The binding arms can be of any length and any symmetry, symmetrical or assymmetrical. B) shows complexities of hammerhead Random Ribozyme Libraries comprising a 6-nt or a 7-nt long binding arms.

Figure 6 is a schematic overview of Target Discovery strategy. An oligonucleotide is prepared in a single reaction vessel in which all 4 standard nucleotides are incorporated in a random fashion in the target binding arm(s) of the ribozyme to produce a pool of all possible ribozymes This pool is cloned into an appropriate vector in a single tube to produce the Random Library expression vector and retroviral vector particles are produced from this pool in a single tube The resulting Random Ribozyme Library retroviral expression vector pool is then used to transduce a cell type of interest Cells exhibiting the desired phenotype are then separated from the rest of the population using a number of possible selection strategies (E and see text). Genes that are critical for expression of the selected phenotype can then be identified by sequencing the target binding arms of ribozymes contained in the selected population SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -13- Figure 7 shows an example of application of Random Ribozyme Libraries to identify genes critical for the induction of ICAM expression. Human Umbilical Vein Endothelial Cells (HUVECs) are transduced with a Random Ribozyme Library ICAM expression is induced using TNF-alpha and cells expressing ribozymes that inhibit ICAM induction are selected from cells expressing ineffective ribozymes by sorting ICAM-low cells Genes critical for ICAM induction are identified by sequencing the binding arms of the ribozymes that inhibit ICAM expression in the ICAM-low cells.

Figure 8 is an example of an efficient cloning strategy for producing a Defined or Random Ribozyme Libraries. DNA oligos encoding ribozyme coding regions and restriction sites for cloning are designed to also contain a stem-loop structure on the 3' ends This stem loop forms an intramolecular primer site for extension to form a double-stranded molecule by DNA polymerase The double-stranded fragment is cleaved with appropriate restriction endonucleases to produce suitable ends for subsequent cloning Figure 9 shows molecular analysis of the PNP-targeted Defined Ribozyme Library: sequence analysis. Plasmid DNA from the PNP-targeted Defined Ribozyme Library was prepared and sequenced as a pool. The sequencing primer used reads the non-coding strand of the region encoding the ribozymes. Note that the sequence diverges at the binding arm, converges at the catalytic domain TTTCGGCCTAACGGCCTCATCAG-3'), and then diverges at the other binding arm.

These results are consistent with those expected for a sequence of a heterogeneous pool of clones containing different sequences at the ribozyme binding arms.

Figure 10 shows molecular analysis of the PNP-targeted Defined Ribozyme Library: sequence analysis after propagation in Sup T1 human T cells and selection in mmol 6-thioguanosine. Sup T1 cells were transduced with retroviral vector-based Defined Ribozyme Library comprised of 40 different PNP-targeted ribozyme oligos cloned into the U6+27 transcription unit (Figure 1 The cells were propagated for 2 weeks following transduction, then subjected to 16 days of selection in 10 mmol 6-thioguanosine.

Surviving cells were harvested, and ribozyme sequences present in the selected population SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -14of cells were amplified and sequenced. Note that, relative to the original Library where sequences of the binding arms were ambiguous due to the presence of 40 different ribozymes (Figure the sequence of the binding arms in the selected population corresponded to only 1 of the 40 ribozymes included in the Library. These results suggest that this ribozyme was the most-potent ribozyme of 40 ribozymes tested.

Figure 11 is a schematic representation of transcription units suitable for expression ribozyme library of the instant invention. A) is a diagrammatic representation of some RNA polymerase (Pol) II and III ribozyme (RZ) transcription units. CMV Promoter Driven is a Pol II transcript driven by a cytomegalovirus promoter; the transcript can designed such that the ribozyme is at the region, 3'-region or some where in between and the transcript optionally comprises an intron. tRNA-DC is a Pol III transcript driven by a transfer RNA (tRNA) promoter, wherein the ribozyme is at the 3'-end of the transcript; the transcript optionally comprises a stem-loop structure 3' of the ribozyme.

U6+27 is a Pol III transcript driven by a U6 small nuclear (snRNA) promoter; ribozyme is 3' of a sequence that is homologous to 27 nucleotides at the 5'-end of a U6 snRNA; the transcript optionally comprise a stem-loop structure at the 3'-end of the ribozyme. is a Pol III transcript driven by an adenovirus VA promoter; ribozyme is 3' of a sequence homologous to 90 nucleotides at the 5'-end of a VAI RNA; the transcript optionally comprises a stem-loop structure at the 3'-end of the ribozyme. VAC is a Pol III transcript driven by an adenovirus VAI promoter; the ribozyme is inserted towards the region of the VA RNA and into a S35 motif, which is a stable greater than or equal to 8 bp long intramolecular stem formed by base-paired inteaction between sequences in the and the 3'-region flanking the ribozyme (see Beigelman et al., International PCT Application No. WO 96/18736); the S35 domain positions the ribozyme away from the main transcript as an independent domain. TRZ is a Pol III transcript diven by a tRNA promoter; ribozyme is inserted in the S35 domain and is positioned away from the main transcript (see Beigelman et al., International PCT Application No. WO 96/18736). B) shows various transcription units based on the U1 small nuclear RNA (snRNA) system.

C) is a schematic representation of a retroviral vectors encoding ribozyme genes. NGFR, nerve growth factor receptor is used as a selectable marker, LTR, long terminal repeat of a SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 retrovirus, UTR, untranslated region. D) shows a U6+27 hammerhead ribozyme transcription unit based on the U6 snRNA. The ribozyme transcript comprises the first 27 nt from the U6 snRNA which is reported to be necessary for the stability of the transcript.

The transcript terminates with a stretch of uridine residues. The hammerhead ribozyme shown in the figure has random binding arm sequence.

Figure 12 is a schematic representation of a combinatorial approach to the screening of ribozyme variants.

Figure 13 shows the sequence of a Starting Ribozyme to be used in the screening approach described in Figure 12. The Starting Ribozyme is a hammerhead (HH) ribozyme designed to cleave target RNA A Position 7 in HH-A is also referred to in this application as position 24 to indicate that U24 is the 24th nucleotide incorporated into the HH-A ribozyme during chemical synthesis. Similarly, positions 4 and 3 are also referred to as positions 27 and 28, respectively, s indicates phosphorothioate substitution. Lower case alphabets in the HH-A sequence indicate 2'-O-methyl nucleotides; uppercase alphabets in the sequence of HH-A at positions 5, 6, 8, 12 and 15.1 indicate ribonucleotides. Positions 3, 4 and 7 are shown as uppercase, large alphabets to indicate the positions selected for screening using the method shown in Figure 12. indicates base-paired interaction. iB represents abasic inverted deoxy ribose moiety.

Figure 14 shows a scheme for screening variants of HH-A ribozyme. Positions 24, 27 and 28 are selected for analysis in this scheme.

Figure 15 shows non-limiting examples of some of the nucleotide analogs that can be used to construct ribozyme libraries. 2'-O-MTM-U represents 2'-O-methylthiomethyl uridine; 2'-O-MTM-C represents 2'-O-methylthiomethyl cytidine; 6-Me-U represents 6methyl uridine (Beigelman et al., International PCT Publication No. WO 96/18736 which is incorporated by reference herein).

Figure 16 shows activity of HH-A variant ribozymes as determined in a cell-based assay. indicates the substitution that provided the most desirable attribute in a ribozyme.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -16- Figure 17A shows the sequence and chemical composition of ribozymes that showed the most desirable attribute in a cell.

Figure 17B shows formulae for four different novel ribozyme motifs.

Figure 18 shows the formula foe a novel ribozyme motif.

Figure 19 shows the sequence of a Starting Ribozyme to be used in the screening approach described in Figure 14. A HH ribozyme targeted against RNA B (HH-B) was chosen for analysis of the loop II sequence variants.

Figure 20 shows a scheme for screening loop-II sequence variants of HH-B ribozyme.

Figure 21 shows the relative catalytic rates (kre) for RNA cleavage reactions catalyzed by HH-B loop-II variant ribozymes.

Figure 22 is a schematic representation of HH-B ribozyme-substrate complex and the activity of HH-B ribozyme with either the 5'-GAAA-3' or the 5'-GUUA-3' loop-II sequence.

Figure 23 shows a scheme for using a combinatorial approach to identify potential ribozyme targets by varying the binding arms.

Figure 24 shows a scheme for using a combinatorial approach to identify novel ribozymes by the varying putative catalytic domain sequence.

Figure 25 shows a table of accessible sites within a Bcl-2 transcript ((975 nucleotides) which were found using the combinatorial in vitro screening process.

Figure 26 shows a table of accessible sites with a Kras transcript (796 nucleotides) which were found using the combinatorial in vitro screening process as well as a graphic depiction of relative activity of ribozymes to those sites.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -17- Figure 27 shows a table of accessible sites with a UPA transcript (400 nucleotides) which were found using the combinatorial in vitro screening process as well as a graphic depiction of relative activity of ribozymes to those sites.

Figure 28 shows a graph displaying data from a ribonuclease protection assay (RPA) after treatment of MCF-7 cells with ribozymes to targeted to site 549 of the transcript (Seq.ID The Bcl-2 mRNA isolated from MCF-7 cells is normalized to GAPDH which was also probed in the RPA. The graph includes an untreated control and an irrelevant ribozyme (no complementarity with Bcl-2 mRNA).

Figure 29 displays a schematic representation of NTP synthesis using nucleoside substrates.

Figure 30 depicts a scheme for the synthesis of a xylo ribonucleoside phosphoramidite.

Figure 31 is a diagrammatic representation of hammerhead (HH) ribozyme targeted against stromelysin RNA (site 617) with various modifications.

Figure 32 is a is a schematic representation of a one pot deprotection of RNA synthesized using RNA phosphoramidite chemistry.

Figure 33 is a comparison of a one-pot and a two-pot process for deprotection of

RNA.

Figure 34 shows the results of a one-pot deprotection with different polar organic reagents.

Figure 35 is a diagrammatic represention of ras signal transduction pathway.

Figure 36 is a diagrammatic representation of hammerhead ribozymes targeted against c-raf RNA.

Figure 37 is a graphical representation of c-raf 2'-C-allyl 1120 hammerhead (HH) ribozyme-mediated inhibition of cell proliferation.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 18- Figure 38 is a graphical representation of inhibition of cell proliferation mediated by c-raf 2'-C-allyl 1120 and 1251 hammerhead (HH) ribozymes.

Figure 39 shows the effects of flt-1 ribozymes (active/inactive) on LLC-HM primary tumor growth in mice.

Figure 40 shows the effects offlt-1 ribozymes on LLC-HM primary tumor volume immediately following the cessation of treatment.

Figure 41 shows the effects offlt-1 ribozymes on lung metastatic indices (number of metastases and lung mass).

Figure 42 shows the effects of flk-1 ribozymes (active/inactive) on LLC-HM primary tumor growth in mice.

Figure 43 shows the effects offlk-1 ribozymes on LLC-HM primary tumor volume immediately following the cessation of treatment.

Figure 44 shows the effects of flk-1 ribozymes on lung metastatic indices (number of metastases and lung mass).

Nucleic Acid Catalysts: Catalytic nucleic acid molecules (ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such nucleic acid catalysts can be used, for example, to target cleavage of virtually any RNA transcript (Zaug et al., 324, Nature 429 1986 Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989). Catalytic nucleic acid molecules mean any nucleotide base-comprising molecule having the ability to repeatedly act on one or more types of molecules, including but not limited to nucleic acid catalysts. By way of example but not limitation, such molecules include those that are able to repeatedly cleave nucleic acid molecules, peptides, or other polymers, and those that are able to cause the polymerization of such nucleic acids and other polymers. Specifically, such molecules SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -19include ribozymes, DNAzymes, external guide sequences and the like. It is expected that such molecules will also include modified nucleotides compared to standard nucleotides found in DNA and RNA.

Because of their sequence-specificity, trans-cleaving nucleic acid catalysts show promise as therapeutic agents for human disease (Usman McSwiggen, 1995 Ann. Rep.

Med Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).

Nucleic acid catalysts can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited. In addition, nucleic acid catalysts can be used to validate a therapeutic gene target and/or to determine the function of a gene in a biological system (Christoffersen, 1997, Nature Biotech. 15, 483).

There are at least seven basic varieties of enzymatic RNA molecules derived from naturally occurring self-cleaving RNAs (see Table Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a substrate/target RNA. Such binding occurs through the substrate/target binding portion of an nucleic acid catalyst which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic and selective cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and thus can repeatedly bind and cleave new targets.

In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R.

Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Breaker, 1997, Nature Biotech. 15, 427).

There are several reports that describe the use of a variety of in vitro and in vivo selection strategies to study structure and function of catalytic nucleic acid molecules (Campbell et al., 1995, RNA 1, 598; Joyce 1989, Gene, 82,83; Lieber et al., 1995, Mol Cell Biol. 15, 540; Lieber et al., International PCT Publication No. WO 96/01314; Szostak 1988, in Redesigning the Molecules of Life, Ed. S. A. Benner, pp 87, Springer-Verlag, Germany; Kramer et al., U.S. Patent No. 5,616.459; Draper et al., US Patent No.

5,496,698; Joyce, U.S. Patent No. 5,595,873; Szostak et al., U.S. Patent No. 5,631,146).

The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme sufficient to effect a therapeutic treatment is generally lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme (enzymatic nucleic acid) molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base-pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.

The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (kcat) of -1 min- 1 in the presence of saturating (10 mM) concentrations of Mg 2 cofactor. However, the rate for this ribozyme in Mg 2 SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -21concentrations that are closer to those found inside cells (0.5 2 mM) can be 10- to 100fold slower. In contrast, the RNase P holoenzyme can catalyze pre-tRNA cleavage with a kcat of-30 min- 1 under optimal assay conditions. An artificial 'RNA ligase' ribozyme (Bartel et al., supra) has been shown to catalyze the corresponding self-modification reaction with a rate of -100 min- 1 In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min- 1 Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain self-cleaving ribozymes may not be optimized to give maximal catalytic activity, or that entirely new RNA motifs could be made that display significantly faster rates for RNA phosphoester cleavage.

By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a sugar moiety. Nucleotide generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; all hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art and has recently been summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines 5-methylcytidine), 5-alkyluridines ribothymidine), SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -22- 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines 6methyluridine) and others (Burgin et al., 1996, Biochemistry, 35, 14090). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used within the catalytic core of the enzyme and/or in the substrate-binding regions.

In another preferred embodiment, catalytic activity of the molecules described in the instant invention can be optimized as described by Draper et al., supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No.

WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules). Modifications which enhance their efficacy in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All of these publications are hereby incorporated by reference herein).

There are several examples in the art describing sugar and phosphate modifications that can be introduced into nucleic acid catalysts without significantly effecting catalysis and with significant enhancement in their nuclease stability and efficacy. Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996 Biochemistry 14090). Sugar modification of nucleic acid catalysts has been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al.

Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317; Usman and SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 23 Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, US Patent No. 5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702; all of the references are hereby incorporated in their totality by reference herein).

Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid catalysts of the instant invention.

In yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity is provided. Such a nucleic acid is also, generally, more resistant to nucleases than the corresponding unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered.

As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such modifications herein are said to "maintain" the enzymatic activity on all RNA ribozyme.

In a preferred embodiment, the nucleic acid catalysts of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. Using the methods described herein, other nucleic acid catalysts that cleave target nucleic acid may be derived and used as described above. Specific examples of nucleic acid catalysts of the instant invention are provided below in the Tables and figures.

Sullivan, et al., WO 94/02595, describes the general methods for delivery of nucleic acid catalysts. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -24ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al., supra and Draper et al., WO 93/23569 which have been incorporated by reference herein.

Such nucleic acid catalysts can be delivered exogenously to specific cells as required. In the preferred hammerhead motif the small size (less than 60 nucleotides, preferably between 30-40 nucleotides in length) of the molecule allows the cost of treatment to be reduced.

Therapeutic ribozymes delivered exogenously must remain stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, ribozymes must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA (Wincott et al., 1995 Nucleic Acids Res. 23. 2677; incorporated by reference herein) have expanded the ability to modify ribozymes by introducing nucleotide modifications to enhance their nuclease stability as described above.

Synthesis, Deprotection. and Purification of Nucleic Acid Catalysts: Generally, RNA molecules are chemically synthesized and purified by methodologies based on the use of tetrazole to activate the RNA phosphoramidite, ethanolic-NH40H to remove the exocyclic amino protecting groups, tetra-nbutylammonium fluoride (TBAF) to remove the 2'-OH alkylsilyl protecting groups, and gel purification and analysis of the deprotected RNA. Examples of chemical synthesis, deprotection, purification and analysis procedures for RNA are provided by Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al. Nucleic Acids Res. 1990, 18, 5433- SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 5341; Perreault et al. Biochemistry 1991, 30 4020-4025; Slim and Gait Nucleic Acids Res.

1991, 19, 1183-1188. All the above noted references are all hereby incorporated by reference herein.

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs antisense oligonucleotides, hammerhead or the hairpin ribozymes) are used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of the mRNA structure. However, these nucleic acid molecules can also be expressed within cells from eukaryotic promoters Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci.USA 83, 399; SullengerScanlon et al., 1991, Proc.

Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992 J. Virol, 66, 1432-41; Weerasinghe et al.. 1991 J. Virol, 65. 5531-4; Ojwang et al., 1992 Proc. Natl. Acad. Sci.USA 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20. 4581-9; Sarver et al., 1990 Science 247, 1222-1225; Thompson et al., 1995 Nucleic Acids Res. 23, 2259). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al., PCT W093/23569, and Sullivan et al., PCT W094/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al., 1992 Nucleic Acids Symp.

Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994 J. Biol. Chem. 269, 25856).

The ribozymes were chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem.

Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'end. Small scale synthesis were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 pmol scale protocol with a 5 min coupling step for alkylsilyl protected nucleotides and 2.5 min coupling step for 2'-O-methylated nucleotides. Table II SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -26outlines the amounts, and the contact times, of the reagents used in the synthesis cycle. A excess (163 iL of 0.1 M 16.3 4mol) of phosphoramidite and a 24-fold excess of S-ethyl tetrazole (238 IL of 0.25 M 59.5 pmol) relative to polymer-bound was used in each coupling cycle. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, were 97.5- 99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc.

synthesizer:detritylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic 2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF (Millipore). B J Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from American International Chemical, Inc.

Deprotection of the RNA was performed as follows. The polymer-bound oligoribonucleotide, trityl-off, was transferred from the synthesis column to a 4mL glass screw top vial and suspended in a solution of methylamine (MA) at 65 °C for 10 min.

After cooling to -20 oC, the supernatant was removed from the polymer support. The support was washed three times with 1.0 mL of EtOH:MeCN:H 2 0/3:1:1, vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder.

The base-deprotected oligoribonucleotide was resuspended in anhydrous TEA*HF/NMP solution (250 LL of a solution of 1.5mL N-methylpyrrolidinone, 750 uL TEA and 1.0 mL TEA-3HF to provide a 1.4M HF concentration) and heated to 65 0 C for h. The resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.

For anion exchange desalting of the deprotected oligomer, the TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB mL), the RNA was eluted with 2 M TEAB (10 mL) and dried down to a white powder.

Deprotection of RNA: SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -27- For high throughput chemical synthesis of oligoribonucleotides, it is important that the two main steps involved in the deprotection of oligoribonucleotides aqueous basic treatment to remove exocyclic amino protecting groups and phosphate protecting groups and fluoride treatment to remove the 2'-OH alkylsilyl protecting groups such as the tButylDiMethylSilyl) are condensed.

Stinchcomb et al., supra describe a time-efficient 2 hrs) one-pot deprotection protocol based on anhydrous methylamine and triethylamine trihydrogen fluoride. Since it has recently been reported that water contamination during fluoride treatment may be detrimental to the efficiency of the desilylation reaction (Hogrefe et al, Nucleic Acids Res.

(1993), 21 4739-4741), it is necessary to use an anhydrous solution of base such as a 33% methylamine in absolute ethanol followed by neat triethylamine trihydrofluoride to effectively deprotect oligoribonucleotides in a one-pot fashion. However it may be cumbersome to apply such a protocol to plate format deprotection where the solid-support is preferentially separated from the partially deprotected oligoribonucleotides prior to the 2'-hydroxyl deprotection. Indeed, because the methylamine solution used is anhydrous, it may not be suitable to solubilize the negatively charged oligoribonucleotides obtained after basic treatment. Therefore, applicant investigated a 1:1 mixture of the ethanolic methylamine solution and different polar additives such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), methanol, hexamethylphosphoramide (HMPA), 1methyl-2-pyrrolidinone (NMP) or 2-methoxyethyl ether (glyme). Of all these additives, dimethylsufoxide is capable of efficiently solubilizing partially deprotected oligoribonucleotides (figure 34). A comparison of the one pot and two pot deprotection methods are outlined and demonstrated in figure 33.

The deprotection process commonly involves the deprotection of the exocyclic amino protecting groups by NH4OH, which is time consuming (6-24 h) and inefficient.

This step is then followed by treatment with TBAF to facilitate the removal of alkylsilyl protecting groups, which again is time consuming and not very effective in achieving efficient deprotection.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -28- A recent modification of this two-step strategy for oligoribonucleotide deprotection has been reported by Wincott et al., (Nucleic Acids Res., 1995, 23, 2677- 2784) and by Vinayak et al., (Nucleic Acids Symposium series, 1995. 33, 123-125). The optimized conditions make use of aqueous methylamine at 65 0 C for 15 minutes in place of the ammonium hydroxide cocktail to remove exocyclic amino protecting groups while the desilylation treatment needed to remove the 2'-OH alkylsilyl protecting groups utilizes a mixture of triethylamine trihydrogen fluoride (TEA.3HF), N-methyl-pyrrolidinone and triethylamine at 65 0 C for 90 minutes, thereby replacing tetrabutyl ammonium fluoride.

Stinchcomb et al., International PCT Publication No. WO 95/23225 describe a process for one pot deprotection of RNA. On page 73, it states that: "In an attempt to minimize the time required for deprotection and to simplify the process of deprotection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol... According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65 °C for 15 minutes and the reaction is allowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 minutes in a TEA*3HF reagent. The reaction is quenched with 16 mM TEAB solution." This invention concerns a one-pot process for the deprotection of RNA molecules.

This invention features a novel method for the removal of protecting groups from the nucleic acid base and 2'-OH groups, which accelerates the process for generating synthetic RNA in a high throughput manner in a 96 well format).

Chemical synthesis of RNA is generally accomplished using a traditional column format on a RNA synthesizer where only one oligoribonucleotide is synthesized at a time.

Simultaneous synthesis of more than one RNA molecule in a time efficient manner requires alternate methods to the traditional column format, such as synthesis in a 96 well plate format where up to 96 RNA molecules can be synthesized at the same time. To expedite this process of simultaneous synthesis of multiple RNA molecules, it is important to accelerate some of the time consuming processes such as the deprotection of RNA following synthesis removal of base protecting group, such as the exocyclic SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -29amino protecting group and the phosphate protecting groups and the removal of 2'-OH protecting groups, such as the tButylDiMethylSilyl). In a preferred embodiment, the invention features a one-pot process for rapid deprotection of RNA.

Stinchcomb et al., supra described a one-pot protocol for RNA deprotection using anhydrous methylamine and triethylamine trihydrogen fluoride. This procedure involves the use of an anhydrous solution of base such as a 33% methylamine in absolute ethanol followed by neat triethylamine trihydrofluoride to effectively deprotect oligoribonucleotides in a one-pot fashion. However such a protocol may be cumbersome for deprotection of RNA synthesized on a plate format, such as a 96 well plate, because it may be necessary to separate the solid-support from the partially deprotected RNA prior to the 2'-hydroxyl deprotection. Also, since the methylamine solution used is anhydrous, it may be difficult to solubilize the negatively charged oligoribonucleotides obtained after basic treatment. So, in a first aspect the invention features the use of a 1:1 mixture of the ethanolic methylamine solution and a polar additive, such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), methanol, hexamethylphosphoramide (HMPA), 1methyl-2-pyrroiidinone (NMP), 2-methoxyethyl ether (glyme) or the like. More specifically, dimethylsufoxide is used to partially deprotect oligoribonucleotides (Figure 32). A comparison of the one pot and two pot deprotection methods are outlined and demonstrated in Figure 33.

This invention also concerns a rapid (high through-put) deprotection of RNA in a 96-well plate format. More specifically rapid deprotection of enzymatic RNA molecules in greater than microgram quantities with high biological activity is featured. It has been determined that the recovery of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its deprotection.

In a preferred embodiment, the invention features a process for one-pot deprotection of RNA molecules comprising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be branched or unbranched, ethyl, propyl or butyl and is preferably methyl, methylamine), trialkylamine (where alkyl can be branched or unbranched, methyl, propyl or butyl and is SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 preferably ethyl, ethylamine) and dimethylsulfoxide, preferably in a 10:3:13, or 1:0.3:1 proportion at temperature 20-30 °C for about 30-100 minutes, preferably minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group 2-cyanoethyl) (vs 4-20 h at 55-65 °C using NH 4 0H/EtOH or

NH

3 /EtOH, or 10-15 min at 65 0 C using 40% aqueous methylamine) under conditions suitable for partial deprotection of the RNA; b) contacting the partially deprotected RNA with anhydrous triethylamine-hydrogen fluoride (3HF-TEA) and heating at about 50-70 oC, preferably at 65 for about 5-30 min, preferably 15 min to remove the 2'-hydroxyl protecting group (vs 8 24 h using TBAF, or TEA-3HF for 24 h (Gasparutto et al. Nucleic Acids Res. 1992, 20, 5159-5166) (Other alkylamine-HF complexes may also be used, trimethylamine or diisopropylethylamine) under conditions suitable for the complete deprotection of the RNA. The reaction can then be quenched by using aqueous ammonium bicarbonate (1.4 Although some other buffers can be used to quench the desilylation reaction triethylammonium bicarbonate, ammonium acetate), the ammonium bicarbonate buffer is perfectly suited to retain the 5'-O-dimethoxytrityl group at the 5'-end of the oligoribonucleotide thereby facilitating a reverse phase-based solidphase extraction purification protocol.

By "one-pot" deprotection is meant that the process of deprotection RNA is carried out in one container instead of multiple containers as in two-pot deprotection.

In another preferred embodiment, the invention features a process for one pot deprotection of RNA molecules comprising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be branched or unbranched, ethyl, propyl or butyl and is preferably methyl, methylamine), and dimethylsulfoxide, preferably in a 1:1 proportion at 20-30 OC temperature for about 100 minutes, preferably 90 minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group 2-cyanoethyl) (vs 4-20 h at 55-65 °C using NH 4 0H/EtOH or NH 3 /EtOH, or 10-15 min at 65 0 C using 40% aqueous methylamine) under conditions suitable for partial deprotection of the RNA; b) contacting the partially deprotected RNA with anhydrous triethylamine-hydrogen fluoride (3HF-TEA) and heating at about 50-70 preferably at 65 for about 5-30 min, SUBSTITUTE SHEET (RULE 26) 31 it may be difficult to solubilize the negatively charged oligoribonucleotides obtained after basic treatment. So, in a first aspect the invention features the use of a 1:1 mixture of the ethanolic methylamine solution and a polar additive, such as dimethylsulfoxide

(DMSO),

N,N-dimethylformamide (DMF), methanol, hexamethylphosphoramide (HMPA), 1methyl-2-pyrrolidinone (NMP), 2-methoxyethyl ether (glyme) or the like. More specifically, dimethylsufoxide is used to partially deprotect oligorbonucleotides (Figure 32). A comparison of the one pot and two pot deprotection methods are outlined and demonstrated in Figure 33.

Sll crc is also disclosed a rapid (high through-put) deprotection of RNA in a 9 6-well plate format. More specifically rapid deprotection of enzymatic RNA molecules n greater than microgram quantities with high biological activity is featured. It has been determined that the recovery of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its deprotection.

prcle n rred aspect of this disclosure features a process for one-pot deprotection oRNA molecules compising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be S branched o unbrancd, ethyl, propyl or buty and is preferably methyl, e.g.

S methylamine), trialkylamine (where alkyl can be branched or unbranched, methyl, propyl 0 or butyl and is preferably ethyl, ethylamine) and dimethylsulfoxide, preferably in:a 10:13, or 1:0.3:1 proportion at temperature 20-30 'C for about 30-100 minutes, Preferably 90 minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group 2 -cyanoethyl) (vs 4-20 h at 55-65 'C using

NH

4 0H/EtOH or NH 3 /EtOH, or 10-15 min at 65 0 C using 40% aqueous methylamine) under conditions suitable for partial deprotection of the RNA; b) contacting the pa-tially deprotected RNA with anhydrous triethylaminehydrogen fluoride (3HF*TEA) and heating at about 50-70 preferably at 65 oC, for about 5-30 min, preferably 15 min to remove the 2 '-hydroxyl protecting group (vs 8 24 h using TBAF, or TEA*3HF for 24 h (Gasparutto et al. Nucleic Acids Res. 1992, 20, 5159-5166) (Other alkylamine*HF complexes may also be used, trimethylamine or diisopropylethylamine) under conditions suitable for the complete deprotection of the RNA. The reaction can then be quenched by using aqueous armonium bicarbonate (1.4 Although some other buffers can be used to quench the desilylation reaction triethylammonium bicarbonate, ammonium acetate), the ammonium bicarbonate buffer is perfectly suited to retain the 5'-O-diethoxytrityl group at the 5'-end of the oligorbonucleotide thereby facilitating reverse phase-based solid-phase extraction purification protocol.

By "one-pol" deprotection is meant that the process of deprotection RNA4 is can-ied out in one container instead of multiple containers as in two-pot deprotection.

1 0 \nother preferred aspect of this disclosure features a process for one pot deprotection ofRNA molecules comprising protecting groups, comprising the steps of: a) contacting the RNA with a mixture of anhydrous alkylamine (where alkyl can be branched or unbranched, ethyl, propyl or butyl and is preferably methyl, e.g., methylamine), and dimethylsulfoxide, preferably in a 1:1 proportion at 20-30 'C 15 temperature for about 30-100 minutes, preferably 90 minutes, to remove the exocyclic amino (base) protecting groups and the phosphate protecting group 2 -cyanoethyl) 4-20 h at 55-65 'C using NH40H/EtOH or NH 3 /EtOH, or 10-15 min at 65 0 C using 40% aqueous methylamine) under conditions suitable for partial deprotection of the N A b) contacting the partially deprotected RNA with anhydrous 0 thyamnehydrogen fluoride (3HF*TEA) and heating at about 50-70 OC, preferably at 65 C, for about 5-30 min, preferably 15 min to remove the 2 '-hydroxyl protecting group (Other alkylamine-HF complexes may also be used, trimethylamine or diisopropylethylamine) under conditions suitable for the complete deprotection of the RNA. The reaction can then be quenched by using aqueous ammonium bicarbonate (1.4 Although some other buffers can be used to quench the desilylation reaction (i.e.

trethylammonium bicarbonate, ammonium acetate), the ammonium bicarbonate buffer is perfectly suited to retain the 5'-O-dimethoxytrityl group at the 5'-end of the oligoribonucleotide thereby facilitating a reverse phase-based solid-phase extraction purification protocol.

lcrie is further disclosed a process for RNA deprotection where the exocycic amino and phosphate deprotection reaction is performed with the ethanolic Smethylamine solution atroom temperature for about 90 nin or at 65 0 C for 15 min or at for 30 min or at 35 0 C for 60 min.

prelrred aspect of this disclosurc is a process for deprotection of RNA of the present Ivention is used to deprotect a ribozyme synthesized using a column format as described in (Scaringe er al., supra; Wicott er al., supra).

Inactive hammerhead ribozymes were synthesized by substituting a U for G5 and aU for Al 1 4 (numbering from Hertel, K. et al., 1992, Nucleic cids Res., 20, 3252).

Teaverage stepwie yiedsweei98 The average stepwise coupling yields were >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684).

Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active rbozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840) Ribozymes are also synthesized from DNA templates using bacteiophage T7 RNA Spolymerase (Milligan and Uhlenbeck, 1989, Methods Enzynol. 180, 51).

:Ribozymes are modified to enhance stability and/or enhance catalytic activity by 4 modification with nuclease resistant groups, for example, 2 '-amino, 2 '-C-allyl, 2 '-flouro, 2 '-O-methyl, 2-H, nucleotide base modifications (for a review sec Usman and Cedergren, 1992 TIBS 17, 34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgih et al.

1996 Biochemistry 6, 14090).

Ribozymes were purified by gel electrophoresis using general methods or are pufied by high pressure liquid chromatography (HPLC; See Stinchcomb et aii., WO 98/50530 PCT/US98/09249 34- Lin et al., 1994, Nucleic Acids Research 22, 5229-5234, reports the incorporation of 2'-NH 2 -CTP and 2'-NH,-UTP into RNA using bacteriophage T7 RNA polymerase and polyethylene glycol containing buffer. The article describes the use of the polymerase synthesized RNA for in vitro selection of aptamers to human neutrophil elastase (HNE).

The invention features NTP's having the formula triphosphate-OR, for example the following formula I: O O O -0 P 0 P 0 P OR I I I 0- O- 0where R is any nucleoside; specifically the nucleosides 2'-O-methyl-2,6-diaminopurine riboside; 2'-deoxy-2'amino-2,.6-diaminopurine riboside; 2'-(N-alanyl) amino-2'-deoxyuridine; 2'-(N-phenylalanyl)amino-2'-deoxy-uridine; 2'-deoxy -2'-(N-p-alanyl) amino 2'deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl uridine; 2'-O-amino-uridine; methylthiomethyl adenosine; 2'-O-methylthiomethyl cytidine 2'-O-methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-Deoxy-2'-(N-histidyl) amino uridine; 2'cytidine; 2'-(N-p-carboxamidine-p-alanyl)amino- 2 -deoxyuridine; 2'-deoxy-2'-(N--alanyl)-guanosine: and 2'-O-amino-adenosine.

In a second aspect, the invention features a process for the synthesis of pyrimidine nucleotide triphosphate (such as UTP, 2'-O-MTM-UTP, dUTP and the like) including the steps of monophosphorylation where the pyrimidine nucleoside is contacted with a mixture having a phosphorylating agent (such as phosphorus oxychloride, phospho-tristriazolides, phospho-tris-triimidazolides and the like), trialkyl phosphate (such as triethylphosphate or trimethylphosphate or the like) and dimethylaminopyridine (DMAP) under conditions suitable for the formation of pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine monophosphate is contacted with a pyrophosphorylating reagent (such as tributylammonium pyrophosphate) under conditions suitable for the formation of pyrimidine triphosphates.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 By "pyrimidines" is meant nucleotides comprising modified or unmodified derivatives of a six membered pyrimidine ring. An example of a pyrimidine is modified or unmodified uridine.

By "nucleotide triphosphate" or "NTP" is meant a nucleoside bound to three inorganic phosphate groups at the 5' hydroxyl group of the modified or unmodified ribose or deoxyribose sugar where the 1' position of the sugar may comprise a nucleic acid base or hydrogen. The triphosphate portion may be modified to include chemical moieties which do not destroy the functionality of the group allow incorporation into an RNA molecule).

In another preferred embodiment, nucleoside triphosphates (NTP's) of the instant invention are incorporated into an oligonucleotide using an RNA polymerase enzyme.

RNA polymerases include but are not limited to mutated and wild type versions of bacteriophage T7, SP6, or T3 RNA polymerases.

In yet another preferred embodiment, the invention features a process for incorporating modified NTP's into an oligonucleotide including the step of incubating a mixture having a DNA template, RNA polymerase, NTP, and an enhancer of modified NTP incorporation under conditions suitable for the incorporation of the modified NTP into the oligonucleotide.

By "enhancer of modified NTP incorporation" is meant a reagent which facilitates the incorporation of modified nucleotides into a nucleic acid transcript by an RNA polymerase. Such reagents include but are not limited to methanol; LiCl; polyethylene glycol (PEG); diethyl ether; propanol; methyl amine; ethanol and the like.

In another preferred embodiment, the modified nucleoside triphosphates can be incorporated by transcription into a nucleic acid molecules including enzymatic nucleic acid, antisense, 2-5A antisense chimera, oligonucleotides, triplex forming oligonucleotide (TFO), aptamers and the like (Stull et al., 1995 Pharmaceutical Res. 12, 465).

By "antisense" it is meant a non-nucleic acid catalyst that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., SUBSTITUTE SHEET (RULE 26) Polyethylene glycol containing buffer. The article describes the use of the polymerase Synlthesized RNA for in vitro selection of aptamners to human neutrophil elastase hIere '1e- also disclosed NTP's having the formula tniphosphate-OR, for example the following formula

L:

-0 P -P-o P

OR

0- U- 0wVhere R is any nucleoside- specifically the nucleosides 2'-O-methyl-2,6-diaminopurine riboside; 2 '-deoxy-2'amino2,6-diaminopurine riboside; 2 '-(A'-alanyl) amino-2 '-deoxy- Unie 2'-(N-phenylalanyl)amino-2'-deox- die 2'-deoxN' 2 '-(N-f3-alanx'l) amino 2'-deoxv-2'. (lysiyl) am'n nriine; 2'-C-allyl uridine; 2 '-O-ami no-rie; 2'-O- ,ethylth i onethvi; adenosine; 2 '--methylthiomethyl cvtidine 2 '-O-methylthiomethy] gunsn; 2 '-O0-methyl thiomnethyl -uridi ne; 2 '-Deoxy-2'-(N-histidyl) amino ur-idine; 2'deoxy-2 )'-amino-5-methyl cytidine; 2'-(N-p-carboxamidinepalany)mino- 2 '-deoxy- Li rdine; 2 '-deoxy-2 3 -alanyl)-guanosine; and 2 '-O-amino-adenosine.

There isalso disclosed a process for the synthesis of pyrimidine nuclcotide triphosphate (such as UTP, 2'-O-MTM-UTP, dUTP and the like) including the steps of monophosphorylation where the pyrimidine nucleoside is contacted with a mixture having a phosphorylating agent (such as phosphor-us oxychloride, phospho-t-isnazolides, phospho-tris-tnimidazo I]des and the like), trialkyl phosphate (such as triethyiphosphate or trim ethyliphosphate or the like) and dimethylaminopyi dine (DM4AP) under conditions suitable for the formation of pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine monophosphate is contacted with a pyrophosphorylating reagent (such as tributylanu-monium pyrophosphate) under conditions suitable for the formation of pyrimidine triphosphates.

By "pyrimidines" s meant nucleotides comprising modified or unmodified derivatives of a six membered pyrimidine ring. An example of a pyrimidine is modified or unmodified uridine.

By "nucleotide triphosphate" or "NTP" is meant a nucleoside bound to three inorganic phosphate groups at the 5' hydroxyl group of the modified or unmodified ribose or deoxyribose sugar where the 1' position of the sugar may comprse a nucleic acid base or hydrogen. The triphosphate portion may be modified to include chemical moieties which do not destroy the functionality of the group allow incorporation into an RNA molecule).

S In another pre'crred embodiment. nucleoside triphosphates (NIP's) Sare incorporated into an oligonucleotide using an RNA polymerase enzyme.

P NA polymerases include but are not limited to mutated and wild type versions of bacteriophage T7, SP6, or T3 RNA polymerases a l c here is also disclosed a process for incorporating modified NTP's into an oligonucleotide including the step of incubating a mixture having a DNA template, RNA polymerase, NTP, and an enhancer of modified NTP incorporation under conditions suitable for the incorporation of the modified

NTP

into the oligonucleotide.

.By"enhaner of modified NTP incorporation" is meant a reagent which facilitates the incorporation of modified nucleotides into a nulec acd transcript by an RNA polymerase. Such reagents include but are not limited to methanol; LiCI; polyethylene glycol (PEG); diethyl ether; propanol; methyl amine; ethanol and the like.

I he modiliced nucleoside triphosphates can also be incorporated by transcription into a nucleic acid molecules including enzymatic nucleic acid, antisense, 2-5A antisense chimera, oligonucleotides, triplex forming oligonucleotide (TFO), aptamers and the like (Stull e al., 1995 Pharmaceutical Res. 12, 465).

PA

By "antisense" it is meant a non-nucleic acid catalyst that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al.

1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004; Agrawal et al., U.S. Patent No. 5,591,721; Agrawal, U.S. Patent No. 5,652,356).

By "2-5A antisense chimera" it is meant, an antisense oligonucleotide containing a phosphorylated 2 '-5'-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2 -5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence e al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).

By "triplex forming oligonucleotides (TFO)" it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504).

By "oligonucleotide" as used herein is meant a molecule having two or more nucleotides. The polynucleotide can be single, double or multiple stranded and may have modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.

:Ilie 11e modified nucleoside triphosphates disclosed herein S: can be used for combinatorial chemistry or in vitro selection of nucleic 20 acid molecules with novel function. Modified oligonucleotides can be enzymatically synthesized to generate libraries for screening.

Nucleoside modifications of bases and sugars, have been discovered in a variety of naturally occurring RNA tRNA, mRNA, rRNA; reviewed by Hall, 1971 The Modified Nucleosides in Nucleic Acids, Columbia University Press, New York; Limbach er al., 1994 Nucleic Acids Res. 22, 2183). In an attempt to understand the biological significance, structural and thermodynamic properties, and nuclease resistance of these nucleoside modifications in nucleic acids, several investigators have chemically WO 98/50530 PCT/US98/09249 -39give a Pv-containing interucleoside linkage. A suitable nucleoside building block may also contain a phosphorus atom at the oxidation state III reacting readily, upon activation, to give a P"-containing intemucleoside linkage that can be oxidized to the desired pVcontaining intemucleoside linkage. Applicant has found that the phosphoramidite chemistry (Pm) is a preferred coupling method for ribozyme library synthesis. There are several other considerations while designing and synthesizing certain ribozyme libraries, such as: a) the coupling efficiencies of the nucleotide building blocks considered for a ribozyme library should not fall below 90% to provide a majority of full-length ribozyme; b) the nucleotide building blocks should be chemically stable to the selected synthesis and deprotection conditions of the particular ribozyme library; c) the deprotection schemes for the nucleotide building blocks incorporated into a ribozyme library, should be relatively similar and be fully compatible with ribozyme deprotection protocols. In particular, nucleoside building blocks requiring extended deprotection or that cannot sustain harsh treatment should be avoided in the synthesis of a ribozyme library. Typically, the reactivity of the nucleotide building blocks should be optimum when diluted to 100 mM to 200 mM in non-protic and relatively polar solvent. Also the deprotection condition using 3:1 mixture of ethanol and concentrated aqueous ammonia at 65 degrees C. for 4 hours followed by a fluoride treatment as exemplified in Wincott et al. supra, is particularly useful for ribozyme synthesis and is a preferred deprotection pathway for such nucleotide building blocks.

In one preferred embodiment, a "nucleotide building block mixing" approach to generate ribozyme libraries is described. This method involves mixing various nucleotide building blocks together in proportions necessary to ensure equal representation of each of the nucleotide building blocks in the mixture. This mixture is incorporated into the ribozyme at position(s) selected for randomization.

The nucleotide building blocks selected for incorporation into a ribozyme library, are typically mixed together in appropriate concentrations, in reagents, such as anhydrous acetonitrile, to form a mixture with a desired phosphoramidite concentration. This approach for combinatorial synthesis of a ribozyme library with one or more random positions within the ribozyme (X as described above) is particularly useful since a standard SUBSTITUTE SHEET (RULE 26) prevent their degradation by serum nbonucleases and/or enhance their enzymatic activity (see Eckstein et International Publication No. WO 92/07065; Perrault e al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., Intemational Publication No.

S WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat,

US

Patent No 5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules) Modifications which enhance their efficacv in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference nerein.).

By "enhanced enzymatic activity" is meant to include activity measured in cells "and/or in vivo where the activity is a reflection of both cataytic activity and ribozyme stability. In this invention, the product of these properties is increased or not significantly S 15 (less that 0 fold) decreased in vivo compared to an all RNA ribozyme.

SIn yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity is provided. Such nucleic S acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced fold (Burgin e al., 1996, Bioche,,istr,, 35, 14090). Such ribozymes herein are said to maintain" the enzymatic activity on all RNA ribozyme.

The present disclosure also features a method of synthesizing nbozyme libraries of various sizes. This disclosure describes methods to chemically synthesize ribozyme libraries of various sizes from suitable nucleoside analogs.

Considerazios for the selection of nucleotide building blocks and determination of coupling efficiency: In addition to structural considerations (hydrogen bond donors and WO 98/50530 PCT/US98/09249 -41positions 2, 3, 8 and 9 have been fixed as 2'-deoxy-thymidine while the X positions 4- 7 correspond to an approximately equimolar distribution of all the nucleotide building blocks that make up the X mixture.

In another preferred embodiment, a "mix and split" approach to generate ribozyme libraries is described. This method is particularly useful when the number of selected nucleotide building blocks to be included in the library is large and diverse (greater than nucleotide building blocks) and/or when the coupling kinetics of the selected nucleotide building blocks do not allow competitive coupling even after relative concentration adjustments and optimization. This method involves a multi-step process wherein the solid support used for ribozyme library synthesis is "split" (divided) into equal portions, (the number of portions is equal to the number of different nucleotide building blocks (n) chosen for incorporation at one or more random positions within the ribozyme). For example, if there are 10 different nucleotide building blocks chosen for incorporation at one or more positions in the ribozyme library, then the solid support is divided into different portions. Each portion is independently coupled to one of the selected nucleotide building blocks followed by mixing of all the portions of solid support. The ribozyme synthesis is then resumed as before the division of the building blocks. This enables the synthesis of a ribozyme library wherein one or more positions within the ribozyme is random. The number of "splitting" and "mixing" steps is dependent on the number of positions that are random within the ribozyme. For example if three positions are desired to be random then three different splitting and mixing steps are necessary to synthesize the ribozyme library.

Random ribozyme libraries are synthesized using a non-competitive coupling procedure where each of the selected nucleotide analogs separately couple to an inverse number of aliquots of solid-support or of a growing ribozyme chain on the solid-support. A very convenient way to verify completeness of the coupling reaction is the use of a standard spectrophotometric DMT assay (Oligonucleotide Synthesis, A Practical Approach, ed. M. Gait, pp 48, IRC Press, Oxford, UK; incorporated by reference herein). These aliquots may be subsequently combined, mixed and split into one new aliquot. A similar approach to making oligonucleotide libraries has recently been SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -42described by Cook et al.. (US Patent No. 5,587,471) and is incorporated by reference herein.

Nucleotide Synthesis Addition of dimethylaminopyridine (DMAP) to the phosphorylation protocols known in the art can greatly increase the yield of nucleoside monophosphates while decreasing the reaction time (Fig. 29). Synthesis of the nucleosides of the invention have been described in several publications and Applicants previous applications (Beigelman et al., International PCT publication No. WO 96/18736; Dudzcy et al., Int. PCT Pub. No.

WO 95/11910; Usman et al., Int. PCT Pub. No. WO 95/13378; Matulic-Adamic et al., 1997, Tetrahedron Lett. 38, 203; Matulic-Adamic et al., 1997, Tetrahedron Lett. 38, 1669; all of which are incorporated herein by reference). These nucleosides are dissolved in triethyl phosphate and chilled in an ice bath. Phosphorus oxychloride (POCI 3 is then added followed by the introduction of DMAP. The reaction is then warmed to room temperature and allowed to proceed for 5 hours. This reaction allows the formation of nucleoside monophosphates which can then be used in the formation of nucleoside triphosphates. Tributylamine is added followed by the addition of anhydrous acetonitrile and tributylammonium pyrophosphate. The reaction is then quenched with TEAB and stirred overnight at room temperature (about 20C). The triphosphate is purified using column purification and HPLC and the chemical structure is confirmed using NMR analysis. Those skilled in the art will recognize that the reagents, temperatures of the reaction, and purification methods can easily be alternated with substitutes and equivalents and still obtain the desired product.

The invention provides nucleoside triphosphates which can be used for a number of different functions. The nucleoside triphosphates formed from nucleosides found in table III are unique and distinct from other nucleoside triphosphates known in the art.

Incorporation of modified nucleotides into DNA or RNA oligonucleotides can alter the properties of the molecule. For example, modified nucleotides can hinder binding of nucleases, thus increasing the chemical half-life of the molecule. This is especially important if the molecule is to be used for cell culture or in vivo. It is known in the art that the introduction of modified nucleotides into these molecules can greatly increase the SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -43stability and thereby the effectiveness of the molecules (Burgin et al., 1996, Biochemistry 14090-14097; Usman et al., 1996, Curr. Opin. Struct. Biol. 6, 527-533).

Modified nucleotides are incorporated using either wild type and mutant polymerases. For example, mutant T7 polymerase is used in the presence of modified nucleotide triphosphate(s), DNA template and suitable buffers. Those skilled in the art will recognize that other polymerases and their respective mutant versions can also be utilized for the incorporation of NTP's of the invention. Nucleic acid transcripts were detected by incorporating radiolabelled nucleotides (X- 32 P NTP). The radiolabeled NTP contained the same base as the modified triphosphate being tested. The effects of methanol, PEG and LiCI were tested by adding these compounds independently or in combination. Detection and quantitation of the nucleic acid transcripts was performed using a Molecular Dynamics Phosphorlmager. Efficiency of transcription was assessed by comparing modified nucleotide triphosphate incorporation with all-ribonucleotide incorporation control. Wild type polymerase was used to incorporate NTP's using the manufacturers buffers and instructions (Boehringer Mannheim).

Transcription Conditions Incorporation rates of modified nucleoside triphosphates into oligonucleotides can be increased by adding to traditional buffer conditions, several different enhancers of modified NTP incorporation. Applicant has utilized methanol and LiCI in an attempt to increase incorporation rates of dNTP using RNA polymerase. These enhancers of modified NTP incorporation can be used in different combinations and ratios to optimize transcription. Optimal reaction conditions differ between nucleoside triphosphates and can readily be determined by standard experimentation. Overall however, inclusion of enhancers of modified NTP incorporation such as methanol or inorganic compound such as lithium chloride, have been shown by the applicant to increase the mean transcription rates.

Administration of Nucleoside mono, di or triphosphates SUBSTITUTE SHEET (RULE 26) 44 number of positions that are random within the ribozyme. For example if three positions are desired to be random then three different splitting and mixing steps are necessary to synthesize the ribozyme library.

Random ribozyme libraries are synthesized using a non-competitive coupling S procedure where each of the selected nucleotide analogs separately couple to an inverse number of aliquots of solid-support or of a growing ribozyme chain on the solid-support. A very convenient way to verify completeness of the coupling reaction is the use of a standard spectrophotometric DMT assay (Oligonucleotide Synthesis,

A

Practical Approach, ed. M. Gait, pp 48, IRC Press, Oxford, UK; incorporated by reference herein). These aliquots may be subsequently combined, mixed and split into one new aliquot. A similar approach to making oligonucleotide libraries has recently been described by Cook e al., (US Patent No. 5,587,471) andi i ncorporated by reference herein.

Nucleotide Synthesis 15 Addition of dimethylaminopyridine (DMAP) to the phosphorylation protocols known in the art can greatly increase the yield of nucleoside monophosphates while decreasing the reaction time (Fig. 29). Synthesis of the nucleosides has been described in several publications and Applicants previous applications (Beigelman et al., International PCT publication No. WO 96/18736; Dudzcy et al., Int. PCT Pub. No.

20 WO 95/11910; Usman et al., Int. PCT Pub. No. WO 95/13378; Matulic-Adamic et al., 1997, Tetrahedron Lett. 38, 203; Matulic-Adamic e, al., 1997, Tetrahedron Lett. 38, 1669; all of which are incorporated herein by reference). These nucleosides are dissolved in trethyl phosphate and chilled in an ice bath. Phosphorus oxychloride (POCI,) is then added followed by the introduction of DMAP. The reaction is then warmed to room temperature and allowed to proceed for 5 hours. This reaction allows the formation of nucleoside monophosphates which can then be used in the formation of nucleoside trphosphates. Tributylamine is added followed by the addition of anhydrous acetonitrile and tributylammonium pyrophosphate. The reaction is then quenched with TEAB and stirred overnight at room temperature (about 20C). The triphosphate is purified using column p cation and HPLC and the chemical structure is confirmed using

NMR

analysis. Those skilled in the art will recognize that the reagents, temperatures of the r e a c t i o n a n d p u mt e a ndo reaction, and purification methods can easily be alternated with substitutes and equivalents and still obtain the desired product.

HIis disclOsuIr provides nucleoside triphosphates which can be used for a number of different functions. The nucleoside triphosphates formed from nucleosides found in table III are unique and distinct from other nucleoside triphosphates known in the art.

Incororation of modified nucleotides into DNA or RNA oligonucleotides can alter the properties of the molecule. For example, modified nucleotides can hinder binding of Snucleases, thus increasing the chemical half-life of the molecule. This is especially important if the molecul s p mportant if the molecule is to be used for cell culture or in vio. It is known in the art 0 that the introduction of modified nucleotides into these molecules can greatly increase the stability and thereby the effectiveness of the molecules (Burgin et al., 1996. Biochemisty 35 14090-14097; Usman et 1996, Curr. Opin. Struct. Biol. 6, 527-533).

Modified nucleotides are incorporated using either wild type and mutant oymerases. For example, mutant T7 polymerase is used in the presence of modified nucleotide triphosphate(s), DNA template and suitable buffers Those skilled in the art will recognize that other polymerases and their respective mutant versions can also be 20 utilized for the incorporation of NTP's of the invention. Nucleic acd transcpts were *detected b -einc crdtranscripts w ere detected by incorporating radiolabelled nucleotides 3 P NTP). The radiolabeled

NTP

contained the same base as the modified triphosphate being tested. The effects of methanol, PEG and LiCI were tested by adding these compounds independently or in combination. Detection and quantitation of the nucleic acid transcrpts was performed using a Molecular Dynamics Phosphormager. Efficiency of transcription was assessed by comparing modified nucleotide triphosphate incorporation with all-ribonucleotide incorporation control. Wild type polymerase was used to incorporate NTP's using the manufacturers buffers and instructions (Boehringer Mannheim).

Transcription Conditions Incorporation rates of modified nucleoside triphosphates into oligonucleotides can be increased by adding to traditional buffer conditions, several different enhancers of modified NTP incorporation. Applicant has utilized methanol and LiCI in an attempt to increase incorporation rates of dNTP using RNA polymerase. These enhancers of modified NTP incorporation can be used in different combinations and ratios to optimize transcription. Optimal reaction conditions differ between nucleoside triphosphates and can readily be determined by standard experimentation Overall however, inclusion of enhancers of modified NTP incorporation such as methanol or inorganic compound such I0 as lithium chloride, have been shown by the applicant to increase the mean transcription rates.

Administration ofNucleoside mono. di or trinphosphates The nucleotide monophosphates, diphosphates, or triphosphates can be used as a therapeutic agent either independently or in combination with other pharmaceutical components. These molecules can be administered to patients using the methods of Sullivan et al., PCT WO 94/02595. hTle imlecules may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation S. Into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and 20 bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vi-vo to cells or tissues with or without the aforementioned vehicles. Alternatively, the modified nucleotide triphosphate, diphosphate or monophosphate/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intrainuscular subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of delivery and administration are provided in Sullivan et supra and Draper et al., PCT W093/23569 which have been incorporated by reference herein.

Here- ,lso diSClo)Sed a C01OLndpuilhvn IheC 1' ru1Inla I I: R 20 hcenRl Is 01-1, O-R3 'where R3 IS Independenrtly aj mIoiety selected from11 opCoistigo alkcyl, ay.akynyl, aryl1, sn Oal. alkylaryl, caroocyclie aryl/, li t C O V c r N 1 rnid e a nrd ester C h r R Is in d e p e n d e n t] l I a m cietv se e c (_71-0111 r iorl all:'? al I carboc~clc ar l hleterocycl ic aIryl, amide and ester,* halo. NHcR 4 (R4alkyl (Cl acx'l (CI-22), substituted Or unSUbstituted aryl), or OCH9-SCH3 (rnethvithionithyl), ONHJR, where

R.

!S independenrtly H, ainnoacyl 2Foup.) p)i~tdv gr17oUno bioriny) groupJ, choleSteryi group, K lpoic acid idu retinoic acid residueC, folic a1c,-sdc t'id residue, ascorbic acid residue, :nicotinic acid residue. 6 -amilnopenic]IJil i c acid resi'dueC 7 -aminocephabosporanic acid :~:residue, alkyl, alkenyl, alkynyl, ar\'l. alky laryl, carbocyclic aryl, heterocyclic aryl, ari'de oester, ONR, where R, is independently pyridoxal residue, residue, l 3 -cis-retinal residue, 9 -cis-retinal residue, alk'yl, alkenyl alkynyl, alkylaryl, a b c c ic al y a y ,o r h t r c c i l y a t alklarylhtrcci a -kyarI- B is independently a nucleotide base orit :analog orhydrogen; X sidpednl a phosphorus-containing group; and R, is idpnetyblocking group or a phshru-otiiggroup.

Specficlly an"alkyl"~ group refers to a saturated aliphatic hydrocarbon, including straight-chaiin branched-chain, and cyclic alkyl groups. Preferably, the alkyl 2( group has I to 12 carbons. More preferably it is a lower alkyl of from I to 7 carbons, more preferably I to 4 carbons. The alk-yl goup may be substituted or unsubstituted.

Mihen Substituted the substituted group(s) is preferably, hydroxy, cyano, alkoxy, N0 2 Or N(Cl1 3 2 amino, or SI-.

The terrm "alken\I!' group refers to unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branchied-chain, and cyclic WO 98/50530 PCT/US98/09249 -48- Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; of the hepatitis d virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci.USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No. WO 96/22689; and of the Group I intron by Cech et al., U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in a nucleic acid catalyst of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

In a preferred embodiment, a polynucleotide of the invention would bear -one or more 2'-hydroxylamino functionalities attached directly to the monomeric unit or through the use of an appropriate spacer. Since oligonucleotides have neither aldehyde nor hydroxylamino groups, the formation of an oxime would occur selectively using oligo as a polymeric template. This approach would facilitate the attachment of practically any molecule of interest (peptides, polyamines, coenzymes, oligosaccharides, lipids, etc.) directly to the oligonucleotide using either aldehyde or carboxylic function in the molecule of interest.

SUBSTITUTE SHEET (RULE 26) 49 An "ester" refers to an where R is either alkyl, aryl, or alkylary.

A "blocking group" is a group which is able to be removed after polynucleotide s \nthesis and/or which is compatible with solid phase polynucleotide synthesis.

A "phosphorus containing group" can include phosphorus in forms such as Sdclithoates, phosphoramidites and/or as part of an oligonucleotide.

I o I IlIIre Is 'urlc disclosCd a process for synthesis of tilhe CoMIpounds of formula

II.

preerred aslpect of this disclosure is a process for the synthesis of a lofuranosyl nucleoside phosphoramidite comprising the steps of: a) oxidation of a 2' and 5'-protected ribonucleoside with a an oxidant such as chromium oidd/pydine/aceticanhydride, diehylsufoxide/acticanhdride, or Dess-Martin .4 di methylsu Ifoxi1de/acetic' v reagent (periodinane) followed by reduction with a reducing agent such as, triacetoxy S sodium borohydride, sodium borohydride, or lithium borohydride, under conditions suitable for the formation of 2' and 5'-protected xylofuranosyl nucleoside; b) phosphitylation under conditions suitable for the formation of xylofuranosyl nucleoside phosphoramidite.

0 9 In yet another preferred embodiment, the invention features the incorporation of 4.09 Sthe compounds of Formula II into polynucleotides These compounds can be p 94 incorporated into polynucleotides enzymatically. For example by using bacteriophage T7 RNA polymerase, these novel nucleotide analogs can be incorporated into RNA at one or more positions (Milligan el al., 1989, Methods Enzymo/.. 180, 51). Altematively, novel nucleoside analogs can be incorporated into polynucleotides using solid phase synthesis (Brown and Brown, 1991, in Oligonucleotides and Analogues: A Practical Approach, p.

1, ed. F. Eckstein, Oxford University Press, New York; Wincott et al., 1995, Nucleic Acids Res., 23, 2677; Beaucage Caruthers, 1996, in Bioorganic Cheisir-: Nucleic Acids, p 36, ed. S. M. Hecht, Oxford University Press, New York).

The compounds of Formula II can be used for chemical synthesis of nucleotide tri-phosphates and/or phosphoramidites as building blocks for selective incorporation into oligonucleotides. These oligonucleotides can be used as an antsense molecule, antsense chimera, triplex forming oligonucleotides (TFO) or as an nucleic acid catalyst.

The oligonucleotides can also be used as probes or primers for synthesis and/or sequencing of RNA or DNA.

IJc cmpoll1)unds olF I or,1la I1 can be readily converted into nucleotide di phosphate and nucleotide triphosphates usine standard protocols (for a review see utinson 991, in Ceist ofNucleosidesad Nucleotides v.pp 81-160, Ed. L. B. 0 Townsend, Plenum Press, New York, USA; incorporated by reference herein).

The compounds of Formula II can also be independently or in combination used as an antiviral, anticancer or an antitumor agent. These compounds can also be ndently or in combinatio used with other antiviral, anticancer or an antitumor gents.

9 S 5 In one of the preferred embodiments of the inventions herein, the nucleic acid catalyst is formed in a hammerhead or hairpin motif, but may also be formed in the motif S of a hepatitis d virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs 0 are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, I83; of harp 20 Retroirses 13; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, !1989 Biochemistry 28, 4929, Feldstein et al. 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; of the hepatitis d virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 199, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci.USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group

II

WO 98/50530 PCT/US98/09249 -51 extracellular systems, because the synthesis of large quantities of RNA by enzymatic or chemical methods prior to assessing the efficacy of the catalytic nucleic acid molecules is not necessary. The invention further describes a rapid method of using catalytic nucleic acid molecule libraries to identify the biological function of a gene sequence inside a cell.

Applicant describes a method of using catalytic nucleic acid molecule libraries to identify a nucleic acid molecule, such as a gene, involved in a biological process; this nucleic acid molecule may be a known molecule with a known function, or a known molecule with a previously undefined function or an entirely novel molecule. This is a rapid means for identifying, for example, genes involved in a cellular pathway, such as cell proliferation, cell migration, cell death, and others. This method of gene discovery is not only a novel approach to studying a desired biological process but also a means to identify active reagents that can modulate this cellular process in a precise manner.

Applicant describes herein, a general approach for simultaneously assaying the ability of one or more members of a catalytic nucleic acid molecule library to modulate certain attributes/process(es) in a biological system, such as plants, animals or bacteria, involving introduction of the library into a desired cell and assaying for changes in a specific "attribute," "characteristic" or "process." The specific attributes may include cell proliferation, cell survival, cell death, cell migration. angiogenesis, tumor volume, tumor metastasis, levels of a specific mRNA(s) in a cell, levels of a specific protein(s) in a cell, levels of a specific protein secreted, cell surface markers, cell morphology, cell differentiation pattern, cartilage degradation, transplantation, restenosis, viral replication, viral load, and the like. By modulating a specific biological pathway using a catalytic nucleic acid molecule library, it is possible to identify the gene(s) involved in that pathway, which may lead to the discovery of novel genes, or genes with novel function.

This method provides a powerful tool to study gene function inside a cell. This approach also offers the potential for designing novel catalytic oligonucleotides, identifying ribozyme accessible sites within a target, and for identifying new nucleic acid targets for ribozyme-mediated modulation of gene expression.

In another aspect the invention involves synthesizing a Random Binding Arm Nucleic Acid Catalyst Library (Random Library) and simultaneously testing all members SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 52 of the Random Library in cells. This library has ribozymes with random substrate binding arm(s) and a defined catalytic domain. Cells with an altered attribute (such as inhibition of cell proliferation) as a result of interaction with the members of the Random Library are selected and the sequences of the ribozymes from these cells are determined. Sequence information from the binding arm(s) of these ribozymes can be used to isolate nucleic acid molecules that are likely to be involved in the pathway responsible for the desired cellular attribute using standard technology known in the art, nucleic acid amplification using techniques such as polymerase chain reaction (PCR). This method is a powerful means to isolate new genes or genes with new function.

By "Random Library" as used herein is meant ribozyme libraries comprising all possible variants in the binding arm of a given ribozyme motif. Here the complexity and the content of the library is not defined. The Random Library is expected to comprise sequences complementary to every potential target sequence, for the ribozyme motif chosen, in the genome of an organism. This Random Library can be used to screen for ribozyme cleavage sites in a known target sequence or in a unknown target. In the first instance, the Random Library is introduced into the cell of choice and the expression of the known target gene is assayed. Cells with an altered expression of the target will yield the most effective ribozyme against the known target. In the second instance, the Random Library is introduced into the cell of choice and the cells are assayed for a specific attribute, for example, survival of cells. Cells that survive the interaction with the Random Library are isolated and the ribozyme sequence from these cells is determined. The sequence of the binding arm of the ribozyme can then be used as probes to isolate the gene(s) involved in cell death. Because, the ribozyme(s) from the Random Library is able to modulate down regulate) the expression of the gene(s) involved in cell death, the cells are able to survive under conditions where they would have otherwise died. This is a novel method of gene discovery. This approach not only provides the information about mediators of certain cellular processes, but also provides a means to modulate the expression of these modulators. This method can be used to identify modulators of any cell process in any organism, including but not limited to mammals, plants and bacteria.

SUBSTITUTE SHEET (RULE 26) Scheme 3. Post synthetic oxyamide bond formation L ONH 2 HO R

O-N-C-R

Target Discover-: Applicant has developed an efficient and rapid method for screening libraries of caai\,tc nucleic acid molecules capable of performing a desired function in a cell. The disclosure also features the use of a catalytic nucleic acid library to modulate certain attributes or processes in a biological system, such as a mammalian cell, and to identify and isolate a) nucleic acid catalysts from the library involved in modulating the cellular process/attribute of interest; and b) modulators of the desired cellular process/attribute using the sequence of the nucleic acid catalyst.

S 0* 00 0 00 00* 0 0000 0 0 0000 00 0 oo 0 0 0 0 0 00 0 0 00 4 oo oo0 oo..

\(Mor specilically. the disclosed method involves designing and constructing a catalytic nucleic acid library, where the catalytic nucleic acid includes a catalytic and a substrate binding domain, and the substrate binding domain (arms) are 15 randomized This library of catalytic nucleic acid molecules with randomized binding arm(s) are used to modulate certain processes/attributes in a biological system. The method described in this application involves simultaneous screening of a library or pool of catalytic nucleic acid molecules with various substitutions at one or more positions and selecting for ribozymes with desired function or characteristics or attributes. This disclosure also features a method for constructing and selecting for catalytic nucleic acid molecules for their ability to cleave a given target nucleic acid molecule or an unknown target nucleic acid molecule RNA), and to inhibit the biological function of that target molecule or any protein encoded by it.

It is not necessary to know either the sequence or the structure of the target nucleic acid molecule in order to select for catalytic nucleic acid molecules capable of cleaving the target in this cellular system. The cell-based screening protocol described in the instant invention one which takes place inside a cell) offers many advantages over extracellular systems, because the synthesis of large quantities of RNA by enzymatic or chemical methods prior to assessing the efficacy of the catalytic nucleic acid molecules is not necessary. The disclosure further descrbes a rapid method of using catalytic nucleic S acid molecule libraries to identify the biological function of a gene sequence inside a cell.

Applicant describes a method of using catalytic nucleic acid molecule libraries to identify a nucleic acid molecule, such as a gene, involved in a biological process; this nucleic acid molecule may be a known molecule with a known function, or a known molecule with a previouslv undefined function or an entirely novel molecule. This is a rapid means for I0 identifying. for example, genes involved in a cellular pathway, such as cell proliferation.

cell migration, cell death, and others. This method of gene discovery is not only a novel approach to studying a desired biological process but also a means to identify active reagents that can modulate this cellular process in a precise manner.

Applicant describes herein, a general approach for simultaneously assaying the 15 ability of one or more members of a catalytic nucleic acid molecule library to modulate certain attributes/process(es) in a biological system, such as plants, animals or bacteria, nvolving introduction of the library into a desired cell and assaying for changes in a specific "attribute," "characteristic" or "process." The specific attributes mav include cell S: 2 proliferation, cell survival, cell death, cell migration, angiogenesis, tumor volume, tumor metastasis, levels of a specific mRNA(s) in a cell, levels of a specific protein(s) in a cell, levels of a specific protein secreted, cell surface markers, cell morphology, cell differentiation pattern, cartilage degradation, transplantation, restenosis, viral replication, viral load, and the like. By modulating a specific biological pathway using a catalytic nucleic acid molecule library, it is possible to identify the gene(s) involved in that pathway, which may lead to the discovery of novel genes, or genes with noveFfunction.

This method provides a powerful tool to study gene function inside a cell. This approach also offers the potential for designing novel catalytic oligonucleotides, identifying ribozyme accessible sites within a target, and for identifying new nucleic acid targets for ribozyme-mediated modulation of gene expression.

\notlhcr method described herein involves synthesizing a Random Binding Aimr Nucleic Acid Catalyst Library (Random Library) and simultaneously testing all members of the Random Library in cells. This library has ribozymes with random substrate binding arm(s) and a defined catalytic domain. Cells with an altered attribute (such as inhibition of cell proliferation) as a result of interaction with the members of the Random Library are selected and the sequences of the rbozymes from these cells are determined Sequence information from the binding arm(s) of these ribozymes can be used to isolate nucleic acid molecules that are likely to be involved in the pathway responsible for the desired cellular attribute using standard technology known in tile art, nucleic acid I amplification using techniques such as polymerase chain reaction (PCR). This method is a powerful means to isolate new genes or genes with new function.

By "Random Library" as used herein is meant ribozyme libraries comprising all S possible variants in the binding arm of a given ribozyme motif Here tile complexity and the content of the library is not defined. The Random Library is expected to comprise sequences complementary to every potential target sequence, for the ribozyme motif S chosen, in the genome of an organism. This Random Library can be used to screen for ribozyme cleavage sites in a known target sequence or in a unknown target. In the first S. instance, the Random Library is introduced into the cell of choice and the expression of the known target gene is assayed. Cells with an altered expression of the target will yield 0 the most effective n bozyme against the known target. In the second instance, the Random Library is introduced into the cell of choice and the cells are assayed for a specific attribute, for example, survival of cells. Cells that survive the interaction with the Random Library are isolated and the ribozyme sequence from these cells is determined.

The sequence of the binding arm of the ribozyme can then be used as probes to isolate the gene(s) involved in cell death. Because, the ribozyme(s) from the Random Library-is able to modulate down regulate) the expression of the gene(s) involved in cell death, the cells are able to survive under conditions where they would have otherwise died. This is a novel method ofgene discovery. This approach not only provides the information about mediators of certain cellular processes, but also provides a means to modulate the expression of these modulators. This method can be used to identify modulators of any cell process in any organism, including but not limited to mammals, plants and bacteria.

li hcr is also provided a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the nuceic acid sequence of a desired D target. The nucleic acid catalyst is preferably targeted to a highly conserved sequence region of a target such that specific diagnosis and/or treatment of a disease or condition can be provided with a single enzymatic nucleic acid.

S\ I aspc[ ol th disclosJ method I) etures a method for identifying one or more nucleic acid molecules, such as gene(s), involved in a process (such as, cell growth, proliferation, apoptosis, morphology, angiogenesis, differentiation, migration, viral I multiplication, drug resistance, signal transduction, cell cycle regulation, temperature .sensitivity, chemical sensitivity and others) in a biological system, such as a cellf The method involves the steps of: a) providing a random library of nucleic acid catalysts, with a sbstrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence, to the biological system under conditions suitable for the process to be altered; b) identifying any nucleic acid catalyst present in that biological system here the process has been altered by any nucleic acid catalyst; and c) determining the Snucleotide sequence of at least a portion of the binding arm of such a nucleic acid catalyst 2 to allow identification of the nucleic acid molecule involved in the process in that biological system.

related aspect of the disclosed method features a method for identification of a nucleic acid molecule capable of modulating a process in a biological system. The method includes: a) introducing a library of nucleic acid catalysts with a substrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence, into the biological system under conditions suitable for modulating the process; and b) determining the nucleotide sequence of at least a portion of the substrate binding domain of any nucleic acid catalyst from a biological system where the process has been 57 modulated to allow said identification of the nucleic acid molecule capable of modulatin said process in that biological system.

sccond aspect of the disclosed cmethod concerns a method for identification of a nucleic acid catalyst capable of modulating a process in a biological system. This involves: a) introducing a library of nucleic acid catalysts with a substrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence, into the biological system under conditions suitable for modulating the process: and b) identif'ig any nucleic acid catalyst from a bioloical system where the process ias been modulated.

10 Bv "enzymatic portion" or "catalytic domain" is meant that portion/region of the i "bo ,se essmeanal t t c-eaval o ribozyme essential for cleavage of a nucleic acid substrate (for example see Figure 3).

By "nucleic acid molecule" as used herein is meant a molecule having nucleotides The nucleic acid can be single, double or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof An example of a nucleic acid molecule according to the invention is a gene which encodes for macromolecule such as a protein.

The "biological system" as used herein may be a eukaryotic system or a prokaryotic system, may be a bacterial cell, plant cell or a mammalian cell, or may be of S plant origin, mammalian origin, yeast origin, Drosophila origin, or archebacterial origin.

n o Ilis invention relates to novel nucleic acid molecules with catalytic activity, which are particularly useful for cleavage of RNA or DNA. The nucleic acid catalysts of the instant invention are distinct from other nucleic acid catalysts known in the art. This invention also relates to a method of screening variants of nucleic acid using standard nucleotides or modified nucleotides. Applicant has determined an efficient method for screening libraries of catalytic nucleic acid molecules, particularly those with chemical modifications at ne or more positions. The method described in this application involves systematic screening of a library or pool of ribozymes with various substitutions at one or more positions and selecting for ribozymes with desired function or characteristic or attribute.

method for idcniti'fin a nucleic acid molecule involved in i p(rcess in a cell is also described including the steps of: a) synthesizing a library of nucleic acid catalysts, having a substrate binding domain and a catalytic domain, where the substrate binding domain has a random sequence; b) testing the library in the cell under conditions suitable to cause the process in the cell to be altered (such as: inhibition of cell proliferation, inhibition of angiogenesis, modulation of growth and /or differentiation, and others); c) isolating and enriching the cell with the altered process; d) identifying and isolating the nucleic acid catalyst in the altered cell; e) using an oligonucleotide, having the sequence homologous to the sequence of the substrate binding domain of the nucleic acid catalyst isolated from the altered cell. as a probe to isolate the nucleic acid molecule from the cell or the altered cell. Those nucleic acid molecules identified using the selection/screening method described above are likely to be involved !5 in the process that was being assayed for alteration by the member(s) of the ribozyme library. These nucleic acid molecules may be new gene sequences, or known gene sequences, with a novel function. One of the advantages of this method is that nucleic acid sequences, such as genes, involved in a biological process, such as differentiation, cell growth, disease processes including cancer, tumor angiogenesis, arthritis, cardiovascular disease, inflammation, restenosis, vascular disease and the like, can be Sreadily identified using the Random Library approach. Thus theoretically, one Random Library for a given ribozyme motif can be used to assay any process in any biological system.

\nother method described herein involves synthesizing a Defined Arm Nucleic Acid Catalyst Library (Defined Library) and simultaneously testing it against known targets in a cell. The library includes ribozymes with binding arm(s) of known complexity (Defined) and a defined catalytic domain. Modulation of expression of the target gene by ribozymes in the library will cause the cells to have an altered WO 98/50530 PCT/US98/09249 -59varying one or more positions in a putative catalytic domain. Applicant describes a method to vary positions within the catalytic domain, without changing positions within the binding arms, in order to identify new catalytic motifs. An example is illustrated in Figure 24. It is unclear how many positions are required to obtain a functional catalytic domain in a nucleic acid molecule, however it is reasonable to presume that if a large number of functionally diverse nucleotide analogs can be used to construct the pools, a relatively small number of positions could constitute a functional catalytic domain. This may especially be true if analogs are chosen that one would expect to participate in catalysis acid/base catalysts, metal binding, etc.). In the example illustrated, four positions (designated 1, 2, 3 and 4) are chosen. In the first step, ribozyme libraries (Class 1) are constructed: position 1 is fixed and positions 2, 3 and 4 are random (X 2

X

3 and

X

4 respectively). In step 2, the pools (the number of pools tested depends on the number of analogs used; n) are assayed for activity. This testing may be performed in vitro or in a cellular or animal model. Whatever assay that is used, the pool with the desired characteristic is identified and libraries (class 2) are again synthesized with position 1 now constant with the analog that was identified in class I. In class 2, position 2 is fixed

(F

2 and positions 3 and 4 are random (X 3 and This process is repeated until every position has been made constant and the chemical composition of the catalytic domain is determined. If the number of positions in the catalytic domain to be varied are large, then it is possible to decrease the number of Classes by testing multiple positions within a single Class. The number of pools within a Class equals the number of nucleotides or analogs in the random mixture n) to the w power, where w equals the number of positions fixed in each Class. The number of Classes that need to be synthesized to optimize the final ribozyme equals the total number of positions to be optimized divided by the number of positions tested within each Class. The number of pools in each Class= nw. The number of Classes= total number of positions /w.

In a preferred embodiment a method for identifying variants of a nucleic acid catalyst is described comprising the steps of: a) selecting at least three positions, preferably 3-12, specifically 4-10, within said nucleic acid catalyst to be varied with a predetermined group of different nucleotides, these nucleotides are modified or SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 unmodified (non-limiting examples of nucleotides that can used in this method are shown in Figure 15); b) synthesizing a first class of different pools of said nucleic acid catalyst, wherein the number of pools synthesized is equal to the number of nucleotides in the predetermined group of different nucleotides (for example if 10 different nucleotides are selected to be in the group of predetermined nucleotides then 10 different pools of nucleic acid catalysts have to be synthesized), wherein at least one of the positions to be varied in each pool comprises a defined nucleotide (fixed position; F) selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides (X positions) selected from the predetermined group of different nucleotides; c) testing the different pools of said nucleic acid catalyst under conditions suitable for said pools to show a desired attribute (including but not limited to improved cleavage rate, cellular and animal efficacy, nuclease stability, enhanced delivery, desirable localization) and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant (Z position) in subsequent steps; d) synthesizing a second class of different pools of nucleic acid catalyst, wherein at least one of the positions to be varied in each of the second class of different pools comprises a defined nucleotide selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides selected from the predetermined group of different nucleotides (this second class of pools therefore has F, X and Z positions); e) testing the second class of different pools of said nucleic acid catalyst under conditions suitable for showing desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant in subsequent steps; and f) this process is repeated until every position selected in said nucleic acid catalyst to be varied is made constant.

In yet another preferred embodiment, a method for identifying novel nucleic acid molecules in a biological system is described, comprising the steps of: a) synthesizing a pool of nucleic acid catalyst with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence; b) testing the SUBSTITUTE SHEET (RULE 26) 61 particular phenotype or attribute (step This process s repeated until all desired positions have been vaned and screened. For example if three positions are chosen for optimization, the synthesis and testing will need to be repeated three times (3 Classes). In the first two Classes, pools will be synthesized; in the final Class, specific ribozymes will b e synthesized and tested. hen the final position is analyzed (step no random positions will remain and therefore only individual ribozymes are synthesized and tested.

The resulting ribozyme or ribozymes (designated "second nbozyme motif') will have a defined chemical composition which will likely be distinct from the Starting Ribozyme motif(first ribozyne motif. This is a rapid method of screening for variability of one or more positions within a ribozyme motif.

\nUthcr method described herein involves screening of chemical odifications a one or ore positions ithin a hammerhead boz e motif. More Sspecifically, the nietdl) involves variability in the catalytic core sequence of a ammerhead ribozyme. Particlarly, the method describes screening for variability of !5 positions 3, 4 and 7 within a hammerhead ribozyme. The disclosure also features Sscreening for optimal loop 11 sequence in a hammerhead ribozyme.

Y c a"""nother mIethod described herein features a rapid method for screening accessible ribozyme cleavage sites within a target sequence. This method *involves screening of all possible sequences in the binding ann of a ribozyme. The 0 sequence of the binding alms determines the site of action of certain ribozymes. The combinatonal approach can be used to identify desirable and/or accessible sites within a target sequence by essentially testing all possible ann sequences. The difficulty with this approach is that ribozymes require a certain number of base pairs (for example, for hammerhead ribozymes the binding arm length is approximately 12-16 nucleotides) in order to bind functionally and sequence-specifically. This would require, for example 12- 16 different groups of hammerhead ribozyme pools; 12-16 positions would have to be optimized which would require 12-16 different groups being synthesized and tested.

Each pool would contain the four different nucleotides C, U and G) or nucleotide analogs (p 4 for nucleotides). It Would be very time consuming to test each group, identify the best pool, synthesize another group of ribozyme pools with one additional position constant, and then repeat the procedure until all 12-16 groups had been tested.

However it is possible to decrease the number of Classes by testing multiple positions within a single Class. In this case, the number of pools within a Class equals the number of nucleotides or analogs in the random mixture n) to the w power, where w equals the number of positions fixed in each Class. The number of Classes that need to be syvnthesized to optimize the final ribozyme equals the total number of positions to be optumized divided by the number of positions tested within each Class. The number o iools in each Class n. The number of Class total number of positions iw.

Ihe i is also disclosed a rapid method of screening for new catalytic nucleic acid motifs by keeping the binding arms constant and S vartVing one or more positions in a putative catalytic domain. Applicant describes a method to vary positions within the catalytic domain, without changing positions within 5 the binding arms, in order to identify new catalytic motifs. An example is illustrated in Figure 24. It is unclear how many positions are required to obtain a functional catalytic domain in a nucleic acid molecule, however it is reasonable to presume that if a large number of functionally diverse nucleotide analogs can be used to construct the pools, a relatively small number of positions could constitute a functional catalytic domain. This may especially be true if analogs are chosen that one would expect to participate in S: catalysis acid/base catalysts, metal binding, etc.). In the example illustrated, four positions (designated 1, 2, 3 and 4) are chosen. In the first step, ribozyme libraries (Class I) are constructed: position I is fixed and positions 2, 3 and 4 are random X. and respectively). In step 2, the pools (the number of pools tested depends on the number of analogs used; n) are assayed for activity. This testing may be performed in vitro or in a cellular or animal model. Whatever assay that is used, the pool with the desired characteristic is identified and libraries (class 2) are again synthesized with position

I

now constant with the analog that was identified in class 1. In class 2, position 2 is fixed and positions 3 and 4 are random and X 4 This process is repeated until WO 98/50530 WO 9850530PCT/US98/09249 63 Formula V 3'

A

A

A

G

C.

(N)o

AQ

5 7 Z3 Z4

G

A

G

Z

7

A

G

(N)n Z3= 2'-O-MTM-U Z4= 2'-O-MTM-C Z7- 6-Methyl-U Formnula VI 3' /Q M3

A

A

G

C.

(N)o

L

Z4

G

A

G

Z

7

A

G

(N)n Z3= 2'-O-MTM-U Z4= 2'-O-MTM-C Z7= 2'-C-Allyl-U SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -64- Formula VII 3M\

/Q

A z3 A Z4 A G A A G G z 7

A

C G Z3= 2'-O-MTM-U (N)o (N)n Z4= 2'-O-MTM-C L Z7= Pyridin-4-One In each of the above formulae, N represents independently a nucleotide or a nonnucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact by forming hydrogen bonds with complementary nucleotides in the target) with a target nucleic acid molecule (the target can be an RNA, DNA or RNA/DNA mixed polymers); preferably the length of Q is greater than or equal to 3 nucleotides and the length of M is preferably greater than or equal to 5 nucleotides; o and n are integers greater than or equal to 1 and preferably less than about 100, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent the molecule is assembled from two separate molecules), but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or doublestranded region; and represents a chemical linkage a phosphate ester linkage, amide linkage or others known in the art). 2'-O-MTM-U and 2'-O-MTM-C refers to 2'- O-methylthiomethyl uridine and 2'-O-methylthiomethyl-cytidine, respectively. A, C, U and G represent adenosine, cytidine, uridine and guanosine nucleotides, respectively. The nucleotides in the formulae are unmodified or modified at the sugar, base, and/or phosphate portions as known in the art.

SUBSTITUTE SHEET (RULE 26) Ihc iilcjlk~n I'cdttiics I 11LCICic lCild I1l(1(2CLIIC WVith C Jtfli tic jC[W\'i hJ'i1 ifc Ilk Ii 1Ihii I I ii Formuila III 0 c- C

G

I

G

G C -Q Fomiula I 3' /Q 0.* 0 0 0 *00 A Z4

G

A

A

G G V

A

C oG (N)o (N)n

L

Z3- 2'-O-MTM-U Z7= 6-Methyl-U WO 98/50530 PCT/US98/09249 -66nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. The terms "abasic" or "abasic nucleotide" as used herein encompass sugar moieties lacking a base or having other chemical groups in place of base at the 1' position.

In preferred embodiments, the enzymatic nucleic acid includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the nonnucleotide moiety. The necessary RNA components are known in the art, see, e.g., Usman, supra. By RNA is meant a molecule comprising at least one ribonucleotide residue.

As the term is used in this application, non-nucleotide-containing enzymatic nucleic acid means a nucleic acid molecule that contains at least one non-nucleotide component which replaces a portion of a ribozyme, but not limited to, a doublestranded stem, a single-stranded "catalytic core" sequence, a single-stranded loop or a single-stranded recognition sequence. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such molecules can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

The specific nucleic acid catalysts described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in a nucleic acid catalyst of this invention is that it has a specific substrate binding site M and/or Q of Formulae III-VII above) which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule.

Vector Expression of Enzymatic Nucleic Acid The nucleic acid catalysts of the instant invention can be expressed within cells from eukaryotic promoters Izant and Weintraub, 1985 Science 229, 345; McGarry and SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -67- Lindquist, 1986 Proc. Natl. Acad. Sci.USA 83, 399; Scanlon et al., 1991, Proc. Natl.

Acad. Sci.USA, 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992 J. Virol, 66, 1432-41; Weerasinghe et al., 1991 J. Virol, 65, 5531-4; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science 247, 1222-1225; Thompson et al., 1995 Nucleic Acids Res. 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; all of the references are hereby incorporated in their totality by reference herein). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994 J Biol. Chem. 269, 25856; all of the references are hereby incorporated in their totality by reference herein).

In another aspect of the invention, nucleic acid catalysts that cleave target molecules are expressed from transcription units (see for example Figure 11) inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors.

Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary.

Once expressed, the ribozymes cleave the target mRNA. The active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind target nucleic acid molecules such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -68into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510).

In a preferred embodiment, an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid catalyst of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.

In one embodiment, the expression vector comprises: a transcription initiation region eukaryotic pol I, II or III initiation region); b) a transcription termination region eukaryotic pol I, II or III termination region); c) a gene encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the gene encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).

Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol RNA polymerase II (pol II), or RNA polymerase III (pol III).

Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl.

Acad. Sci.U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. US A, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci.U S A, 6340-4; L'Huillier et al., 1992 EMBO J 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -69- Acad. Sci. U. S. 90, 8000-4; Thompson et al., 1995 Nucleic Acids Res. 23, 2259; Sullenger Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb. 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et al., 1997, Gene Ther. 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein. Examples of transcription units suitable for expression of ribozymes of the instant invention are shown in Figure 11. The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In a preferred embodiment an expression vector comprising nucleic acid sequence encoding at least one of the catalytic nucleic acid molecule of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region: b) a transcription termination region: c) a gene encoding at least one said nucleic acid molecule: and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another preferred embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 manner which allows expression and/or delivery of said nucleic acid molecule. In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region: c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.

Delivery of Nucleic Acid Catalysts: In a preferred embodiment, the nucleic acid catalysts are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. Using the methods described herein, other nucleic acid catalysts that cleave target nucleic acid may be derived and used as described above.

Specific examples of nucleic acid catalysts of the instant invention are provided below in the Tables and figures.

Sullivan, et al., supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al., supra and Draper et al., supra which have been incorporated by reference herein.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -71 The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., ammonium, sodium, calcium, magnesium, lithium, and potassium salts.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.

By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, NTP's, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as the cancer cells.

The invention also features the use of the a composition comprising surfacemodified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or longcirculating liposomes or stealth liposomes). These formulations offer an method for SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -72increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem.

Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.

Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of drugs, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J.

Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392; all of these are incorporated by reference herein). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. All of these references are incorporated by reference herein.

The present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.

Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. Id. at 1449. These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 73 physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize.

Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer. In a one aspect, the invention provides nucleic acid catalysts that can be delivered, exogenously to specific cells as required.

Local ribozyme administration offers the advantages of achieving high tissue concentrations of ribozymes and limiting their exposure to catabolic and excretory mechanisms. Although local routes of administration provide access to pathologies involving a number of organ systems, systemic administration would make ribozyme treatment of several other major human diseases feasible.

It has been demonstrated that certain tissues accumulate oligonucleotides and/or oligonucleotide formulations following systemic administration. These tissues include sites of inflammation (Wu et al. 1993, Cancer Res. 53: 3765-3767), solid tumors (Yuan et al. 1994, Cancer Res. 54: 3352-3356), kidney (Cossum et al. 1993, J. Pharmaco. and Exp.

Ther. 267: 1181-1190), brain (Wu et al. 1996, J. Pharmacol. Exp. Ther. 276: 206-11) and those rich in reticulo-endothelial cells (liver, spleen, lymphatics; Litzinger et al. 1994, Biochim. Biophys. Acta 1190: 99-107; Agrawal et al. 1991, Proc. Natl. Acad. Sci.USA 88: 7595-7599; Agrawal et al. 1995, Clin. Pharmacology 28: 7-16; Sands et al. 1994, Molecular Pharmacol. 45: 932-943; Saijo et al. 1994, Oncology Research 6: 243-249).

The kidney, as well as organs of the reticulo-endothelial system (RES), are mainly responsible for clearance of ribozymes following intravenous administration.

Diseases involving these tissues are good candidates for systemic ribozyme therapy by virtue of their tendency to accumulate ribozymes.

In one preferred embodiment, the invention features method of treating inflammation using ribozymes. Inflammatory processes underlie the pathology of a large number of human diseases. Many of these processes can be blocked by inhibiting the expression of inflammatory mediators and/or their receptors (Cohen et al. 1995, Am. J. Med. 99: 52S). Systemic administration of monoclonal antibodies specific to these mediators have SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -74been shown to be efficacious in animal models of rheumatoid arthritis, inflammatory bowel disease, and acute respiratory distress syndrome (Arend et al. 1990, Arthritis and Rheumatism 33: 305-315). One potential way for systemic administration of ribozymes to impact systemic inflammatory disease is through inhibition of TNF-a production by macrophages. TNF-a has been shown participate in a variety of inflammatory processes and is produced mainly by macrophages which are known to accumulate cationic lipidformulated ribozymes (Masahiro et al. 1990, J. Immunology. 144: 1425-1431). Antimouse TNF- a ribozymes were effective in cell culture, thus, it may be possible that systemic delivery of ribozymes by a liposome formulation could be an effective therapeutic in the above mentioned inflammatory disease states.

In another preferred embodiment, the invention features methods of treating diseases involving RES using ribozymes. A number of studies have shown that systemically administered oligonucleotides distribute to RES tissues (liver, spleen and lymphatics).

Several studies with cationic lipid complexed oligonucleotides have also shown specific biodistribution to these. Pathology involving the RES includes a number of infectious diseases of major importance, such as human immunodeficiency virus (HIV), mycobacterium infections including tuberculosis avium, and leprae (leprosy). These diseases are all associated with, for example, overproduction of interleukin-10 (IL-10). a potent immunosuppressive cytokine (Barnes et al. 1993, Infect. Immun. 61: 3482-9).

Some of these infections can potentially be ameliorated by administration of neutralizing antibodies to In yet another preferred embodiment, the invention features method of treating cancer using ribozymes. As evidence of the potential use of systemic oligonucleotides as anticancer agents, antisense phosphorothioates have been have been reported to exhibit antitumor efficacy in a murine model of Burkitt's lymphoma (Huang et al. 1995, Mol.

Med. 1: 647-658). The molecular targets of systemic antineoplastic ribozymes could include oncogenes, protooncogenes, or angiogenic factors and receptors. Although the link between oncogenes and tumorigenesis is now well established, the specific mutations that lead to activation of a proto-oncogene can be widely diverse. Upregulation of protooncogene products is also common in human cancer. Reducing the levels of these SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 gene products may be beneficial in treatment of cancer. In addition, since many tumors are highly vascularized, angiogenic factors or receptors may provide good alternate or adjunct targets to oncogenes for the therapy of solid tumors and their metastases.

Applicant, in a non-limiting example infra, show ribozymes targeting angiogenic mediators.

The potential number of molecular targets in cancer is quite large. Among these targets are oncogenes, protooncogenes, metalloproteinases, growth factors, and angiogenic factors. However, a common denominator in many forms of metastatic solid tumors is extensive vascularization of the tumor. As tumors exceed about 1 mm in diameter, they require neovascularization for continued growth (Gimbrone et al., 1972, J. Exp. Med., 136, 261). In addition, the appearance of new blood vessels within a tumor correlates with the initiation of the process of metastasis (Martiny-Baron and Marme, 1995). It is possible that by using a systemically administered ribozyme targeting a key player in the process of angiogenesis would reduce both primary tumor growth, tumor progression and tumor metastasis.

"Angiogenesis" refers to formation of new blood vessels from existing blood vessels which is an essential process in reproduction, development and wound repair.

"Tumor angiogenesis" refers to the induction of the growth of blood vessels from surrounding tissue into a solid tumor. Tumor growth and tumor metastasis are dependent on angiogenesis (for a review see Folkman, 1985, Nature Med. 1: 27-31; Folkman 1990 J.

Natl. Cancer Inst., 82, 4; Folkman and Shing, 1992 J. Biol. Chem. 267, 10931).

"Tumor metastasis" refers to the transfer and/or migration of tumor cells, originating from a primary tumor, from one part of the body or organ to another. Most malignant tumors have the capacity to metastasize.

"Tumor" refers to a new growth of tissue wherein the cells multiply, divide and grow uncontrolled.

In a preferred embodiment, the invention features a method of treating non-hepatic ascites using ribozymes. Nonhepatic ascites or peritoneal fluid accumulation resulting SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -76from abdominal cancer and ovarian hyperstimulation syndrome (OHSS) can result in significant fluid loss from the intravascular space and hypovolemia. If ascites volumes are large, abdominal pain, hypovolemic hypotension, electrolyte abnormalities and respiratory difficulties can ensue. Thus, if ascites is left untreated, it can be life threatening. Evidence is now accumulating that nonhepatic ascites may be induced, at least in part, by vascular endothelial growth factor (VEGF). For this reason, nonhepatic ascites may be a potential therapeutic indication for ribozymes directed against vascular endothelial growth factor (VEGF) receptors delivered either systemically or regionally to the peritoneum.

Ovaries can be overstimulated by hormonal therapy during fertility treatment. As a result, women can experience ovarian hyperstimulation syndrome which is associated with grossly enlarged ovaries and extreme ascites fluid accumulation. This fluid accumulation is thought to be induced by the release of a vascular permeability agent which may interact with vessels of the peritoneal cavity leading to plasma extravasation. Abramov and coworkers (1997, Fertil. Steril. 67: 261) have shown that plasma VEGF levels are elevated in OHSS and return to normal upon resolution of the syndrome. An earlier study has shown that VEGF is elevated in the serum and follicular fluid of OHSS patients and that the source of this VEGF may be the luteinizing granulosa cells of the ovary (Krasnow et al., 1996, Fertil. Steril. 65: 552). McClure et al. (1994, Lancet 344. 235) concluded that VEGF is the key mediator of OHSS ascites production since rhVEGF increases OHSS ascites but not liver ascites and that this increase is reversible by rhVEGF antiserum.

Thus, reducing the expression of VEGF receptors in the vasculature of the peritoneum may have a therapeutic benefit in OHSS by substantially reducing OHSS-stimulated ascites production. Since VEGF can interact with VEGF receptors on vessels throughout the peritoneum from ovarian release of VEGF into systemic circulation, systemic treatment may represent the best option for treating this syndrome.

Malignant ascites: Another form of ascites can be induced by malignancies of the peritoneum including breast, pancreatic, uterine and colorectal cancers. It is thought that certain cancers produce factors which influence peritoneal vascular permeability leading to plasma extravasation (Garrison et al., 1986; Ann. Surg. 203: 644; Garrison et al., 1987, J.

Surg. Res. 42: 126; Nagy et al., 1993, Cancer Res. 53: 2631). Several solid tumors SUBSTITUTE SHEET (RULE 26) 1994, Biochim. Biophys. Acta 1190: 99-107; Agrawal et al. 1991, Proc. Natl cad.

Sc. USA 88: 7595-7599; Agrawal et al 1995, Clin. Pha, o 28: Sands e a iyi, n. Pharmacology 28: 7-16; Sd, a 1994, folecular Pharmacol. 45: 932-943; Saijo et a. 1994, Oncology Researc 6: 243- 249).

The kidney, as well as organs of the reticulo-endothelia system (RES), are mainly responsible for clearance of ribozymes followin2 intravenous administration Diseases involving these tissues are good candidates for systemic ribozvnie therapy by \'iilue of their tendency to accumulate ribozymes.

\eI n sl pcci of this disclosure is a method of treating inflammation using ribozymes. Inflammatory processes underlie the pathology of a large number of human diseases. Many of these processes can be blocked b inhibiting the expression o inflammatory mediators and/or their receptors (Cohen et al. 1995, Am. J. Med. 99: ystemic administration of monoclonal antibodies specific to these mediators have been shown to be efficacious in animal models of rheumatoid arthritis, inflammatory bowel disease, and acute respiratory distress syndrome (Arend et al. 1990, Arthritis and Rheumatism 33: 305-315). One potential way for ystemic adinistratin o bozymes iIpaco system f y m es S to impact systemic inflamatory disease is through inhibition of TNF-a production by Smacrophages. TNF-a has been shown participate in a variety of inflammatory processes and is produced mainly by macrophages which are known to accumulate cat formulated ribozymes (Masahiro et al. 1990, J ImmunologDy 144: 1425-1431) Ant mouse TNF- a ribozymes were effective in cell culture, thus, it may be possible that systemic delivery of ribozymes by a liposome formulation could be an effective therapeutic in the above mentioned inflammatory disease states.

d\ nothcer aspect ol this disclosure is method of treating diseases involving RES using ribozymes. A number of studies have shown that systemically administered oligonucleotides distribute to RES tissues (liver, spleen and lymphatics) Several studies with cationic lipid complexed oligonucleotides have also shown specific biodistribution to these. Pathology involving the RES includes a number of infectious diseases of major importance, such as human immunodeficiency virus

(HIV),

mycobacterium infections including tuberculosis avium, and leprae (leprosy).

These diseases are all associated with, for example, overproduction ofinterleukin-10

(IL-

a potent immunosuppressive cytokine (Barnes er al. 1993, Infect. Ihnmun. 61: 3482- Some of these infections can potentially be ameliorated by administration of neutralizing antibodies to 'urther aspect of this disclosure is a method of treatine cancer using ribozymes. As evidence of the potential use of systemic oligonucleotides as anticancer agents, antisense phosphorothioates have been have been reported to exhibit antitumor efficacy in a murine model of Burkitt's lymphoma (Huang et al. 1995, Mo/.

Med. 1: 647-658). The molecular targets of systemic antineoplastic ribozvmes could Include oncogenes, protooncogenes, or angiogenic factors and receptors. Although the S link between oncogenes and tumorigenesis is now well established, the specific mutations that lead to activation of a proto-oncogene can be widely diverse. Upregulation of S protooncogene products is also common in human cancer. Reducing the levels of these gene products may be beneficial in treatment of cancer. In addition, since many tumors are highly vascularized, angiogenic factors or receptors may provide good alternate or adjunct targets to oncogenes for the therapy of solid tumors and their metastases.

Applicant, in a non-limiting example inf-a, show nrbozymes targeting angiogenic :20 mediators.

SThe potential number of molecular targets in cancer is quite large. Among these targets are oncogenes, protooncogenes, metalloproteinases, growth factors, and angiogenic factors. However, a common denominator in many forms of metastatic solid tumors is extensive vascularization of the tumor. As tumors exceed about i mm in diameter, they require neovascularization for continued growth (Gimbrone et al., 1972, Exp. Med., 136, 261). In addition, the appearance of new blood vessels within a tumor correlates with the initiation of the process of metastasis (Martiny-Baron and Marmi.

1995). It is possible that by using a systemically administered ribozyme targeting a key 79 player in the process of angiogenesis would reduce both primary tumor growth, tumor progression and tumor metastasis.

"Angiogenesis" refers to formation of new blood vessels from existing blood vessels which is an essential process in reproduction, development and wound repair.

"Tumor angiogenesis" refers to the induction df the growth of blood vessels from surrounding tissue into a solid tumor. Tumor growth and tumor metastasis are dependent on angiogenesis (for a review see Folkman, 1985, Nature Med. 1: 27-3 1; Folkman 1990 .na. Cancer inst.. 82, 4: Folkman and Shing, 1992 J Biol. Chem. 267, 1093 1).

"Tumor metastasis" refers to the transfer and/or migration of tumor cells.

originating from a primary tumor, from one pan of the body or organ to another. Most malignant tumors have the capacity to metastasize.

"Tumor" refers to a new growth of tissue wherein the cells multiply, divide and grow uncontrolled.

Yet a further aspect of this disclosure is a method of treating non-hepatic 5 ascites using ribozymes. Nonhepatic ascites or peritoneal fluid accumulation resulting from abdominal cancer and ovarian hyperstimulation syndrome (OHSS) can result in significant fluid loss from the intravascular space and hypovolemia. If ascites volumes are large, abdominal pain, hypovolemic hypotension, electrolyte abnormalities and S respiratory difficulties can ensue. Thus, if ascites is left untreated, it can be life threatening. Evidence is now accumulating that nonhepatic ascites may be induced, at least in part, by vascular endothelial growth factor (VEGF). For this reason, nonhepatic ascites may be a potential therapeutic indication for ribozymes directed against vascular endothelial growth factor (VEGF) receptors delivered either systemically or regionally to the peritoneum.

Ovaries can be overstimulated by hormonal therapy during fertility treatment. As a result, women can experience ovarian hyperstimulation syndrome which is associated WO 98/50530 PCT/US98/09249 VEGF receptors of both subtypes and their expression are upregulated in the lung under conditions of hypoxia (Tuder et al. 1994, J. Clin. Invest. 95: 1798-1807). This may lead to neovascularization which provides the means by which tumor cells gain access to circulation (Mariny-Baron and Marme, 1995). Thus, VEGF and its receptors may be important targets in the treatment of metastatic disease.

Applicant has shown that a catalytically active ribozyme targetingflt-1 RNA inhibits VEGF-induced neovascularization in a dose-dependent manner in a rat corneal model of angiogenesis. Testing with cytotoxic agents in combination with antiangiogenic ribozymes may also prove useful.

Anti-K- and H-ras ribozymes Mutations involving ras underlie a number of human cancers. Ras also plays a role in metastatic potential (Shekhar and Miller, 1994, Invasion Metastasis 14: 27-37) and may do so, in part, by influencing endothelial cell migration (Fox et al. 1994, Oncogene 9: 3519-26). With regard to lung cancer, ras has been shown to induce abnormal mitoses in lung fibroblasts (Lyubuski et al. 1994, Cytobios 80: 161-178) and is a clinical marker in non-small cell lung tumors (Niklinski and Furman, 1995, Eur. J. Cancer Prev. 4: 129- 138). Studies in cells cultured from human small cell lung tumor xenografts demonstrated overexpression of K-ras (Arvelo et al. 1994, Anticancer Res. 14: 1893-1901). This evidence provides ample support for the systemic testing of ribozymes directed against Hand K-ras in the murine cancer models (primary and secondary metastasis) discussed above.

Four of the current synthetic ribozymes directed against human K- ras will cleave homologous mouse K-ras targets at four sites and inhibit cultured rat aortic smooth muscle cell proliferation.

Anti-c-fos ribozymes The protein product of the proto-oncogene c-fos is a nuclear transcription factor which is involved in tumorigenesis. In support of the possible use of systemically administered ribozymes directed against c-fos, null mouse mutations of c-fos have been SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -81 shown to result in viable mice. Using this mouse model, it has been shown that c-fos is important in malignant conversion of papillomas. Additionally, c-fos has been shown to up-regulate tumor metalloproteinases (Schonthal et al. 1988, Cell 54: 325-334). It is possible that c-fos may play a role in tumor angiogenesis as evidenced by VEGF mRNA levels being significantly reduced in c-fos deficient tumors. It has also been shown that cfos is highly expressed in some B-16 cell and human melanoma cell lines (Kroumpouzos et al. 1994, Pigment Cell Res. 7: 348-353; Nakayama et al. 1995, J. Dermatol. 22: 549- 559; Peris el al. 1991, Arch. Dermatol. Res. 283: 500-505). The expression of c-fos may be directly proportional to metastatic potential in B-16 melanoma cell lines. With this evidence, it is reasonable to conclude that c-fos represents a suitable systemic ribozyme target in either the Lewis lung, B-16 melanoma, or human melanoma models.

Delivery of ribozymes and ribozvme formulations in the Lewis lung model Several ribozyme formulations, including cationic lipid complexes which may be useful for inflammatory diseases DIMRIE/DOPE, etc.) and RES evading liposomes which may be used to enhance vascular exposure of the ribozymes, are of interest in cancer models due to their presumed biodistribution to the lung. Thus, liposome formulations can be used for delivering ribozymes to sites of pathology linked to an angiogenic response.

The sequences of the ribozymes that are chemically synthesized, useful in this study, are non-limiting examples. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables IV CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. Preferably, no more than 200 bases are inserted at these locations. The sequences listed in Tables III and IV may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -82ribozymes (which have enzymatic activity) are equivalent to the ribozymes described specifically in the Tables.

Target sites Targets for useful ribozymes can be determined as disclosed in Draper et al., WO 93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., US Patent No. 5,525,468 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein.

The sequence of human c-raf mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables XII-XIX (All sequences are 5' to 3' in the tables) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

Because Raf RNAs are highly homologous in certain regions, some ribozyme target sites are also homologous (see Table XVIII and XIX). In this case, a single ribozyme will target different classes of Raf RNA. The advantage of one ribozyme that targets several classes of Raf RNA is clear, especially in cases where one or more of these RNAs may contribute to the disease state.

Hammerhead or hairpin ribozymes were designed that could bind and were individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 83 able to bind to, or otherwise interact with, the target RNA. Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above.

Examples The following are non-limiting examples showing the selection, isolation, synthesis and activity of enzymatic nucleic acids of the instant invention.

The following examples demonstrate the selection of ribozymes that cleave c-raf RNA. The methods described herein represent a scheme by which ribozymes may be derived that cleave other RNA targets required for cell division. Also provided is a description of how such ribozymes may be delivered to cells. The examples demonstrate that upon delivery, the ribozymes inhibit cell proliferation in culture and modulate gene expression in vivo. Moreover, significantly reduced inhibition is observed if mutated ribozymes that are catalytically inactive are applied to the cells. Thus, inhibition requires the catalytic activity of the ribozymes.

Example 1: Identification of Potential Ribozyme Cleavage Sites in Human c-rafRNA The sequence of human c-raf RNA was screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and contained potential hammerhead and/or hairpin ribozyme cleavage sites were identified. The sequences of these cleavage sites are shown in tables XII-XIX.

Example 2: Selection of Ribozvme Cleavage Sites in Human c-rafRNA To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in c-raf RNA, 20 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by analyzing genomic sequences of human craf (GenBank Accession No. X03484; Bonner et al., 1986, Nucleic Acids Research, 14, 1009-1015) and prioritizing the sites on the basis of folding. Hammerhead ribozymes were designed that could bind each target (see Figure 1) and were individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -84et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Ribozyme target sites within A-Raf were chosen by analyzing genomic sequences of human A-raf-1 (GenBank Accession No. X04790; Beck et al., 1987, Nucleic Acids Research, 115, 595-609). Ribozyme target sites within B-Raf were chosen by analyzing genomic sequences of human B-raf-1 (GenBank Accession No. M95712 M95720 X54072; Sitanandam et al., 1990, Oncogene, 5, 1775- 1780).

Example 3: Chemical Synthesis and Purification of Ribozvmes for Efficient Cleavage of c-rafRNA Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the RNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al.. (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.

The average stepwise coupling yields were >98%.

Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes were modified to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'- O-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table XII-

XIX.

Example 4: Ribozvme Cleavage of c-raf RNA Target in vitro Ribozymes targeted to the human c-raf RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vitro, for example using the following procedure. The target sequences and the nucleotide location within the c-raf mRNA are given in Table XII.

Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a- 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'- 3 2 P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCl, pH at 37°C, 10 mM MgC12) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37 C using a final concentration of either 40 nM or 1 mM ribozyme, ribozyme excess.

The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95 0 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -86- Example 5: Ability of c-raf Ribozymes to Inhibit Smooth Muscle Cell Proliferation.

Ribozymes targeting sites in c-Raf mRNA were synthesized using modifications that confer nuclease resistance (Beigelman, 1995, J. Biol. Chem. 270. 25702). The ribozymes were screened for their ability to inhibit cell proliferation in serum-starved primary rat aortic smooth muscle cells as described by Jarvis et al. (1996, RNA 2, 419; incorporated by reference herein). The ribozyme targeting site represented by Seq ID Nos 175 and 198 showed particularly high activity in inhibiting cell proliferation. An inactive control ribozyme was synthesized which had identical substrate binding arms but contained mutations in the catalytic core that eliminate cleavage activity. Inhibition of cell proliferation by active versus inactive c-Raf ribozymes is shown in Figures 37 and 38.

The data are presented as proliferation relative to the serum-stimulated untreated control cells. Clearly the active ribozyme is showing substantial inhibition relative to both the untreated control and its corresponding inactive control, thus indicating that the inhibition of proliferation is mediated by ribozyme-mediated cleavage of c-Raf.

In several other systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, C. et al., 1992, Mol.

Pharmacology, 41, 1023-1033). In many of the following experiments, ribozymes were complexed with cationic lipids. The cationic lipid, Lipofectamine (a 3:1 (w/w) formulation of DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1-propanaminium trifluoroacetate) and dioleoyl phosphatidylethanolamine (DOPE)), was purchased from Life Technologies, Inc. DMRIE (N-[1-(2,3-ditetradecyloxy)propyl]-N,Ndimethyl-N-hydroxyethylammonium bromide) was obtained from VICAL. DMRIE was resuspended in CHC13 and mixed at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). The CHC13 was evaporated, the lipid was resuspended in water, vortexed for 1 minute and bath sonicated for 5 minutes. Ribozyme and cationic lipid mixtures were prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives was warmed to room temperature (about 0 cationic lipid was added to the final desired concentration and the solution was vortexed briefly. RNA oligonucleotides were added to the final desired concentration and the solution was again vortexed briefly and incubated for 10 minutes at room temperature.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -87- In dose response experiments, the RNA/lipid complex was serially diluted into DMEM following the 10 minute incubation.

Serum-starved smooth muscle cells were washed twice with PBS, and the RNA/lipid complex was added. The plates were incubated for 4 hours at 37°C. The medium was then removed and DMEM containing 10% FBS, additives and 10 pM bromodeoxyuridine (BrdU) was added. In some wells, FBS was omitted to determine the baseline of unstimulated proliferation.

The plates were incubated at 37 0 C for 20-24 hours, fixed with 0.3% H 2 0 2 in 100% methanol, and stained for BrdU incorporation by standard methods. In this procedure, cells that have proliferated and incorporated BrdU stain brown; nonproliferating cells are counter-stained a light purple. Both BrdU positive and BrdU negative cells were counted under the microscope. 300-600 total cells per well were counted. In the following experiments, the percentage of the total cells that have incorporated BrdU cell proliferation) is presented. Errors represent the range of duplicate wells. Percent inhibition then is calculated from the cell proliferation values as follows: inhibition 100 100((Ribozyme 0% serum)/(Control 0% serum)).

From this initial screen, hammerhead ribozyme targeted against c-raf site 1120 (Figure 36) was further tested. The active ribozyme was able to inhibit proliferation of smooth muscle cell, whereas, the control inactive ribozyme, that cannot cleave c-raf RNA due to alterations in their catalytic core sequence, fails to inhibit smooth muscle cell proliferation (Figure 37). Thus, inhibition of cell proliferation by these hammerhead sequences is due to their ability to cleave c-raf RNA, and not because of any nonribozyme activity.

Example 6: Oligonucleotide design and preparation for cloning Defined and Random Libraries The DNA oligonucleotides used in this study to construct Defined and Random Ribozyme Libraries were purchased from Life Technologies (BRL). A schematic of the oligonucleotide design used to construct said Defined or Comprehensive Ribozyme SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -88- Libraries is shown in Figure 8. This example is meant to illustrate one possible means to construct such libraries. The methods described herein are not meant to be inclusive of all possible methods for constructing such libraries. The oligonucleotides used to construct the hammerhead ribozyme libraries were designed as follows: 5'-CGAAATCAATTG-(NI),- {CatalyticCore}-(N2),-CGTACGACACGAAAGTATCG-3' Where N1 the Stem I target-specific binding arm of length x, Catalytic Core the hammerhead catalytic domain 5'-CTGATGAGGCCGUUAGGCCGAAA-3', and N2 the Stem III target specific binding arm of length x. The oligonucleotides were designed to self-prime via formation of a stem-loop structure encoded at the 3' ends of the oligos (Figure 8A). This intramolecular interaction favored an unbiased extension of complex pools of ribozyme-encoding oligonucleotides. In the case of Defined Ribozyme Library described below (Figures 9-10), NI and N2 were 8 nt each and were designed to be complimentary to the RNA encoded by the purine nucleoside phosphorylase (PNP) gene.

In the case of Random Ribozyme Libraries, N1 and N2 were randomized during synthesis to produce a single pool of all possible hammerhead ribozymes.

In the example shown (Figures 9-10), oligonucleotides encoding different PNP-specific hammerhead ribozymes (greater than 40 ribozymes can be used) were pooled to a final concentration of 1 gM total oligonucleotides (2.5 nM each individual oligo). Oligos were heated to 68°C for 30 min and then cooled to ambient temperature to promote formation of the 3' stem-loop for self-priming (Figure 8A). The 3' stem loop was extended (Figure 8B) using Klenow DNA polymerase (1 p.M total oligonucleotides in 1 ml of 50 mM Tris pH 7.5, 10mM MgC12, 100 pg/ml BSA. 25 pg M dNTP mix, and 200 U Klenow) by incubating for 30 min at 37°C. The reaction mixtures were then heated to 65 0 C for 15 min to inactivate the polymerase. The double-stranded oligos (approximately 30 pg) were digested with the 100 U of the 5' restriction endonuclease Mfe I (NEB) as described by the manufacturer, then similarly digested with the 3' restriction endonuclease BsiWI (Figure 8C). To reduce the incidence of multiple ribozyme inserts during the cloning steps, the cleaved products were treated with Calf Intestinal Phosphatase (CIP, Boehringer Mannheim) as described by the manufacturer to remove the phosphate groups at the 5' ends. This step inhibits intra- and intermolecular SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -89ligation of the ribozyme-encoding fragments. Full-length product corresponding to the double-stranded, restriction digested and phosphatase-treated products was gel-purified following electrophoresis through 10% non-denaturing acrylamide gels prior to cloning to enrich for full-length material.

Example 7: Cloning of Defined and Random Libraries The cloning vectors used contained the following cloning sites: MfeI Cla I BsiWI Vectors were digested with Mfe I and BsiWI prior to use. Thus, vectors cleaved with both enzymes should lack the Cla I site present between the sites, while vectors cleaved with only one of the enzymes should still retain the Cla I site. Pooled oligos were ligated to vector using a 2:1 or 5:1 molar ratio of double-stranded oligo to vector in 50-mL reactions containing 500 ng vector and 5 U ligase in lx ligase buffer (Boehringer Mannheim). Ligation reactions were incubated over night at 16 0 C, then heated to 65°C 10 min to inactivate the ligase enzyme. The desired products contain a single ribozyme insert and lack the original Cla I site included between the Mfe I and BsiWI cloning sites. Any unwanted, background vector lacking ribozyme inserts and thus still containing the Cla I sites were inactivated by cleaving the product with 5 U of the restriction endonuclease Cla I for 1 h at 37 0 C. Approximately 150 ng of ligated vector was used to transform 100 .1 XL-2 Blue competent bacteria as described by the supplier (Stratagene).

Example 8: Simultaneous screening of 40 different ribozvmes targeting PNP using Defined Ribozyme Libraries.

A Defined Ribozyme Library containing 40 different hammerhead ribozymes targeting PNP was constructed as described above (Figures 8-10). PNP is an enzyme that plays a critical role in the purine metabolic/salvage pathways. PNP was chosen as a target because cells with reduced PNP activity can be readily selected from cells with wild-type activity levels using the drug 6-thioguanosine. This agent is not toxic to cells until it is converted to 6-thioguanine by PNP. Thus, cells with reduced PNP activity are more resistant to this drug and can be selectively grown in concentrations of 6-thioguanosine that are toxic to cells with wild-type activity levels.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 The PNP-targeted Defined Ribozyme Library expression vectors were converted into retroviral vector particles, and the resulting particles were used to transduce the Sup T1 human T cell line. A T-cell line was chosen for study because T lymphocytes are more dependent on the purine salvage pathway and thus are highly susceptible to 6thioguanosine killing. Two weeks after transduction, the cells were challenged with mmol 6-thioguanosine. Resistant cells began to emerge two weeks after initiation of selection. 6-Thioguanosine-resistant cells were harvested, and the ribozyme-encoding region of the expression vector was amplified using PCR and sequenced. The sequence pattern of the ribozyme region in the selected cells was significantly different from that produced from the starting library shown in Figure 9. In the original library, sequences of the binding arms were ambiguous due to the presence of all 40 PNP-targeted ribozymes (Figure However, the sequence of the ribozyme-encoding regions from the 6thioguanosine selected cells was clearly weighted towards one of the ribozymes contained in the original pool the ribozyme designed to cleave at nucleotide #32 of PNP mRNA.

These data suggests that the ribozyme targeting position 32 of the PNP mRNA appears to be more active than the other 39 PNP-targeted ribozymes included in the pool.

Example 9: Optimizing Loop II sequence of a Hammerhead Ribozvme (HH-B) for Enhanced Catalytic Rates To test the feasibility of the combinatorial approach described in Figure 12 approach, Applicant chose to optimize the sequence of loop-II of a hammerhead ribozyme (HH-B) (see Figure 22). Previous studies had demonstrated that a variety of chemical modifications and different sequences within loop-II may have significant effects on the rate of cleavage in vitro, despite the fact that this sequence is not phylogenetically conserved and can in fact be deleted completely. According to the standard numbering system for the hammerhead ribozyme, the four positions within loop II are numbered 12.1, 12.2, 12.3, and 12.4. The Starting Ribozyme (HH-B) contained the sequence G 2.1 A12.2 A 2.3 A 2.4. For simplicity, the four positions will be numbered 5' to G 12.1= 1; A 12.2= 2; A 12.3=3; A 12.4= 4. The remainder of the hammerhead ribozyme "template" remained constant and is based on a previously described hammerhead motif (Draper et al., International PCT Publication No. WO 95/13380, incorporated by reference herein).

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -91 A strategy for optimizing the four (number of Classes 4) loop-II positions is illustrated in Figure 180. The four standard ribose nucleotides C, U and G) were chosen to construct the ribozyme pools (n In the first step, four different pools were synthesized by the nucleotide building block mixing approach described herein. Applicant first chose to "fix" (designated F) position 3 because preliminary experiments indicated that the identity of the base at this position had the most profound effects on activity; positions 1, 2 and 4 are random. The four pools were assayed under stoichiometric conditions (1gM ribozyme; 1ptM substrate), to help ensure that the entire population of ribozymes in each pool was assayed. Substrate and ribozyme were pre-annealed and the reactions were initiated with the addition of 10mM MgCIl. The rate of cleavage for each library was derived from plots of fraction of substrate cleaved as a function of time.

Reactions were also performed simultaneously with the starting ribozyme homogenous, loop-II GAAA). The relative rate of cleavage for each library (kre,) was calculated by dividing the observed rate of the library by the rate of the control/starting ribozyme and is plotted in Figure 21. The error bars indicate the standard error derived from the curve fits. The results show that all four pools had similar rates however, the library possessing at position 3 was slightly faster.

Ribozyme pools were again synthesized (Class 2) with position 3 being made constant position 4 was fixed (F 4 and positions 1 and 2 were random The four pools were assayed as before; the pool containing at position 4 was identified as the most desirable pool. Therefore, during the synthesis of the next pool (Class positions 3 and 4 were constant with U 3 and A 4 position 2 was fixed and position 1 was random The four pools were again assayed; all four pools showed very similar, but substantially elevated rates of cleavage. The pool containing U at position 2 was identified as the fastest. Therefore, during the synthesis of the final four ribozymes (Class 4), position 3, 4 and 2 were made constant with U 3

A

4 and position 1 was fixed with A, U, C or G. The final ribozyme containing G at position 4 was clearly identified as the fastest ribozyme, allowing the identification of G2.1 U 1 2.

2

U

1 2.

3

A,.

4 as the optimized ribozyme motif.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -92- To confirm that the final ribozyme (G 1 2.

1 U12.

2 U2.3 A2.4) was indeed faster that the starting ribozyme (G,2.1 A 1

A

1 3 A,2.

4 we compared the two ribozymes (illustrated in Figure 22) under single-turnover conditions at saturating ribozyme concentrations. The observed rates should therefore measure the rate of the chemical step, The fraction of substrate remaining uncleaved as a function of time is shown in Figure 22 (lower panel), and the derived rate contents are shown. The results show that the optimized ribozyme cleaves >10 times faster (3.7 min' vs. 0.35 min') than the starting ribozyme.

Example 10: Optimizing Core Chemistry of a Hammerhead Ribozyme (HH-A) To further test the feasibility of the approach described in Figure 12, we chose to optimize the three pyrimidine residues within the core of a hammerhead ribozyme (HH- These three positions (shown in Figure 13 as U7, U4 and C3) were chosen because previous studies indicated that these positions are critical for both stability (Beigelman et al., 1995, supra) and activity (Ruffner et 1990, supra; Burgin et al., 1996, supra) of the ribozyme. According to the standard numbering system for the hammerhead ribozyme, the three pyrimidine positions are 7, 4 and 3. For construction of the libraries.

the ribozyme positions are numbered 3' to position 24 7, position 27 4, and position 28 3 (see Figure 13). The remainder of the hammerhead ribozyme "template" remained constant and is based on a previously described hammerhead motif (Thompson et al., US Patent No. 5,610,052, incorporated by reference herein). The starting ribozyme template is targeted against nucleotide position 823 of k-ras mRNA (Site Down regulation of this message, as a result of ribozyme action, results in the inability of the cells to proliferate. Therefore in order to optimize a ribozyme, we chose to identify "variants" which were successful in inhibiting cell proliferation.

Cell Culture Assay: Ribozyme:lipid complex formation Ribozymes and LipofectAMINE were combined DMEM at final concentrations of 100 nM and 3.6 iM, respectively. Complexes were allowed to form for 15-min at 37 C in the absence of serum and antibiotics.

Proliferation Assay SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -93- Primary rat aortic smooth muscle cells (RASMC) were seeded at a density of 2500 cells/well in 48 well plates. Cells were incubated overnight in DMEM, supplemented with fetal bovine serum (FBS), Na-pyruvate, penicillin (50 U/ml), and streptomycin ug/ml). Subsequently cells were rendered quiescent by a 48 h incubation in DMEM with 0.5% FBS.

Cells were incubated for 1.5 h with serum-free DMEM ribozyme:lipid complexes.

The medium was replaced and cells were incubated for 24 h in DMEM with 0.25% FCS.

Cells were then stimulated with 10% FBS for 24 h. 3 H-thymidine (0.3 p.Ci//well) was present for the last 12 h of serum stimulation.

At the end of the stimulation period the medium was aspirated and cells were fixed in icecold TCA for 15 min. The TCA solution was removed and wells were washed once with water. DNA was extracted by incubation with 0.1 N NaOH at RT for 15 min.

Solubilized DNA was quantitatively transferred to minivials. Plates were washed once with water. Finally, 3 H-thymidine incorporation was determined by liquid scintillation counting.

A strategy for optimizing the three (number of Class 3) pyrimidine residues is illustrated in Figure 20. Ten different nucleotide analogs (illustrated in Figure 15) were chosen to construct the ribozyme library (n 10). In the first step, ten different pools (Class 1) were synthesized by the mix and split approach described herein. Positions 24 and 27 were random and position 28 was fixed with each of the ten different analogs. The ten different pools were formulated with a cationic lipid (Jarvis et al., 1996, RNA, 2,419; incorporated by reference herein), delivered to cells in vitro, and cell proliferation was subsequently assayed (see Figure 16). A positive control (active ribozyme) inhibited cell proliferation by -50% and an inactive control (inactive) resulted in a less than reduction in cell proliferation. The ten ribozyme pools resulted in intermediate levels of reduction. However, the best pool could be identified as X 24

X

2 2'-MTM-U 28 (positions 24 and 27 random; 2'-O-MTM-U at position 28). Therefore, a second ribozyme library (Class 2) was synthesized with position 28 constant position 24 was random (X 2 4 and position 27 was fixed with each of the ten different analogs (F 27 Again, SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -94the ten pools were assayed for their ability to inhibit cell proliferation. Among Class 2, two pools inhibited proliferation equally well: X, 2 2'-C-allyl-U2 2'-O-MTM-U, and X 24 2'-O-MTM-C 2 7 2'-O-MTM-U 28 Because a single "winner" could not be identified in Class 2, position 27 was made constant with either 2'-C-allyl-U or with 2'-O-MTM-C and the ten analogs were placed individually at position 24 (Class Therefore in Class 3, twenty different ribozymes were assayed for their ability to inhibit cell proliferation.

Because both positions 27 and 28 are constant, the final twenty ribozymes contain no random positions. Thus in the final group (Class pure ribozymes and not pools were assayed. Among the final groups four ribozymes inhibited cell proliferation to a greater extent than the control ribozyme (Figure 22). These four winners are illustrated in Figure 23A. Figure 23B shows general formula for four different motifs. A formula for a novel ribozyme motif is shown in Figure 18.

Example 11: Identifying Accessible Sites for Ribozyme Action in a target In the previous two examples (9 and 10), positions within the catalytic domain of the hammerhead ribozyme were optimized. The number of groups that needed to be tested equals the total number of positions within the ribozyme that were chosen to be tested.

A similar procedure can be used on the binding arms of the ribozyme. The sequence of the binding arms determines the site of action of the ribozyme. The combinatorial approach can be used to identify those sites by essentially testing all possible arm sequences. The difficulty with this approach is that ribozymes require a certain number of base pairs (12- 16) in order bind tightly and specifically. According to the procedure outlined above, this would require 12-16 different groups of ribozyme pools; 12-16 positions would have to be optimized which would require 12-16 different groups being synthesized and tested. Each pool would contain the four different nucleotides C, U and G) or nucleotide analogs (n It would be very time consuming to test each group, identify the best pool, synthesize another group of ribozyme pools with one additional position constant, and then repeat the procedure until all 12-16 groups had been tested. However it is possible to decrease the number of groups by testing multiple positions within a single group. In this case, the number of pools within a group equals the number of nucleotides or analogs in the random mixture n) to the w power, where w equals the number of positions fixed SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 in each group. The number of groups that need to be synthesized to optimize the final ribozyme equals the total number of positions to be optimized divided by the number of positions tested within each group. The number of pools in each group nw. The number of groups total number of positions w.

For example, Figure 23 illustrates this concept on a hammerhead ribozyme containing 12 base pair binding arms. Each of the two binding arms form 6 base pairs with it's corresponding RNA target. It is important to note that for the hammerhead ribozyme one residue (A15.1) must remain constant; A15.1 forms a base pair with a substrate nucleotide (U16.1) but is also absolutely required for ribozyme activity. It is the only residue within the hammerhead ribozyme that is part of both the catalytic domain, and the binding domain (arms). In the example this position is not optimized. In the first Group, three positions are fixed (designated F) with the four different 2'-O-methyl nucleotides C, U and The 2'-O-methyl modification stabilizes the ribozyme against nuclease degradation and increases the binding affinity with it's substrate. The total number of pools in each group does not equal n, as in the previous examples. The number of pools in each group equals 43 (four analogs)^(number of positions fixed; 3) 64. In all 64 pools, all other positions in the arm are made random (designated X) by the nucleotide mixing building block approach. The catalytic domain is not considered in this example and therefore remains part of the ribozyme template constant).

In the first step, all 64 ribozyme pools are tested. This test may be cleavage in vitro (see Example or efficacy in a cellular (see Example 10) or animal model, or any other assayable end-point. This end-point however, should be specific to a particular RNA target. For example, if one wishes to identify accessible sites within the mRNA of GeneB, a suitable end-point would be to look for decreased levels of GeneB mRNA after ribozyme treatment. After a winning pool is identified, since each pool specifies the identity of three positions three positions can be made constant for the next group (Class Class 2 is synthesized containing 64 different pools; three positions that were fixed in Class 1 are now constant (designated three more positions are fixed and the remaining positions are a random mix of the four nucleotides. The 64 pools are assayed as before, a winning pool is identified, allowing three more positions to be constant in the SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -96next Class ofribozyme pools (Class 3) and the process is repeated again. In the final Class of ribozymes (Class only two positions are fixed, all other positions have been previously fixed. The total number of ribozymes is therefore nw 42 16; these ribozymes also contain no random positions. In the final step (step the 16 ribozymes are tested; the winning ribozyme defines the sequence of the binding arms for a particular target.

Fixing multiple positions within a single group it is possible to decrease the overall number of groups that need to be tested. As mentioned, this is particularly useful when a large number of different positions need to be optimized. A second advantage to this approach is that it decreases the complexity of molecules in each pool. If one would expect that many combinations within a given pool will be inactive, by decreasing the number of different ribozymes in each pool, it will be easier to identify the "winning" pool. In this approach, a larger number of pools have to be tested in each group, however, the number of groups is smaller and the complexity of each ribozyme pool is smaller.

Finally, it should be emphasized there is not a restriction on the number of positions or analogs that can be tested. There is also no restriction on how many positions are tested in each group.

Example 12: Identifving new RNA targets for Ribozymes As described above for identifying ribozyme-accessible sites, the assayed used to identify the "winning" pool of ribozymes is not defined and may be cleavage in vitro (see Example or efficacy in a cellular (see Example 9) or animal model, or any other assayable end-point. For identifying accessible sites, this end-point should be specific to -a particular RNA target mRNA levels). However, the end-point could also be nonspecific. For example, one could choose a disease model and simply identify the winning ribozyme pool based on the ability to provide a desired effect. In this case, it is not even necessary to know what the cellular target that is being acted upon by the ribozyme is. One can simply identify a ribozyme that has a desired effect. The advantage to this approach is that the sequence of the binding arms will be complementary to the RNA target. It is therefore possible to identify gene products that are involved in a disease SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -97process or any other assayable phenotype. One does not have to know what the target is prior to starting the study. The process of identifying an optimized ribozyme (arm combinatorial) identifies both the drug (ribozyme) and the RNA target, which may be a known RNA sequence or a novel sequence leading to the discovery of new genes.

Example 13: Identifying New Ribozvme Catalytic Domains In the previous two examples, positions within the binding domain of the hammerhead ribozyme were varied and positions within the catalytic domain were not changed. Conversely, it is possible to vary positions within the catalytic domain, without changing positions within the binding arms, in order to identify new catalytic motifs. An example is illustrated in Figure 24. The hammerhead ribozyme, for example comprises about 23 residues within the catalytic domain. It is unclear how many of these 23 positions are required to obtain a functional catalytic domain, however it is reasonable to presume that if a large number of functionally diverse nucleotide analogs can be used to construct the pools, a relatively small number of positions could constitute a functional catalytic domain. This may especially be true if analogs are chosen that one would expect to participate in catalysis acid/base catalysts, metal binding, etc.). In the example illustrated in Figure 24, four positions (designated 1, 2, 3 and 4) are chosen. In the first step, ribozyme libraries (Class 1) are constructed: position 1 is fixed and positions 2, 3 and 4 are random (X2, X 3 and respectively). In step 2, the pools (the number of pools tested depends on the number of analogs used; n) are assayed for activity. This testing may be performed in vitro or in a cellular or animal model. Whatever assay that is used, the pool with the most activity is identified and libraries (class 2) are again synthesized with position 1 now constant with the analog that was identified in class 1. In class 2, position 2 is fixed (F 2 and positions 3 and 4 are random (X 3 and X 4 This process is repeated until every position has been made constant, thus identifying the catalytic domain or a new motif.

EXAMPLE 14: Determination of Coupling Efficiency of the phosphoramidite derivatives of 2'-C-allyl-uridine, 1: 4'-thio-cvtidine. 2: 2'-methylthiomethyl-uridine. 3, 2' methvlthiomethvl-cvtidine. 4: 2'-amino-uridine. 5: N3-methvl-uridine, 6: 1-b-D- SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -98- (ribofuranosyl)-pyridin-4-one. 7: 1-b-D-(ribofuranosvl)-pvridin-2-one, 8: 1-b-D- (ribofuranosvl)-phenvl, 9: 6-methvl-uridine, 10 to be used in a split and mix approach.

The determination of the coupling efficiency of amidites 1 to 10 was assessed using ten model sequences agacXGAuGa (where upper case represents ribonucleotide residues, lower case represents 2'-O-methyl ribonucleotide residues and X is amidites 1 to to be used in the construction of a hammerhead ribozyme library wherein the modified amidites 1 to 10 would be incorporated. Ten model sequences were synthesized using ten 0.112 g aliquots of 5'-O-DMT-2'-O-Me-Adenosine Polystyrene (PS) solid-support loaded at 22.3 imol/g and equivalent to a 2.5 pmol scale synthesis. Synthesis of these ten decamers were performed on ABI 394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standard nucleic acid synthesis reagents and synthesis protocols, with the exception of an extended (7.5 min) coupling time for the ribonucleoside phosphoramidites and phosphoramidites 1, 2, 3, 4, 6, 7, 8, 9, 10, 12.5 min coupling time for the 2'-aminouridine phosphoramidite, amidite 5 and 2.5 min coupling time for the 2'-O-methyl nucleoside phosphoramidites.

Oligomers were cleaved from the solid support by treatment with a 3:1 mixture of ammonium hydroxide:absolute ethanol at 65 degree C for 4 hrs followed by a desilylation treatment and butanol precipitation as described in Wincott et al. (Wincott et al, Nucleic Acids Res, 1995, 23, 2677-2684; incorporated by reference herein). Oligonucleotides were analyzed directly on an anion-exchange HPLC column (Dionex, Nucleopac, PA-100, 4x250 mm) using a gradient of 50% to 80% of B over 12 minutes (A 10 mM sodium perchlorate, 1 mM Tris, pH 9.43; B 300 mM sodium perchlorate, 1 mM Tris, pH 9.36) and a Hewlett-Packard 1090 HPLC system.

The average stepwise yield (ASWY), indicating the coupling efficiency of phosphoramidites, 1 to 10, were calculated from peak-area percentages according to the equation ASWY where FLP% is the percentage full-length product in the crude chromatogram and n the number of synthesis cycles. ASWY ranging from of 96.5% to 97.5% were obtained for phosphoramidites, 1 to 10. The experimental coupling efficiencies of the phosphoramidites 1 to 10, as determined using a standard SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -99spectrophotometric dimethoxytrityl assay were in complete agreement with the ASWY and were judged satisfactory to proceed with the X24, X27, X28 ribozyme library synthesis.

EXAMPLE 15: Determination of optimal relative concentration of a mixture of 2'-Omethyl-guanosine. cytidine. uridine and adenosine providing equal representation of the four nucleotides.

A mixture N, composed of an equimolar mixture of the four 2'-O-Me- nucleoside phosphoramidites (mG=2'-O-methyl guanosine; mA=2'-O-methyl adenosine; mC=2'-Omethyl cytidine; mU=2'-O-methyl uridine) was used in the synthesis of a model sequence TTXXXXTTB, where T is 2'-deoxy-thymidine and B is a 2'-deoxy-inverted abasic polystyrene solid-support as described in Example 14. After standard deprotection (Wincott et al., supra), the crude nonamer was analyzed on an anion-exchange HPLC column (see example From the HPLC analysis, an averaged stepwise yield (ASWY) of 99.3% was calculated (see example 14) indicating that the overall coupling efficiency of the mixture N was comparable to that of 2'-deoxythymidine. To further assess the relative incorporation of each of the components within the mixture, N, the full-length product TTXXXXTTB (over 94.3% at the crude stage) was further purified and subjected to base composition analysis as described herein. Purification of the FLP from the failures is desired to get accurate base composition.

Base composition analysis summary: A standard digestion/HPLC analysis was performed: To a dried sample containing A.sub.260 units of TTXXXXTTB, 50 l.1 mixture, containing 1 mg of nuclease P1 (550 units/mg), 2.85 ml of 30 mM sodium acetate and 0.3 ml of 20 mM aqueous zinc chloride, was added. The reaction mixture was incubated at 50 degrees C overnight. Next, 50 .tl of a mixture comprising 500 L1 of alkaline phosphatase (1 units/pl), 312 p.1 of 500 mM Tris pH 7.5 and 2316 p1 water was added to the reaction mixture and incubated at 37 degrees C for 4 hours. After incubation, the samples were centrifuged to remove sediments and the supernatant was analyzed by HPLC on a reversed-phase C18 column equilibrated with SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -100mM KH2PO4. Samples were analyzed with a 5% acetonitrile isocratic gradient for 8 min followed by a 5% to 70% acetonitrile gradient over 8 min.

The HPLC percentage areas of the different nucleoside peaks, once corrected for the extinction coefficient of the individual nucleosides, are directly proportional to their molar ratios.

The results of these couplings are shown in Table IV.

Nucleoside dT 2'-OMe-C 2'-OMe-U 2'-OMe-G 2'-OMe-A 0.1 M 0.025M 0.025M 0.025M 0.025M area 43.81 6.04 14.07 18.54 17.54 Epsilon 260 nm 8800 7400 10100 11800 14900 moles 0.00498 0.00082 0.00139 0.00157 0.00118 equivalent 4 0.656 1.119 1.262 0.946 As can be seen in Table IV, the use of an equimolar mixture of the four 2'-Omethyl phosphoramidites does not provide an equal incorporation of all four amidites, but favors 2'-O-methyl-U and G and incorporates 2'-O-methyl-A and C to a lower efficiency.

To alleviate this, the relative concentrations of 2'-O-methyl-A, G, U and C amidite were adjusted using the inverse of the relative incorporation as a guide line. After several iterations, the optimized mixture providing nearly identical incorporation of all four amidites was obtained as shown in Table V below. The relative representation do not exceed 12% difference between the most and least incorporated residue corresponding to a 6% deviation from equimolar incorporation.

Nucleoside dT 2'-OMe-C 2'-OMe-U 2'-OMe-G 2'-OMe-A 0.1M 0.032M 0.022M 0.019M 0.027M area 44.47 8.91 11.81 15.53 19.28 Epsilon 260 nm 8800 7400 10100 11800 14900 moles 0.00505 0.00120 0.00117 0.00132 0.00129 equivalent 4 0.953 0.926 1.042 1.024 SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 101 EXAMPLE 16: A Non-competitive coupling method for the preparation of the X24. X27 and N28 ribozvme library aaag aFX GAXGasg cg aaa gcc Gaa Age ccu cB -3' wherein 2'-C-allvl-uridine. 1: 4'-thio-cvtidine. 2: 2'-methvlthiomethvl-uridine, 3: 2'methylthiomethvl-cvtidine. 4: 2'-amino-uridine, 5: N3-methvl-uridine. 6; 1-b-D- (ribofuranosvl)-pvrimidine-4-one. 7: 1-b-D-(ribofuranosvl)-pvrimidine-2-one. 8; 1-b-D- (ribofuranosvl)-phenvl, 9: and/or 6-methyl-uridine, 10 are incorporated at the X24. X27 and F28 positions through the mix and split approach.

The synthesis of ten different batches of 2.5 imol scale Gag gcg aaa gcc Gaa Age ccu cB sequence was performed on 2'-deoxy inverted abasic polystyrene solid support B on a 394 ABI DNA synthesizer (Applied Biosystems, Foster City, CA). These ten aliquots were then separately reacted with phosphoramidite building blocks I to according to the conditions described in example 11. After completion of the individual incorporation of amidites 1 to 10, their coupling efficiencies were determined to be above as judged by trityl monitoring. The 10 different aliquots bearing the ten different sequences were mixed thoroughly and divided into ten equal subsets. Each of these aliquots were then successively reacted with ribo-A, ribo-G amidites and one of the amidites 1 to 10. The ten aliquots were combined, mixed and split again in 10 subsets. At that point, the 10 different polystyrene aliquots, exhibiting the following sequence: X GAX Gag gcg aaa gcc Gaa Age ccu cB, were reacted again with amidites 1 to 10 separately.

The aliquots were not mixed, but kept separate to obtain a unique residue at the 28th position of each of the ten pools. The ribozyme synthesis was then finished independently to yield ten random ribozymes pools. Each pool comprises a 3'-terminal inverted abasic residue B, followed by the sequence Gag gcg aaa gcc Gaa Age ccu c, followed with one random position X in the 24th position corresponding to a mixture of amidites 1 to followed by the sequence GA, followed one random position X in the 27th position corresponding to a mixture of amidites 1 to 10, followed by a fixed monomer F (one of the amidites 1 to 10) in the 28th position and finally the 5'-terminal sequence ascsasasa g a.

This is represented by the sequence notation ascsasasag aFX GAX Gag gcg aaa gcc Gaa Age ccu cB-3', in which X are random positions and F is a unique fixed position. The SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 102total complexity of such a ribozyme library was 103 or 1,000 members separated in pools of 100 different ribozyme sequences each.

EXAMPLE 17: Competitive coupling method (monomer mixing approach) for the preparation of the and X 30 .3 5 "binding arms" ribozme library Synthesis of 5'-xxx xFF cuG Au G Agg ccg uua ggc cGA AAF xxx xB-3' is described, with F being a defined 2'-O-methyl-ribonucleoside chosen among 2'-O-methylribo-adenosine -guanosine -cytidine -uridine (mU) and x being an equal mixture of 2'-O-methyl-ribo-adenosine, -guanosine, -cytidine, -uridine.

The syntheses of this ribozyme library was performed with an ABI 394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standard nucleic acid synthesis reagents and synthesis protocols, with the exception of an extended (7.5 min) coupling time for the ribonucleoside phosphoramidites (upper case) and 2'-amino-uridine phosphoramidite, u, (2.5 min) coupling time for the 2'-O-methyl-ribonucleoside phosphoramidites (lower case) and the 2'-O-methyl-ribonucleoside phosphoramidites mixture, n.

Sixty four (64) batches of 0.086 g aliquots of 3'-O-DMT-2'-deoxy-inverted abasic- Polystyrene solid-support loaded at 29 jtmol/g and equivalent to a 2.5 pmol scale synthesis were individually reacted with a 27:32:19:22 v:v:v:v mixture, x, of mA:mC:mG:mU diluted in dry acetonitrile to 0.1 M as described in example 7. This synthesis cycle was repeated for a total of four times. The 64 aliquots were then grouped into four subsets of sixteen aliquots (Class 1) that were reacted with either mA, mG, mC, mU to synthesize the n6 position. This accomplished, the sequence: cuG Au G Agg ccg uua ggc cGA AA was added onto the 6 position of the 64 aliquots constituting Class 1.

Each subset of Class 1 was then divided into four subsets of four aliquots (Class 2) that were reacted with either mA, mG, mC, mU to synthesize the F30 position. Each subset of Class 2 was then divided into four subsets of one aliquot (Class 3) that were reacted with either mA, mG, mC, mU to synthesize the F31 position. Finally, the random sequence xsxsx x was added onto each of the 64 aliquots.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 103- The ribozyme library yielded sixty four random ribozymes pools each having an equal mixture of the four 2'-O-methyl-nucleoside at the position x2 to 6 and x30 to and a defined 2'-O-methyl-nucleoside chosen among mA, mC, mG, mU at the positions F6, F30 and F31. The total complexity of such a "binding arms" ribozyme library was 4 or 4,194,304 members separated in 64 pools of 65,536 different ribozyme sequences each.

EXAMPLE 18: Competitive coupling method (monomer mixing approach) for the preparation of the position 15 to 18 "loop II" ribozyme library Synthesis of 5' UCU CCA UCU GAU GAG GCC XXF XGG CCG AAA AUC CCU 3' is described, with F being a defined ribonucleoside chosen among adenosine guanosine cytidine uridine and X being an equal mixture of adenosine guanosine cytidine uridine The syntheses of this ribozyme library was performed with an ABI 394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standard nucleic acid synthesis reagents and synthesis protocols, with the exception of an extended (7.5 min) coupling time for the ribonucleoside phosphoramidites G, C, U) and the ribonucleoside phosphoramidite mixture, X.

Four batches of 2.5 uimol scale of GG CCG AAA AUC CCU sequence were synthesized on 0.085 g samples of 5'-O-DMT-2'-O-TBDMS-3'-succinyl-uridine- Polystyrene solid-support loaded at 29.8 tmol/g. To synthesize the position X15, the four aliquots of solid-supports were individually reacted with a 30:26:24:20 v:v:v:v mixture, X, of A:C:G:U diluted in dry acetonitrile to 0.1 M according to the optimized conditions for the DNA phosphoramidites mixed-base coupling as described in the DNA Synthesis Course Manual published by Perkin-Elmer-Applied Biosystem Division. (DNA Synthesis Course Manual Evaluating and isolating synthetic oligonucleotides, the complete guide, p. 2-4, Alex Andrus, August 1995). The four aliquots of solid-supports were then individually reacted with either of the four ribonucleoside phosphoramidites (A, G, C, U) to create the F16 position. The position X17 and X18 were then added onto the SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -104- F16 (either A, G, C or U) of the four aliquots of solid-supports by repeating twice the same procedure used for the position The synthesis of the ribozyme library was then ended by adding the sequence UCU CCA UCU GAU GAG GCC on the position X18 of each of the four subsets of the ribozyme library. The ribozyme library yielded four random ribozymes pools that each have an equal mixture of the four ribonucleoside G, C and U) at the position X15, X17 and X18, and a discrete ribonucleoside chosen among A, C, G or U at the positions F16.

The total complexity of such a loop II ribozyme library was 256 members separated in 4 pools of 64 different ribozyme sequences.

Example 19: Arm-Combinatorial Library Screening For Bcl-2, K-ras and Urokinase plasminogen Activator (UPA) Substrate synthesis through in vitro transcription: Run-off transcripts for Bcl-2 and Kras were prepared using linearized plasmids (975 and 796 nucleotides respectively).

Transcripts for UPA were produced from a PCR generated DNA fragment containing a T7 promoter (400 nucleotides). Transcription was performed using the T7 Megascript transcription kit (Ambion, Inc.) with the following conditions: a 50ul reaction volume containing 7.5mM each of ATP, CTP, UTP, and GTP, 2mM guanosine, 5ul 10x T7 reaction buffer, 5ul T7 enzyme mix, and 0.5ug of linearized plasmid or PCR'd DNA template. The mixture was incubated at 37C for 4 hours (6 hours for transcripts 500 bases). Guanosine was added to the transcription reactions so that the final transcript could be efficiently 5'-end labeled without prior phosphatase treatment. Transcription volume was then increased to 200ul with buffer containing 50mM TRIS pH 7.5, 100mM KC1, and 2mM MgCl 2 and spin column purified over Bio-Gel P-60 (BioRad) equilibrated in the same buffer. 100ul of transcript was then applied to 750ul of packed resin. Spin column flow-through was used directly in a 5'-end labeling reaction as follows (100ul final volume): 82ul of P-60 spin column purified transcript, 10ul 10x polynucleotide kinase buffer, 4ul 10U/ul Polynucleotide Kinase (Boehringer/Mannheim) and 4ul 150uCi/ul Gamma-32P-ATP (NEN) were incubated together at 37 0 C for one hour. The reaction volume was increased to 200ul with buffer containing 50mM TRIS pH 100mM KC1 and 2mM MgCl 2 and the sample was then purified over Bio-Gel P-60 packed SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -105spin column as described above. Approximate specific activities of the 5'-end labeled transcripts were determined via BioScan and stored frozen at -20 0

C.

Synthesis ofRibozyme pools: In vitro ribozyme-transcript cleavage reactions: Cleavage reactions were carried out as follows: 5'-end labeled transcript x 104 dpm/ul final) was incubated with ribozyme pool in 50mM TRIS pH 7.5, 50mM NaC1, 2mM MgC12 and 0.01% SDS for 24- 48 hours at room temperature (-22 0 An equal volume of gel loading dye formamide, 0.01M EDTA, 0.0375% bromophenol blue, and 0.0375% xylene cyanol) was added to stop the reaction and the samples are heated to 95 0 C. Reactions (1-2 x 105 dpm per lane) were run on a 5% denaturing polyacrylamide gel containing 7M urea and lx TBE. Gels are dried and imaged using the Phosphorlmager system (Molecular Dynamics). Ambion, Inc. RNA Century Marker Plus RNA standards body labeled in a T7 Megascript reaction as described above using 3ul of 0lmCi/ml Alpha- 32 P-ATP (BioRad) and 0.5ug Century RNA template and subsequently spin column purified over Bio-Gel P-6 (Bio-Rad) were used as a size reference on the gel. Cleavage product sizes were determined using the RNA standards which provided an approximate site of cleavage (est.

Size in Figure). Because each of the ribozyme pools has three positions within the binding arms fixed, it is possible to identify all of the potential ribozyme sites that can potentially be cleaved by that pool. The estimated size of the cleavage product is therefore compared with the potential sites to identify the exact site of cleavage.

This protocol has been completed on three different transcripts: Bcl-2 (figure 25), Kras (figure 26), and UPA (figure 27). The data is summarized in the figures. All potential hammerhead ribozyme cleavage sites are indicated in the graph with a short vertical line.

The actual sites identified are indicated in the graph. The size of the bar reflects the intensity of the cleavage product from the cleavage reaction. The combinatorial pool used to identify each site, the actual sequence of each site, the position of cleavage within the transcript (based on the known sequence), and the estimated size of the cleavage product (based on gel analysis) are listed.

Example 20: Reduction of Bcl-2 mRNA using Optimized Ribozvmes Two ribozymes targeted against the same site in the bcl-2 transcript (Seq.ID#9, figure 25) were synthesized, but the two ribozymes were stabilized using two different SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -106chemistries (U4/U7 amino and U4 c-allyl). Ribozymes (200 nM) were delivered using lipofectamine (7.2 mM) for 3 hours into MCF-7 cells (50% confluency). Cellular RNA was harvested 24 hours after delivery, analyzed by RNase protectection analysis (RPA) and normalized to GAPDH mRNA in triplicate samples. Both ribozymes gave a reduction in bcl-2 mRNA (see Figure 28). A ribozyme targeted against an irrelevant mRNA (c-myb) had no effect on the ratio of bcl-2 mRNA to GAPDH mRNA. All reduction of bcl-2 RNA was statistically significant with respect to untreated samples and samples treated with the irrelevant ribozyme.

Example 21: Synthesis of purine nucleoside triphosphates: triphosphate 2'-O-methyl guanosine nucleoside (0.25 grams, 0.84 mmol) was dissolved in triethyl phosphate ml by heating to 100 C for 5 minutes. The resulting clear, colorless solution was cooled to 0 C using an ice bath under an argon atmosphere. Phosphorous oxychloride (1.8 eq., 0.141 ml) was then added to the reaction mixture with vigorous stirring. The reaction was monitored by HPLC, using a sodium perchlorate gradient.

After 5 hours at 0 C, tributylamine (0.65 ml) was added followed by the addition of anhydrous acetonitrile (10.0 ml), and after 5 minutes (reequilibration to 0 C) tributylammonium pyrophosphate (4.0 eq., 1.53 g) was added. The reaction mixture was quenched with 20 ml of 2M TEAB after 15 minutes at 0 C (HPLC analysis with above conditions showed consumption of monophosphate at 10 minutes) then stirred overnight at room temperature, the mixture was evaporated in vacuo with methanol co-evaporation (4x) then diluted in 50 ml 0.05M TEAB. DEAE sephadex purification was used with a gradient of 0.05 to 0.6 M TEAB to obtain pure triphosphate (0.52 g, 66.0% yield) (elutes around 0.3M TEAB); the purity was confirmed by HPLC and NMR analysis.

Example 22: Synthesis of Pvrimdine nucleoside triphosphates: 2'-O-methylthiomethyl- 2'-O-methylthiomethyl uridine nucleoside (0.27 grams, 1.0 mmol) was dissolved in triethyl phosphate (5.0 ml). The resulting clear, colorless solution was cooled to 0 C with an ice bath under an argon atmosphere. Phosphorus oxychloride (2.0 eq., 0.190 ml) was then added to the reaction mixture with vigorous stirring. Dimethylaminopyridine (DMAP, 0.2eq., 25 mg) was added, the solution warmed to room temperature and the SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -107reaction was monitored by HPLC, using a sodium perchlorate gradient. After 5 hours at C, tributylamine (1.0 ml) was added followed by anhydrous acetonitrile (10.0 ml), and after 5 minutes tributylammonium pyrophosphate (4.0 eq., 1.8 g) was added. The reaction mixture was quenched with 20 ml of 2M TEAB after 15 minutes at 20 C (HPLC analysis with above conditions showed consumption of monophosphate at 10 minutes) then stirred overnight at room temperature. The mixture was evaporated in vacuo with methanol coevaporation (4x) then diluted in 50 ml 0.05M TEAB. DEAE fast flow Sepharose purification with a gradient of 0.05 to 1.0 M TEAB was used to obtain pure triphosphate (0.40 g, 44% yield) (elutes around 0.3M TEAB) as determined by HPLC and NMR analysis.

Example 23: Utilization of DMAP in Uridine 5'-Triphosphate Synthesis The reactions were performed on 20 mg aliquots of nucleoside dissolved in 1 ml of triethyl phosphate and 19 ul of phosphorus oxychloride. The reactions were monitored at 40 minute intervals automatically by HPLC to generate yield-of-product curves at times up to 18 hours. A reverse phase column and ammonium acetate/ sodium acetate buffer system (50mM 100mM respectively at pH 4.2) was used to separate the 2' monophosphates (the monophosphates elute in that order) from the triphosphate and the starting nucleoside. The data is shown in table VI. These conditions doubled the product yield and resulted in a 10-fold improvement in the reaction time to maximum yield (1200 minutes down to 120 minutes for a 90% yield). Selectivity for monophosphorylation was observed for all reactions. Subsequent triphosphorylation occurred in nearly quantitative yield.

Materials Used in Bacteriophage T7 RNA Polymerase Reactions BUFFER 1: Reagents are mixed together to form a 10X stock solution of buffer 1 (400 mM Tris-Cl (pH 200 mM MgCl,, 100 mM DTT, 50 mM spermidine, and 0.1% triton X-100. Prior to initiation of the polymerase reaction methanol, LiCl is added and the buffer is diluted such that the final reaction conditions for condition 1 consisted of: tris pH 20mM MgC1 2 10 mM DTT, 5 mM spermidine, 0.01% triton X-100, methanol, and 1 mM LiCI.

BUFFER 2: Reagents are mixed together to form a 10X stock solution of buffer 2(400 mM Tris-Cl (pH 200 mM MgCl 2 100 mM DTT, 50 mM spermidine, and 0.1% triton SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 108- X-100. Prior to initiation of the polymerase reaction PEG, LiCl is added and the buffer is diluted such that the final reaction conditions for buffer 2 consisted of 40mM tris pH 20mM MgC1, 10 mM DTT, 5 mM spermidine. 0.01% triton X-100, 4% PEG, and 1 mM LiC1.

BUFFER 3: Reagents are mixed together to form a 10X stock solution of buffer 3 (400 mM Tris-Cl (pH 120 mM MgCl 2 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG is added and the buffer is diluted such that the final reaction conditions for buffer 3 consisted of 40mM tris pH 12 mM MgC1 2 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, and 4% PEG.

BUFFER 4: Reagents are mixed together to form a 10X stock solution of buffer 4 (400 mM Tris-Cl (pH 120 mM MgCl 2 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG, methanol is added and the buffer is diluted such that the final reaction conditions for buffer 4 consisted of tris pH 12 mM MgCl 2 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, methanol, and 4% PEG.

BUFFER 5: Reagents are mixed together to form a 10X stock solution of buffer 5 (400 mM Tris-Cl (pH 120 mM MgCl 2 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG, LiCI is added and the buffer is diluted such that the final reaction conditions for buffer 5 consisted of 40mM tris pH 12 mM MgCI 2 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, 1 mM LiCl and 4% PEG.

BUFFER 6: Reagents are mixed together to form a 10X stock solution of buffer 6 (400 mM Tris-Cl (pH 120 mM MgCI 2 50 mM DTT, 10 mM spermidine and 0.02% triton X-100. Prior to initiation of the polymerase reaction PEG, methanol is added and the buffer is diluted such that the final reaction conditions for buffer 6 consisted of tris pH 12,mM MgCl 2 5 mM DTT, 1 mM spermidine, 0.002% triton X-100, methanol, and 4% PEG.

Example 24: Screening of Modified Nucleoside triphosphates with Mutant T7 RNA Polymerase Each modified nucleotide triphosphate was individully tested in buffers 1 through 6 at two different temperatures (25 and 37 0 Buffers 1-6 tested at 25 0 C were designated SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -109conditions 1-6 and buffers 1-6 test at 37°C were designated conditions 7-12 (table VII).

In each condition, Y639F mutant T7 polymerase (Sousa and Padilla, Supra) (0.3-2 ml reaction), NTP's (2 mM each), DNA template (10 pmol), inorganic pyrophosphatase and a- 32 P NTP(0.8 mCi/pmol template) were combined and heated at the designated temperatures for 1-2 hours. The radiolabeled NTP used was different from the modified triphosphate being testing. The samples were resolved by polyacrylamide gel electrophoresis. Using a phosphorlmager (Molecular Dynamics, Sunnyvale, CA), the amount of full-length transcript was quantified and compared with an all-RNA control reaction. The data is presented in Table VIII; results in each reaction is expressed as a percent compared to the all-ribonucleotide triphosphate (rNTP) control. The control was run with the mutant T7 polymerase using commercially available polymerase buffer (Boehringer Mannheim, Indianapolis, IN).

Example 25: Incorporation of Modified NTP's using Wild-type T7 RNA polvmerase Bacteriophage T7 RNA polymerase was purchased from Boehringer Mannheim at 0.4 U/ tL concentration. Applicant used the commercial buffer supplied with the enzyme and 0.2 uCi alpha- 3 2 P NTP in a 50 uL reaction with nucleotides triphosphates at 2 mM each. The template was double-stranded PCR fragment, which was used in previous screens. Reactions were carried out at 37 0 C for 1 hour. 10 uL of the sample was run on a analytical PAGE and bands were quantitated using a Phosphorlmager. Results are calculated as a comparison to an "all ribo" control (non-modified nucleoside triphosphates) and the results are in Table IX.

Example 26: Incorporation of Multiple Modified Nucleoside triphosphates Into Oligonucleotides Combinations of modified nucleoside triphosphates were tested with the transcription protocol described in example 9, to determine the rates of incorporation of two or more of these triphosphates. Incorporation 2'-Deoxy-2'-(L-histidine) amino uridine (2'-his-NH 2 -UTP) was tested with unmodified cytidine nucleoside triphosphates, rATP and rGTP in reaction condition number 9. The data is presented as a percentage of incorporation of modified NTP's compared to the all rNTP control and is shown in Table Xa.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -110- Two modified cytidines (2'-NH2-CTP or 2'dCTP) were incorporated along with 2'his-NH,-UTP with identical efficiencies. 2'-his-NH 2 -UTP and 2'-NH,-CTP were then tested with various unmodified and modified adenosine triphosphates in the same buffer (Table Xb). The best modified adenosine triphosphate for incorporation with both 2'-his- NH,-UTP and 2'-NH,-CTP was 2'-NH 2

-DAPTP.

EXAMPLE 27: Optimization of Reaction conditions for Incorporation of Modified Nucleotide Triphosphate The combination of 2'-his-NH,-UTP, 2'-NH2-CTP, 2'-NH,-DAP, and rGTP was tested in several reaction conditions (Table XI) using the incorporation protocol described in example 14. The results demonstrate that of the buffer conditions tested, incorporation of these modified nucleoside triphosphates occur in the presence of both methanol and LiCI.

Example 28: Deprotection of Ribozvme in a 96 Well Plate A ribozyme sequence (200nmole) was synthesized as described herein on a polystyrene solid support in a well of a 96 well plate. A 10:3:13 mixture (800 pL) of anhydrous methylamine (308uL), triethylamine (92tL) and dimethylsulfoxide (DMSO) (400 iL) was prepared of which half (400 pL) was added to the well and incubated at room temperature for 45 minutes. Following the reaction the solution was replaced with the remaining 400 pL and incubated as before. At the end of the reaction, the solid support was filtered off, all 800 iL of MA/TEA/DMSO solution was collected together and 100 puL of TEA.3HF was added. The reaction was then heated at 65 0 C for 15 minutes and then cooled to room temperature. The solution was then quenched with aqueous NH 4 'HC03 (ImL) (see Figure 30). HPLC chromatography of the reaction mixture afforded 32 0.

D.u 26 nm of which 46% was full length ribozyme.

Example 29: Column Deprotection of Ribozyme A ribozyme was synthesized using the column format as described herein. The polystyrene solid-support with protected oligoribonucleotide or modified oligoribonucleotide (200 nmole) was transferred into a glass vial equipped with a screw cap. A 10:3:13 mixture of anhydrous methylamine 308 pL), triethylamine (92 pL) and dimethylsulfoxide (DMSO) (400 LL) was added followed by vortexing of the glass vial.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 111 After allowing the reaction for 1.5 hours, the solid support was filtered off. 100 1 L of TEA.3HF was added at room temperature to the vial and the reaction was mixed causing the solution to gel. The reaction was then heated at 65 °C for 15 minutes and then cooled to room temperature. The solution was then quenched with 1.5 M aqueous NH 4

'HCO

3 (lmL). HPLC chromatography of the reaction mixture afforded 32 0. D.u 6 0 o m of which 46% was full length ribozyme.

Example 30: Column Deprotection of Ribozvme with anhydrous ethanolic methvlamine A ribozyme was synthesized using the column format as described herein. The polystyrene solid-support with protected oligoribonucleotide or modified oligoribonucleotide (200 nmole) was transferred into a glass vial equipped with a screw cap. A 1:1 mixture of anhydrous ethanolic methylamine 400 4L) and dimethylsulfoxide (DMSO) (400 L) was added followed by vortexing of the glass vial. After allowing the reaction for 1.5 hours, the solid support was filtered off. 100 pL of TEA.3HF was added at room temperature to the vial and the reaction was mixed causing the solution to gel.

The reaction was then heated at 65 °C for 15 minutes and then cooled to room temperature. The solution was then quenched with 1.5 M aqueous NH4'HCO 3 (lmL).

HPLC chromatography of the reaction mixture afforded 32 0. D.u 2 60 nm of which 46% was full length ribozyme.

Example 31. Large-scale One-Pot Deprotection of Ribozvme A ribozyme was synthesized at the 0.5 mmol scale using the column format as described herein. The polystyrene solid-support (24 grs) with protected oligoribonucleotide or modified oligoribonucleotide (500 umole) was transferred into a 1L Schott bottle equipped with a screw cap. A 1:1.3 mixture of anhydrous ethanolic methylamine 150 mL) and dimethylsulfoxide (DMSO) (200 mL) was added followed by vortexing (200 rpm) of the glass bottle for 1.5 hours. The reaction mixture was then frozen at -70 °C for 30 minutes. 50 mL of neat TEA.3HF was then added at room temperature to the reaction mixture and the reaction was placed in a shaking oven (200 rpm) where it was heated at 65 OC for 60 minutes and subsequently frozen at -70 OC for minutes. The solution was then quenched with 1.5 M aqueous NH 4 HC03 (200 mL). The SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -112reaction mixture was separated from the polystyrene solid-support by filtration on a sintered glass funnel (10-20 j.m porosity). U.V. spectrophotometric quantification and HPLC chromatography of the reaction mixture afforded 160,000 O.D.u 260 m of which 46.4% was full length ribozyme. After allowing the reaction for 1.5 hours, the solid support was filtered off Example 32: Antitumor and antimetastatic efficacy of ribozvmes directed against the mRNA encoding the two VEGF receptor subtypes, fit-1 and flk-1 in the mouse Lewis lung-HM carcinoma model of primary tumor growth and metastasis The Lewis lung carcinoma (LLC) model is a syngeneic mouse model of metastatic cancer commonly used for antitumor agent efficacy screening. According to Folkman (1995, supra), primary tumor growth and metastasis in this model is dependent upon VEGF-induced angiogenesis. Two variants of the LLC model exist. The low metastatic form involves the implantation of a tumor, usually subcutaneous, which sends micrometastases to the lungs whose growth is suppressed by the presence of the primary tumor. The highly metastatic (HM) form differs from the low metastatic variant in that the growth of metastases is not suppressed by the presence of the primary tumor. Thus, the HM form is a model in which it is possible to measure pharmacologic efficacy on both primary tumor growth and metastasis in the same mouse without excision of the primary tumor.

Applicant selected the highly metastatic variant of the Lewis lung model for antitumor/metastatic screening of ribozymes directed against VEGF receptor (flt-1 andflk- 1) mRNA. These ribozymes have been shown to reduce VEGF binding and VEGFstimulated proliferation in cultured MVEC's as well as VEGF-induced neovascularization of the rat cornea (Cushman et al., 1996, Angiogenesis Inhibitors and Other Novel Therapeutic Strategies for Ocular Diseases of Neovascularization, IBC Conference Abstract). Pharmacokinetically, Applicant has found that ribozymes distribute systemically following continuous i.v. infusion (via Alzet osmotic minipumps) at significant concentrations within most tissues including subcutaneously implanted tumors.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -113- This study examines the antitumor/antimetastatic efficacy of flt-I and flk-1 ribozymes continuously infused i.v. in the LLC-HM mouse model.

Methods Ribozymes The ribozymes used in this study were hammerhead ribozymes comprising a 4 base pair stem II, four phosphorothioate linkages at the 5'-end, a 2'-C-allyl substitution at position 4 (see Figure and an inverted abasic nucleotide substitution at the 3'-end. The catalytically active and inactive ribozymes were RPI.4610/4611 (active/inactive) and RPI.4733/4734 directed against flt-1 and flk-1 messages, respectively. Ribozymes solutions were prepared in normal saline (USP).

Test solutions (ribozymes or saline control) were dispensed into Alzete osmotic minipumps (Model 1002--total volume capacity including excess 200 pl) which dispense 0.5 pl/h at 37 °C when exposed to interstitial water. Pumps were either filled with normal saline (USP) or 167.0, 50.0, 16.7, 5.0, or 1.7 mg/ml ribozyme solutions. Prior to animal implantation, osmotic minipumps were placed in 37 °C sterile water for at least four hours to activate pumping.

Tumor inoculation All animal procedures in this study were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (1996), USDA regulations, and the policies and procedures of the RPI Institutional Animal Care and Use Committee. A total of 210 female C57BL/6J mice weighing between 20-25 g were used in this study. All animals were housed under 12 h on/12 h off light cycles and received ad libitum food and water.

Highly metastatic variant Lewis lung carcinoma (LLC-HM) tumors were propogated in vivo from an LLC-HM cell line. These tumors needed to be propogated in vivo because they can revert to the low metastatic phenotype in culture. LLC-HM cells were initially cultured in DMEM 10% FCS 1 GPS. For in vivo propogation, 5 X 10 6 cells were injected subcutaneously in mice. Tumors were allowed to grow for 25 days at which time SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -114animals were euthanized by CO 2 inhalation and lung macrometastases were counted.

Animals with the most macrometastases (approximately 15-20) were selected for preparation of tumor breis and propogation. When tumors in animals selected for propogation reached a volume of approximately 1500 mm 3 animals were euthanized by

CO

2 inhalation and tumors were excised. Tumors were seived through a 100 mrn pore size sterile nylon mesh. LLC-HM cells were resuspended in normal saline to a final concentration of 5 x 10 6 viable cells/ml (via hemocytometer). Three days prior to ribozyme dosing, all animals were subcutaneously inoculated on the right flank with 5 x 105 cells (in a volume of 100 utl).

Ribozyme or saline dosing Each ribozyme solution was prepared to deliver 100, 30, 10, 3, or 1 mg/kg/day in a volume of 12 ml. A total of 10 animals per dose or saline control group were surgically implanted on the left flank with osmotic minipumps pre-filled with the respective test solution three days following tumor inoculation. Pumps were attached to indwelling jugular vein catheters. The specifications for the model #1002 Alzet osmotic minipump show that they accurately deliver aqueous solutions at 0.5 ptl/h for 14 days. Table III summarizes the experimental groups.

Tumor volume and metastatic index quantitation Beginning four days and ending 24 days days following tumor inoculation, the length and width of all primary tumors were measured every other day using microcalipers. Tumor volumes were calculated using the standard formula for an elipsoid volume, (L W 2 Tumor volumes were calculated in triplicate for each animal. A mean tumor volume was calculated for each animal. Group means and standard error of the group means were calculated from individual animal mean tumor volumes.

Twenty-five days following tumor inoculation, all animals were euthanized by CO 2 inhalation and lungs and primary tumors harvested. Lung macrometastases were counted under a dissecting microscope (2.5 X magnification). Lungs and primary tumors were also SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -115weighed on an analytical balance. Lung weights served as an index of total lung metastatic burden.

Statistical analysis For all treatment groups, group tumor volume means on day 18 (end of treatment) as well as means of primary tumor and lung weight and numbers of lung metastases were evaluated for normality and subjected to analysis of variance. Statistical differences between group means were evaluated using the Tukey-Kramer post-hoc test (alpha 0.05). Comparisons with the control group (saline control) were made using the Dunnett's test (alpha 0.05).

Results Fit-i The effects of several doses of active and inactive Flt-] ribozymes (RPI.4610/4611, respectively) on primary LLC-HM tumor growth are summarized in Figure 39 At the lowest dose (Figure 39A), both active and inactive reduce primary tumor growth similarly throughout the entire time course compared to saline controls. However, with increasing dose, active ribozyme reduces primary tumor growth to a greater extent than the inactive ribozyme, with the largest difference observed at 30 mg/kg/day (Figure 39D).

The magnitude of the maximal reduction compared to saline was approximately four fold with the active ribozyme RPI.4610 at 30 mg/kg/day. It should be noted that this observed four fold reduction is still present at day 24 even though treatment ended 7 days earlier.

The growth curve data was subjected to exponential regression. The curve fits show that the tumor growth data fits an exponential curve with a high correlation coefficient Thus, there appears to be no long lasting toxic effect on tumor growth. Since the calculated slope of the exponential curve at any point indicates the rate of tumor growth, it should be possible to compare rates of growth between treatments. Since the curve fits do not assume that the tumor growth starts from the same point (which is a correct assumption since the all tumors start with' the same tumor cell inoculum concentration), an accurate calculation of the slope of the exponential curve is not possible SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -116since the curve fitting algorithm extrapolates a t 0 tumor size which is then used to calculate the slope. In the analysis, the saline tumor size at t 0 is much greater than the other treatment groups, thus comparisons with saline are not necessarily accurate. If the curve fit algorithm is restricted to the same tumor size, a dose-dependent reduction in the rate of tumor growth is observed with the active ribozyme. However, the curve fits show lower correlation coefficients in some cases.

In order to see whether a the ribozyme treatments statistically reduce primary tumor growth, primary tumor volume measurements at each dose immediately following treatment (day 18) were compared (Figure 40). Active ribozyme RPI.4610 produced a statistically significant (p 0.05) and dose-dependent reduction in primary tumor volume.

Although the inactive ribozyme (RPI.4611) showed some reduction in primary tumor volume at the lowest and highest doses, there was no dose-dependent reduction observed.

At doses between 3 and 30 mg/kg/day, the inactive ribozyme showed no significant reduction in primary tumor volume. There was a significant difference (p 0.05) between active and inactive ribozymes (Tukey-Kramer test) at doses of 10 and 30 mg/kg/day.

Applicant has also observed that the active ribozyme RPI.4610 produced a significant reduction in primary tumor mass at all doses tested (1-100 mg/kg/day) 25 days following inoculation.

Figures 41 A and B illustrate that the active ribozyme reduced both the number of lung metastases and lung mass in a dose-dependent manner. The active flt-1 ribozyme showed a significant reduction (p 0.05 by Dunnetts) in the number of lung metastases at the 30 and 100 mg/kg/day doses compared to saline. There was also a significant difference between active and inactive ribozymes at these doses (p 0.05 by Student's t).

RPI.4610 reduced the lung weight to almost normal levels at the highest dose (100 mg/kg/day). There was no observable dose-related effect of the inactive ribozyme on either the number of lung metastases or lung weight. A significant reduction (p 0.05, Student's t) in lung mass, an index of metastatic burden, was observed between saline and the active ribozyme. The lack of significance using more stringent statistical tests (Dunnet's or Tukey-Kramer), which take into account the variance within all groups, was SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 117due to high variability, especially in the inactive ribozyme group. However, since five doses were tested, it is possible to say that there is a dose-dependent trend in the reduction of lung metastases/lung weight.

Example 33: Effects of flk-1 ribozvmes (active/inactive) on LLC-HM primary tumor growth in mice.

The dose-related effects of active and inactive flk-1 directed ribozymes (RPI.4733/4734, respectively) on primary LLCare shown in Figure 38 A-E.

The dose-related effects of active and inactive flk-1 directed ribozymes (RPI.4733/4734, respectively) on primary LLCare shown in Figure 42 A-E. At the lowest dose, there was no observable effect on primary tumor growth with the active flk-1 ribozyme (Figure 42A). The inactive ribozyme showed a modest reduction in primary tumor growth. At higher doses (3-100 mg/kg/day, Figure 42B-E), the active flk-1 ribozyme reduced primary tumor growth while the inactive ribozyme showed little, if any, antitumor efficacy over the dose range between 10 and 100 mg/kg/day (Figures 42C-E).

The antitumor efficacy of both active and inactive flk-1 ribozymes are similar at 3 mg/kg/day (Figure 42B).

As in the case of the fit-1 ribozymes, tumor growth followed exponential growth kinetics. Again, since the t 0 tumor size could not accurately be estimated by the curve fit program, it is not possible to calculate the slope of the exponential curve fits for theflk- 1 ribozymes.

Immediately following the cessation of treatment (day 18), the activeflk-1 ribozyme showed a significant reduction in primary tumor volume from 3 to 100 mg/kg/day (Figure 43). The magnitude of the reduction is approximately four fold and appeared to be maximal at 3 mg/kg/day. The lowest dose had no significant effect on primary tumor volume. The inactive flk-1 ribozyme had a significant antitumor effect at doses of 1 and 3 mg/kg/day; however, this effect disappeared between 10 and 100 mg/kg/day.

The antimetastatic effects of theflk-1 ribozymes are illustrated in Figure 44 A and B.

Although neither ribozyme showed a statistically significant effect on the number of lung SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -118metastases at any dose, it appears that the active flk-1 ribozyme showed a significant reduction in lung mass over the dose range between 3 and 100 mg/kg/day.

Applicant has further observed that the lung mass was reduced to normal over the entire dose range. The inactive ribozyme reduced lung mass at 1 and 3 mg/kg/day (Figure 41C); however, this trend was not observed at higher doses (3-100 mg/kg/day).

Example 34: Ribozvme-mediated decrease in vascularitv of tumor Three tumors from each of three treatment groups (saline controls, inactive RPI.4611 and active RPI.4610, 30 mg/kg/day dose only) were analyzed for vascularity using an immunohistochemical assay which stains endothelial cells for CD31 (PECAM).

The vascularity was quantitated in a blinded fashion. From the raw data the average number of vessels per high magnification field (400X) were calculated. They are as follows: SALINE CONTROL 24.1; RPI.4611 (Inactive) 27.6; RPI.4610 (Active) 16.0.

It is suggestive that ribozyme-specific antiangiogenic effect is exhibited by the active Flt-1 ribozyme in Lewis lung tumors. Thus, the mechanism of action for the observed reduction in the primary tumor volumes may be due to an antiangiogenic effect.

Similar delivery strategies can be used to deliver c-raf ribozymes to treat a variety of diseases.

Use of Ribozymes Targeting c-raf Overexpression of the c-raf oncogene has been reported in a number of cancers (see above). Thus, inhibition of c-raf expression (for example using ribozymes) can reduce cell proliferation of a number of cancers, in vitro and in vivo and can reduce their proliferative potential. A cascade of MMP and serine proteinase expression is implicated in the acquisition of an invasive phenotype as well as in angiogenesis in tumors (MacDougall Matrisian, 1995, Cancer Metastasis Reviews 14, 351;Ritchlin Winchester, 1989, Springer Semin Immunopathol., 11, 219).

A number of human diseases are characterized by the inappropriate proliferation of cells at sites of injury or damage to the normal tissue architecture. These diseases include SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -119restenosis, caused by the local proliferation of medial smooth muscle cells at sites of arterial wall disruption by surgery; psoriasis, caused by proliferation of keratinocytes at regions of endothelial cell damage in the skin, and various fibrosis, caused by the inappropriate replication of cells during wound healing processes. In certain inflammatory processes, cell proliferation may not be causative, yet it exacerbates the disease pathology.

For example, in rheumatoid arthritis, synovial hyperplasia leads to accelerated cartilage damage due to secretion of proteases by the expanding population of synovial fibroblasts.

Any number of these diseases and others which involve cellular proliferation or the loss of proliferative control, such as cancer, could be treated using ribozymes which inhibit the expression of the cellular Raf gene products. Alternatively, ribozyme inhibition of the cellular growth factor receptors could be used to inhibit downstream signalling pathways.

The specific growth factors involved would depend upon the cell type indicated in the proliferative event.

Ribozymes, with their catalytic activity and increased site specificity (see above), are likely to represent a potent and safe therapeutic molecule for the treatment of cancer. In the present invention, ribozymes are shown to inhibit smooth muscle cell proliferation.

From those practiced in the art, it is clear from the examples described, that the same ribozymes may be delivered in a similar fashion to cancer cells to block their proliferation.

Diagnostic uses Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of c-rafRNA in a cell. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -120treatment of the disease progression by affording the possibility of combinational therapies multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNAs associated with crafrelated condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype c-ra]) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

Additional Uses Potential usefulness of sequence-specific nucleic acid catalysts of the instant invention might have many of the same applications for the study of RNA that DNA SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -121 restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev.

Biochem. 44:273). For example, the pattern of restriction fragments could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence.

The use of NTP's described in this invention have several research and commercial applications. These modified nucleoside triphosphates can be used for in vitro selection (evolution) of oligonucleotides with novel functions. Examples of in vitro selection protocols are incorporated herein by reference (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442).

Additionally, these modified nucleoside triphosphates can be employed to generate modified oligonucleotide combinatorial chemistry libraries. Several references for this technology exist (Brenner et al., 1992, PNAS 89, 5381-5383, Eaton, 1997, Curr. Opin.

Chem. Biol. 1, 10-16) which are all incorporated herein by reference.

Nucleic acid molecules of the instant invention might have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence. Nucleic acid molecules ribozymes) of the invention can be used, for example, to target cleavage of virtually any RNA transcript (Zaug et al., 324, Nature 429 1986 Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989). Such nucleic acids can be used as a therapeutic or to validate a therapeutic gene target and/or to determine the function of a gene in a biological system (Christoffersen, 1997, Nature Biotech. 15, 483).

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -122- Various ligands can be attached to oligonucleotides using the componds containing zylo modification for the purposes of cellular delivery, nuclease resistance, cellular trafficking and localization, chemical ligation of oligonucleotide fragments. Incorporation of one or more compounds of Formula II into a ribozyme may increase its effectiveness.

Compounds of Formula II can be used as potential antiviral agents.

Other embodiments are within the following claims.

TABLE I Characteristics of naturally occurring ribozymes Group I Introns Size: -150 to 1000 nucleotides.

Requires a U in the target sequence immediately 5' of the cleavage site.

Binds 4-6 nucleotides at the 5'-side of the cleavage site.

Reaction mechanism: attack by the 3'-OH of guanosine to generate cleavage products with 3'-OH and Additional protein cofactors required in some cases to help folding and maintainance of the active structure.

S Over 300 known members of this class. Found as an intervening sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others.

Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies Complete kinetic framework established for one ribozyme 1. Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 5-7.

2 Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17.

S Herschlag, Daniel; Cech, Thomas Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCTIUS98/09249 -123- Studies of ribozyme folding and substrate docking underway Chemical modification investigation of important residues well established The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" P3galactosidase message by the ligation of new (3-galactosidase sequences onto the defective message RNAse P RNA (M1 RNA) Size: -290 to 400 nucleotides.

RNA portion of a ubiquitous ribonucleoprotein enzyme.

the active site. Biochemistry (1990), 29(44), 10159-71.

4 Herschlag, Daniel; Cech, Thomas Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry (1990), 29(44), 10172-80.

S Knitt, Deborah Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5), 1560-70.

6 Bevilacqua, Philip Sugimoto, Naoki; Turner, Douglas A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58.

S Li, Yi; Bevilacqua, Philip Mathews, David; Turner, Douglas Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9.

8. Banerjee, Aloke Raj; Turner, Douglas The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12.

Zarrinkar, Patrick Williamson, James The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8.

Strobel, Scott Cech, Thomas Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D. (1995), 267(5198), 675-9.

1. Strobel, Scott Cech, Thomas Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11.

12. Sullenger, Bruce Cech, Thomas Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371(6498), 619-22.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -124 Cleaves tRNA precursors to form mature tRNA Reaction mechanism: possible attack by M2+-OH to generate cleavage products with 3'-OH and S RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.

S Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA [14,15] S Important phosphate and 2' OH contacts recently identified [6,17] Group II Introns Size: 1000 nucleotides.

Trans cleavage of target RNAs recently demonstrated [18,19].

Sequence requirements not fully determined.

Reaction mechanism: 2'-OH of an internal adenosine generates cleavage products with 3'- OH and a "lariat" RNA containing a and a branch point.

S Only natural ribozyme with demonstrated participation in DNA cleavage [20,21] in addition to RNA cleavage and ligation.

13. Robertson, Altman, Smith, J.D. J. Biol. Chem., 247, 5243-5251 (1972).

14. Forster, Anthony Altman, Sidney. External guide sequences for an RNA enzyme.

Science (Washington, D. 1883-) (1990), 249(4970), 783-6.

5 i. Yuan, Hwang, E. Altman, S. Targeted cleavage of mRNA by human RNase P. Proc.

Natl. Acad. Sci. USA (1992) 89, 8006-10.

16. Harris, Michael Pace, Norman Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 210-18.

17 Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2'-hydroxylbase contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U. S. A. (1995), 92(26), 12510-14.

8. Pyle, Anna Marie; Green, Justin Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716-25.

19. Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77.

Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip Lambowitz, SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 125- S Major structural features largely established through phylogenetic comparisons [22].

Important 2' OH contacts beginning to be identified 23 Kinetic framework under development [24] Neurospora VS RNA Size: -144 nucleotides.

S Trans cleavage of hairpin target RNAs recently demonstrated Sequence requirements not fully determined.

Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.

Binding sites and structural requirements not fully determined.

Only 1 known member of this class. Found in Neurospora VS RNA.

Hammerhead Ribozyme (see text for references) Size: -13 to 40 nucleotides.

Requires the target sequence UH immediately 5' of the cleavage site.

Binds a variable number nucleotides on both sides of the cleavage site.

Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.

Alan A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38.

2. Griffin, Edmund Jr.; Qin, Zhifeng; Michels, Williams Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70.

22. Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev.

Biochem. (1995), 64, 435-61.

23. Abramovitz, Dana Friedman, Richard Pyle, Anna Marie. Catalytic role of 2'-hydroxyl groups within a group II intron active site. Science (Washington, D. (1996), 271(5254), 1410-13.

24. Daniels, Danette Michels, William Jr.; Pyle, Anna Marie. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J.

Mol. Biol. (1996), 256(1), 31-49.

Guo, Hans C. Collins, Richard Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -126- 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent.

Essential structural features largely defined, including 2 crystal structures [6,27] Minimal ligation activity demonstrated (for engineering through in vitro selection) [28] Complete kinetic framework established for two or more ribozymes [29].

S Chemical modification investigation of important residues well established Hairpin Ribozyme Size: -50 nucleotides.

Requires the target sequence GUC immediately 3' of the cleavage site.

Binds 4-6 nucleotides at the 5'-side of the cleavage site and a variable number to the 3'-side of the cleavage site.

S Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.

S 3 known members of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent.

S Essential structural features largely defined [31,32,33,34] 26 Scott, Finch, Aaron,K. The crystal structure of an all RNA hammerhead ribozyme:Aproposed mechanism for RNA catalytic cleavage. Cell, (1995), 81, 991-1002.

27. McKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403.

28. Long, Uhlenbeck, Hertel, K. Ligation with hammerhead ribozymes. US Patent No.

5,633,133.

29. Hertel, Herschlag, Uhlenbeck, O. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry, (1994) 33, 3374-3385.Beigelman, et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.

0 Beigelman, et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.

3. Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. 'Hairpin' catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res.

(1990), 18(2), 299-304.

32 Chowrira, Bharat Berzal-Herranz, Alfredo; Burke, John Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -127- S Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection Complete kinetic framework established for one ribozyme [36].

Chemical modification investigation of important residues begun [37,38].

Hepatitis Delta Virus (HDV) Ribozyme Size: -60 nucleotides.

Trans cleavage of target RNAs demonstrated [39].

Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.

Only 2 known members of this class. Found in human HDV.

33 Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat Butcher, Samuel E.; Burke, John Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73.

3 Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat Butcher, Samuel E..

Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 130-8.

35 Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes Dev.

(1992), 129-34.

36. Hegg, Lisa Fedor, Martha Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28.

37 Grasby, Jane Mersmann, Karin; Singh, Mohinder; Gait, Michael Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA.

Biochemistry (1995), 34(12), 4068-76.

38. Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim; Sorensen, Ulrik Gait, Michael Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure. Nucleic Acids Res. (1996), 24(4), 573-81.

Perrotta, Anne Been, Michael Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis .delta. virus RNA sequence. Biochemistry (1992), 31(1), 16-21.

Perrotta, Anne Been, Michael A pseudoknot-like structure required for efficient selfcleavage of hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434-6.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -128- Circular form of HDV is active and shows increased nuclease stability [41] Table II: 2.5 timol RNA Synthesis Cycle Reagent Equivalents Amount Wait Time* Phosphoramidites 6.5 163 lL S-Ethyl Tetrazole 23.8 238 pL Acetic Anhydride 100 233 piL 5 sec N-Methyl Imidazole 186 233 gIL 5 sec TCA 83.2 1.73 mL 21 sec Iodine 8.0 1.18 mL 45 sec Acetonitrile NA 6.67 mL NA Wait time does not include contact time during delivery.

41. Puttaraju, Perrotta, Anne Been, Michael A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 129- TABLE 111. NUCLEOSIDES USED FOR CHEMICAL SYNTHESIS OF MODIFIED NUCLEOTIDE TRIPHOSPHATES SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 -130- SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTJUS98/09249 131 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 132- SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 133) Table V1. PHOSPHORYLATION OF URIDINE IN THE PRESENCE OF DMAP 0 equiv. DMAP 0.2 equiv. DMAP 0.5 equiv. DMIAP 1.0 equiv. DMAP Time Product Time Product Time Product Time Product (min) (min) %(min) %(min) 0 1 0 0 0 0 0 0 7 10 8 20 27 30 74 10 50 24 60 46 70 77 120 12 90 '3 3 100 57 110 84 160 14 130 39140 63 150 83 200 17 170 43 1 180 63 190 84 240 19 210 47 220 64 230 77 1 320 20 250 48 260 68 270 79 1130 48 290 49 300 64 310 77 1200 46 1140 68 1150 76 1160 72 1210 69 1220 76 1230 74 SUBSTITUTE SHEET (RULE 26) Condition TRIS-HCL 1MgCI 2 DTT Spermidine Triton tMETHANOL LiCI PEG Temp(*C) No. (mM) 1(m M) (mM) (mM) X-100 N% (mm) N% 1 40 (pHB8.0) 20 10 5 0.01 10 1 2 40(pH 8.0) 20 10 5 0.01 10 1 4 3 0(H81 2 51 0.002 4 4 0(H81 2 51 0.002 10 4 0(H81 2 51 0.002 1 4 6 0(H81 2 51 0.002 10 1 4 7 40 (pH 8.0) 20 10 5 0.01 10 1 -37 8 40 (pH 8.0) 20 10 5 0.01 10 1 4 37 9 40 (pH 8.1) 12 5 1 0.002 4 37 40 (pH 8.1) 12 5 1 0.002 10 4 37 11 40 (pH 8.1) 12 5 1 0.002 1 4 37 12 40 (pH 8. 1) 12 5 1 0.002 10 1 4 37 Table VII. Detailed Description of the NTP Incorporation Reaction Conditions Modification COND# COND# COND# COND# COND# COND# COND# COND# COND# COND# COND# COND# 1 2 3 4 5 6 7 8 9 10 11 12 2'-NH 2 -ATP 1 2 3 5 2 4 1 2 10 11 5 9 NH,-CTP 11 37 45 64 25 70 26 54 292 264 109 244

NH

2 -GTP 4 7 6 14 5 17 3 16 10 21 9 16

NH

2 -UTP 14 45 4 100 85 82 48 88 20 418 429 440 2'-dATP 9 3 19 23 9 24 6 3 84 70 28 51 2-dCTP 1 10 43 46 35 47 27 127 204 212 230 235 2'-dGTP 6 10 9 15 9 12 8 34 38 122 31 46 2'-dTTP 9 9 14 18 13 18 8 15 116 114 59 130 2'-O-Me-ATP 0 0 0 0 0 0 1 1 2 2 2 2 2'-O-Me-CTP no data compared to ribo; incorporates at low level 2'-O-Me-GTP 4 3 4 4 4 4 2 4 4 5 4 2'-O-Me-UTP 55 52 39 38 41 48 55 71 93 103 81 77 2'-O-Me-DAP 4 4 3 4 4 5 4 3 4 5 5 2'-NH 2 -DAP 0 0 1 1 1 1 1 0 0 0 0 0 ala-2'-NH 2 -UTP 2 2 2 2 3 4 14 18 15 20 13 14 phe-2'-NH,-UTP 8 12 7 7 8 8 4 10 6 6 10 6 2'-B NH 2 -ala-UTP 65 48 25 17 21 21 220 223 265 300 275 248 2'-F-ATP 227 252 98 103 100 116 288 278 471 198 317 185 2'-F-GTP 39 44 17 30 17 26 172 130 375 447 377 438 2'-C-allyl-UTP 3 2 2 3 3 2 3 3 3 2 3 3 2'-O-NH 2 -UTP 6 8 5 5 4 5 16 23 24 24 19 24 2'-O-MTM-ATP 0 1 0 0 0 0 1 0 0 0 0 0 2'-O-MTM-CTP 2 2 1 1 1 1 3 4 5 4 5 3 2'-O-MTM-GTP 6 1 1 3 1 2 0 1 1 3 1 4 Table VIII. INCORPORATION OF MODIFIED NUCLEOTIDE TRIPHOSPHATES WO 98/50530 WO 9850530PCTIUS98/09249 -136- Table IX: INCORPORATION OF MODIFIED NUCLEOTIDE TRIPHOSPHATES USING WILD TYPE BACTERIOPHAGE T7 POLYMERASE Modification [label ribo control 2'-NH.,-GTP ATP 4% 2'-dGTP ATP 3% 2'-O-Me-GTP ATP 3% 2'-F-GTP ATP 4% 2'-O-MTM-GTP ATP 3% 2' -NH.-UTP ATP 39% 2'-dTTP ATP 2'-O-Me-UTP ATP 3% ala-2'-NH-,-UTP ATP 2% phe-2'-NH 2 UTP ATP 1% 2'-p-a1a-NH 2 .UTP ATP 3% 2'-C-aIlyl-UTP ATP 2% 2'-O-NH,-UTP ATP 1% 2'-O-MTM-UTP ATP 64% 2'-NH,-ATP GTP 1% 2'-O-MTM-ATP GTP 1% 2'-NI-L-CTP GTP 59% 2' -dCTP GTP SUBST!TUE SHEET (RULE 26) WO 98/50530 WO 9850S30PCT/US98/09249 137- Table Xa: Incorporation of 2'-his-UTP and Modified CTP's modification 12'-his-U TP frUTP CTP 16.1 100 2'-amino-CTP 9.5* 232.7 2'-deoxy-CTP 9.6* 130.1 2'-OMe-CTP 1.9 6.2 2'-MTM-CTP 5.9 5.1 control Table Xb: Incorporation of 2'-his-UTP, 2-amino CTP, and Modified ATP's 12'-his-UTP and moifcaion 2'-amino-CTP JrUTP and rCTP ATP 15.7 100 2'-amino-ATP 2.4 28.9 2'-deoxy-ATP 2.3 146.3 2'-OMe-ATP 2.7 2'-F-ATP 4 222.6 2'-MTM-ATP 4.7 15.3 2'-OMe-DAP 1.9 5.7 2'-amino-DAP 8.9* 9.6 Numbers shown are a percentage of incorporation control compared to the all-RNA -Bold number indicates incorporation best observed rate of modified nucleotide triphosphate SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 138- Table XI. INCORPORATION OF 2'-his-UTP, 2'-NH 2 -CTP, 2'-NH 2

-DAP,

and rGTP USING VARIOUS REACTION CONDITIONS Conditions compared to all rNTP 7 8.7* 8 7* 9 2.3 2.7 11 1.6 12 Numbers shown are a percentage of incorporation compared to the all-RNA control Two highest levels of incorporation contained both methanol and LiC1 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 -139- Table X1I: Human C-raf Hammerhead Ribozyme and Target Sequences nt Target SEQ 1D., Ribozyme SEQ position Site No. Sequence ID. No.

17 GACCGCCUC CCGCUCCC 1 GGGAGCGG CUGAUGAG X CGAA AGGCGGUC 502 23 CUCCCGCUC CCUCACCC 2 GGGUGAGG CUGAUGAG X CGAA AGCGGGAG 503 27 CGCEJCCCUC ACCCGCCG 3 CGGCGGGU CUGAUGAG X CGAA AGGGAGCG 504 82 CAGGACGUU GGGGCGGC 4 GCCGCCCC CUGAUGAG X CGAA ACGUCCUG 505 97 GCCUGGCUC CCUCAGGU 5 ACCUGAGG CUGAUGAG X CGAA AGCCAGGC 506 101 GGCUCCCUC AGGUUUAA 6 UUAAACCU CUGAUGAG X CGAA AGGGAGCC 507 106 CCUCAGGUU UAAGAAUU 7 AAUUCUIUA CUGAUGAG X CGAA ACCUGAGG 508 107 CUCAGGUUU AAGAAUUG 8 CAAUUCUU CUGAUGAG X CGAA AACCUGAG 509 108 UCAGGUUUA AGAAUUGU 9 ACAAUUCU CUGAUGAG X CGAA AAACCUGA 510 114 tJUAAGAAUU GUUUAAGC 10 GCUTUAAAC CUGAUGAG X CGAA AUUCUUAA 511 117 AGAAUUGUTU UAAGCUGC 11 GCAGCUUA CUGAUGAG X CGAA ACAAUUCU 512 118 GAAUUGUUU AAGCUGCA 12 UGCAGCUTJ CUGAUGAG X CGAA AACAAUUC 513 119 AAUEJGUUUA AGCUJGCAU 13 AUGCAGCU CUGAUGAG X CGAA AAACAAUU 514 128 AGCUGCAUC AAUGGAGC 14 GCUCCAUU CUGAUGAG X CGAA AUGCAGCU 515 141 GAGCACAUA CAGGGAGC 15 GCUCCCUG CUGAUGAG X CGAA AUGUGCUC 516 151 AGGGAGCUU GGAAGACG 16 CGUCUUCC CUGAUGAG X CGAA AGCUCCCU 517 162 AAGACGAUC AGCAAUGG 17 CCAUUGCU CUGAUGAG X CGAA AUCGUCTJU 518 172 GCAAUGGUUL UUGGAUUC 18 GAAUCCAA CUGAUGAG X CGAA ACCAUUGC 519 173 CAAUGGUUU UGGAUU1CA 19 UGAAUCCA CUGAUGAG X CGAA AACCAUUG 520 174 AAUGGUUUEJ GGAUUCAA 20 UUGAAUCC CUGAUGAG X CGAA AAACCAUU 521 179 UUUUGGAUU CAAAGAUG 21 CAUCUUUG CUGAUGAG X CGAA AUCCAAAA 522 180 UUUGGAUUC AAAGAUGC 22 GCAUCUUU CUGAUGAG X CGAA AAUCCAAA 523 194 UGCCGUGUU UGAUGGCU 23 AGCCAUCA CUGAUGAG X CGAA ACACGGCA 524 195 GCCGUGUUU GAUGGCUC 24 GAGCCAUC CUGAUGAG X CGAA AACACGGC 525 203 UGAUGOCUC CAGCUGCA 25 UGCAGCUG CUGAUGAG X CGAA AGCCAUCA 526 213 AGCUGCAUC UCUCCUAC 26 GUAGGAGA CUGAUGAG X CGAA AUGCAGCU 527 215 CUGCAUCUC UCCUACAA 27 UUGUAGGA CUGAUGAG X CGAA AGAUGCAG 528 217 GCAUCUCUC CUACAAUA 28 UAUUGUAG CUGAUGAG X CGAA AGAGAUGC 529 220 UCUCUCCUA CAAUAGUU 29 AACUAUUG CUGAUGAG X CGAA AGGAGAGA 530 225 CCUACAAUA GUUCAGCA 30 UGCUGAAC CUGAUGAG X CGAA AUUGUAGG 531.

228 ACAAUAGUU CAGCAGUU 31 AACUGCUG CUGAUGAG X CGAA ACUAUUGU 532 229 CAAUAGUUC AGCAGUUU 32 AAACUGCU CUGAUGAG X CGAA AACUAUUG 533 236 UCAGCAGTJU UGGCUAUC 33 GAUAGCCA CUGAUGAG X CGAA ACUGCUGA 534 237 CAGCAGUUU GGCUAUCA 34 UGAUAGCC CuGAUGAG X CGAA AACUGCUG 535 242 GUUUGGCUA UCAGCGCC 35 GGCGCUGA CUGAUGAG X CGAA AGCCAAAC 536 244 UUGGCUAUC AGCGCCGG 36 CCGGCGCU CUGAUGAG X CGAA AUAGCCAA 537 257 CCGGGCAUC AGAUGAUG 37 CAUCAUCU CUGAUGAG X CGAA AUGCCCGG 538 273 GGCAAACUC ACAGAUCC 38 GGAUCUGU CUGAUGAG X CGAA AGUUUGCC 539 280 UCACAGAUC CUUCUAAG 39 CUUAGAAG CUGAUGAG X CGAA AUCUGUGA 540 283 CAGAUCCUU CUAAGACA 40 UGUCUAG CUGAUGAG X CGAA AGGAUCUG 541 284 -AGAUCCUUC UAAGACAA 41 UUGUCUA CUGAUGAG X CGAA AAGGAUCU 542 286 AUCCUUCUA AGACAAGC 42 GCUUGUCU CUGAUGAG X CGAA AGAAGGAU 543 301 GCAACACUA UCCGUGUJU 43 AACACGGA CUGAUGAG X CGAA AGUGUUGC 544 303 AACACUAUC CGUGUUUU 44 AAAACACG CUGAUGAG X CGAA AUAGUGUU 545 309 AUCCGUGUU UUCUUGCC 45 GGCAAGAA CUGAUGAG X CGAA ACACGGAU 546 310 UCCGUGUEJU UCUUGCCG 46 CGGCAAGA CUGAUGAG X CGAA AACACGGA 547 311 CCGUGUUUU CUUGCCGA 47 UCGGCAAG CUGAUGAG X CGAA AAACACGG 548 312 CGUGUUUUC UUGCCGAA 48 UEJCGGCAA CUGAUGAG X CGAA AAAACACG 549 314 UGUUUUCUU GCCGAACA 49 UGUUCGGC CUGAUGAG X CGAA AGAAAACA 550 339 ACAGUGGUC AAUGUGCG 50 CGCACAUU CUGAUGAG X CGAA ACCACUGU 551 362 AAUGAGCUU GCAUGACU 51 AGUCAUGC CUGAUGAG X CGAA AGCUCAUU 552 375 GACUGCCUU AUGAAAGC 52 GCUUUC-AU CUGAUGAG X CGAA AGGCAGUC 553 376 ACUGCCEJUA UGAAAGCA 53 uGCUUUCA CUiGAUGAG X CGAA AAGGCAGU 554 3871 AAAGCACUC AAGUGAG1 54 CUCACCUU CU-GAUGAG X CGAA AGUGCtJUU 555 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 -140nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

425 UGCAGUGUU CAGACUUC 55 GAAGUCUG CUGAUGAG X CGAA ACACUGCA 556 426 GCAGUGUUC AGACUUCU 56 AGAAGUCU CUGAtIGAG X CGAA AACACUGC 557 432 UUCAGACUU CUCCACGA 57 UCGUGGAG CUGAUGAG X CGAA AGUCUGAA 558 433 UCAGACUUC UCCACGAA 58 UUCGUGGA CuGAIJGAG X CGAA AAGUCUGA 559 435 AGACUUCUC CACGAACA 59 UGUUCGUG CUGAUGAG X CGAA AGAAGUCU 560 451 ACAAAGGUA AAAAAGCA 60 UGCUUUUU CUGAUGAG X CGAA ACCUUUGU 562.

464 AGCACGCUU AGAUUGGA 61 UCCAAuCU CUGAUGAG X CGAA AGCGUGCU 562 465 GCACGCtJUA GAUUGGAA 62 UUCCAAUC CUGAUGAG X CGAA AAGCGUGC 563 469 GCUUAGAUU GGAAUACU 63 AGUAUUCC CUGAUGAG X CGAA AUCUAAGC 564 475 AUUGGAAUA CUGAUGCU 64 AGCAUCAG CUGAUGAG X CGAA AUUCCAAU 565 488 UGCUGCGUC UUUGAUUG 65 CAAUCAAA CUGAUGAG X CGAA ACGCAGCA 566 490 CUGCGUCUU UGAUUGGA 66 UCCAAUCA CUGAUGAG X CGAA AGACGCAG 567 492. UGCGUCUUU GAUUGGAG 67 CUCCAAUC CUGAUGAG X CGAA AAGACGCA 568 495 UCUUUJGAUU GGAGAAGA 68 UCUUCUCC CUGAUGAG X CGAA AUCAAAGA 569 507 GAAGAACUU CAAGUAGA 69 UCUACUUG CUGAUGAG X CGAA AGUUCUUC 570 508 AAGAACUUC AAGUAGAU 70 AUCUACUU CUGAUGAG X CGAA AAGUUCUU 572.

513 CUUCAAGUA GAUUUCCU 72. AGGAAAUC CUGAUGAG X CGAA ACUUGAAG 572 517 AAGUAGAUU UCCUGGAU 72 AUCCAGGA CUGAUGAG X CGAA AUCUACtJTJ 573 518 AGUAGAUUU CCUGGAUC 73 GAUCCAGG CUGAUGAG X CGAA AAUCUACU 574 519 GUAGAUUUC CUGGAUCA 74- UGAUCCAG CUGAUGAG X CGAA AAAUCUAC 575 526 UCCUGGAUC AUGUUCCC 75 GGGAACAU CUGAUGAG X CGAA AUCCAGGA 576 531 GAUCAUGUU CCCCUCAC 76 GUGAGGGG CUGAUGAG X CGAA ACAUGAUC 577 532 AUCAUGUUC CCCUCACA 77 UGUGAGOG CUGAUGAG X CGAA AACAUGAU 578 537 GUUJCCCCUC ACAACACA 78 UGUGUUGU CUGAUGAG X CGAA AGGGGAAC 579 551 ACACAACUU UGCUCGGA 79 UCCGAGCA CUGAUGAG X CGAA AG-UUGUGU 580 552 CACAACUUU GCUCGGAA 80 UUCCGAGC CUGAUGAG X CGAA AAGUUGUG 581 556 ACUUUGCUC GGAAGACG 81 CGUCUUCC CUGAUGAG X CGAA AGCAAAGU 582 566 GAAGACGUU CCUGAAGC 82 GCUUCAGG CUGAUGAG X CGAA ACGUCUUC 583 567 AAGACGUUC CUGAAGCU 83 AGCUUCAG CUGAUGAG X CGAA AACGUCUU 584 576 CUGAAGCUU GCCUUCUG 84 CAGAAGGC CUGAUGAG X CGAA AGCUUCAG 585 582. GCUTUGCCtJU CUGUGACA 85 UGUCACAG CUGAUGAG X CGAA AGGCAAGC 586 582 CUUGCCUJUC UGUGACAU 86 AUGUCACA CUGAUGAG X CGAA AAGGCAAG 587 592. UGUGACAUC UGUCAGAA 87 UUCUGACA CUGAUGAG X CGAA AUGUCACA 588 595 ACAUCUGUCAGAAAUUC 88 GAAUUUCU CUGAtJGAG X CGAA ACAGAUGU 589 602 UCAGAAAUU CCUGCUCA 89 UGAGCAGG CUGAUGAG X CGAA AUUUCUGA 590 603 CAGAAAUtJC CUGCUCAA 90 UUGAGCAG CUGAUGAG X CGAA AAUUUCUG 591 609 UUCCUGCUC AAUGGAUU 91 AAUCCAUU CUGAUGAG X CGAA AGCAGGAA 592 62.7 CAAUGGAUU UCGAUGUC 92 GACAUCGA CUGAUGAG X CGAA AUCCAUUG 593 618 AAUGGAUUU CGAUGUCA 93 UGACAUCG CUGAUGAG X CGAA AAUCCAUU 594 619 AUGGAUUUC GAUGUCAG 94 CUGACAUC CUGAUGAG X CGAA AAAUCCAU 595 625 UUCGAUGUC AGACUUGU 95 ACAAGUCU CUGAUGAG X CGAA ACAUCGAA 596 631 GUCAGACUU GUGGCUAC 96 GUAGCCAC CUGAUGAG x CGAA AGUCUGAC 597 638 UUGUGGCUACAAAIJUUC 97 GAAAUUUG CUGAUGAG X CGAA AGCCACAA 598 644 CUACAAAUU UCAUGAGC 98 GCUCAUGA CUGAUGAG X CGA.A A UUUGUAG 599 645 UACAAAUUU CAUGAGCA 99 UGCUCAUG CUGAUGAG X CGAA AAtJUUGUA 600 646 ACAAAUUUC AUGAGCAC 100 GUGCUCAU CUGAUGAG x CGAA AAAUUUGU 601 658 AGCACUGUA GCACCAAA 101 UUUGGUGC CUGAUGAG X COAA ACAGUGCU 602 669 ACCAAAGUA CCUACUAU 102 AUAGUAGG CUGAUGAG X CGAA ACEJUUGGU 603 673 AAGUACCUA CUAUGUGU 103 ACACAUAG CUGAUGAG X CGAA AGGUACUU 604 676 UACCUACUA UGUGUGUG 104 CACACACA CtJGAUGAG X CGAA AGUAGGUA 605 694 ACUGGAGUA ACAUCAGA 105 UCUGAUGU CUGAUGAG X CGAA ACUCCAGU 606 699 AGUAACAUC AGACAACtJ 106 AGUUGUCU CUGAUGAG X CGAA AiUGUUACU 607 708 AGACAACUC UUAUUGUU 107 AACAAUAA CUGAUGAG X CGAA AGUUGUCU 608- 710 ACAACUCUU AUUGUUUC 108 GAAACAAU CUGAUGAG X CGAA AGAGUUGU 609 711 CAACUCUUA UUGUUUCC 109 GGAAACAA CUGAUGAG x CGAA AAGAGUUG 610 713 ACUCUUAUU GUEJUCCAAj 110 UUGGAAAC cuGAUGAG x CG AA AUAAGAGU 611 SUBSTITUJTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 141 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

716 CUUAUUGUU UCCAAAUU '111 AAUUUGGA CUGAUGAG X CGAA ACAAUAAG 612 717 UUAUTUGUUU CCAAAUUC 112 GAAUUUGG CUGAUGAG X CGAA AACAAUAA 613 718 UAUUGUUUC CAAAUUCC 113 GGAAUUUG CUGAUGAG X CGAA AAACAAUA 614 724 UUCCAAAUU CC-ACUAUU 114 AAUAGUGG CUGAUGAG X CGAA AUUUGGAA 615 725 UCCAA.AUUC C-ACUAUUG 115 CAAUAGUG CUGAUGAG X CGAA AAUEJUGGA 616 730 AUtJCCACtJA UUGGUGAU 116 AUCACCAA CUGAUGAG X CGAA AGUGGAAU 617 732 UCCACUAUEJ GGUGAUAG 117 CUAUCACC CUGAUGAG X CGAA AUAGUGGA 618 739 UUGGUGAUA GUGGAGUC 118 GACUCCAC CUGAUGAG X CGAA AUCACCAA 619 747 AGUGGAGUC CCAGCACU 119 AGUGCUGG CUGAUGAG X CGAA ACUCCACU 620 756 CCAGCACUA CCUUCUUU 120 AAAGAAGG CUGAUGAG X CGAA AGUGCUGG 621 760 CACUACCUU CUTUUGACU 121 AGUCAAAG CUGAUGAG X CGAA AGGUAGUG 622 761 ACUACCUUC UUEJGACUA 122 UAGUCAAA CUGAUGAG X CGAA AAGGUAGU 623 763 UACCUUCUU UGACUAUG 123 CAUAGUcA CUGAUGAG X CGAA AGAAGGUA 624 764 ACCUUCUUU GACUAUGC 124 GCAUAGUC CUGAUGAG X CGAA AAGAAGGU 625 769 CUUGACUA UGCGUCGU 125 ACGACGCA CUGAUGAG X CGAA AGUCAAAG 626 775 CtAUGCGUC GEJAUGCGA 126 UCGCAUAC CUGAUGAG X CGAA ACGCAUAG 627 778 UGCGUCGUA UGCGAGAG 127 cuCUCGCA CUGAUGAG X CGAA ACGACGCA 628 788 GCGAGAGUC UGUUUCCA 128 uGGAAAC-A CUGAUGAG X CGAA ACUCUCGC 629 792 GAGUCUGUU UCCAGGAU 129 AUCCUGGA CUGAUGAG X CGAA ACAGACUC 630 793 AGUCUGUUU CCAGGAUG 130 CAUCCUGG CUGAUGAG X CGAA AACAGACU 631 794 GUCUGUUUC CAGGAUGC 131 GCAUCCUG CUGAUGAG X CGAA AAACAGAC 632 807 AUGCCUGUU AGUUCUCA 132 UGAGAACU CUGAUGAG X CGAA ACAGGCAU 633 808 UGCCUGUUA GtJUCUCAG 133 CUGAGAAc CUGAUGAG X CGAA AACAGGCA 634 811 CUGUUAGUU CUCAGCAC 134 GUGCUGAG CUGAUGAG X CGAA ACUAACAG 635 812 UGUUAGUUC UCAGCACA 135 UGUGCUGA CUGAUGAG X CGAA AACUAACA 636 814 UTJAGUUCUC AGCACAGA 136 UCUGUGCU CUGAUGAG X CGAA AGAACUAA 637 824 GCACAGAUA UtJCUACAC 137 GUGUAGAA CUGAUGAG X CGAA AUCUGUGC 638 826 ACAGAUAtJU CUACACCU 138 AGGUGUAG CUGAUGAG X CGAA AUAUCUGU 639 827 CAGAUAUUC UACACCUC 139 GAGGUGUA CUGAUGAG X CGAA AAUAUCUG 640 829 GAUAUUCUA CACCUCAC 140 GUGAGGUG CUGAUGAG x CGAA AGAAUAUC 641 835 CUACACCUC ACGCCUUC 141 GAAGGCGU CUGAUGAG X CGAA AGGUGUAG 642 842 UCACGCCUU CACCUUUA 142 UAAAGGUG CUGAUGAG X CGAA AGGCGUGA 643 843 CACGCCUUC ACCUUUAMA 143 UTJAAAGGU CUGAUGAG X CGAA AAGGCGUG 644 848 CUUCACCUU UAACACCU 144 AGGUGUUA CUGAUGAG X CGAA AGGUGAAG 645 849 UtJCACCtJUU AACACCUC 145 GAGGUGUU CUGAUGAG X CGAA AAGGUGAA 646 850 UCACCLJUUA ACACCUCC 146 GGAGGUGU CUGAUGAG X CGAA AAAGGUGA 647 857 UAACACCUC CAGUCCCU 147 AGGGACUG CUGAUGAG x CGAA AGGUGUUA 648 862 CCUCCAGUC CCUCAUCU 148 AGAUGAGG CUGAUGAG X CGAA ACUGGAGG 649 866 CAGUCCCUC AUCUGAAG 149 CuEJCAGAU CUGAUGAG X CGAA AGGGACUG 650 869 UCCCUCAUC UGAAGGUU 150 AACCUUCA CUGAUGAG X CGAA AUGAGGGA 651 877 CUGAAGGUU CCCUCUCC 151 GGAGAGGG CUGAUGAG X CGAA ACCUUCAG 652 878 UGAAGGt5UC CCtJCUCCC 152 GGGAGAGG CUGAUGAG x cGAA AACCuuCA 653 882 GGUUCCCtJC UCCCAGAG 153 CUCUGGGA CUGAUGAG X CGAA AGGGAACC 654 884 UUCCCUCUC CCAGAGGC' 154 GCCUCUGG CUGAUGAG X CGAA AGAGGGAA 655 899 GCAGAGGUC GACAUCCA 155 UGGAUGUC CUGAUGAG X CGAA ACCUCUGC 656 905 GUCGACAUC CACACCUA 156 UAGGUGUG CUGAUGAG X CGAA AUGUCGAC 657 913 CCACACCUA AUGUCCAC -157 GUGGACAU CUGAUGAG X CGAA AGGUGUGG 658 918 CCUAAUGUC CACAUGGU 158 ACCAUGUG CUGAUGAG X CGAA ACAUUAGG 659 927 CACAUGGUC AGCACCAC 159 GUGGUGCU CUGAUGAG X CGAA ACCAUGUG 660 960 AGGAUGAtJU GAGGAUGC 160 GCAUCCUC CUGAUGAG X CGAA AUCAUCCU 661 972 GAUGCAAUU CGAAGUCA 161 UGACUUCG CUGAUGAG X CGAA AtJUGCAUC 662 973 AUGCAAUUC GAAGUCAC 162 GUGACUC CUGAUGAG X CGAA AAUUGCAU 663 979 UUCGAAGUC ACAGCGAA 163 UUCGCUGU CUGAUGAG X CGAA ACLUJCGAA 664 989 CAGCGAAUC AGCCUCAC 164 GUGAGGCU CUGAUGAG X CGAA AUJUCGCUG 665 995 AUCAGCCUC ACCUUCAG 165 CUGAAGGU CUGAUGAG X CGAA AGGCUGAU 666 1000 CCUCACCUU CAGCCCUG 166 CAGGGCUG CUGAUGAG X CGAA AGGUGAGG 667 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 142 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

1001 CUCACCUUC AGCCCUGU 167 ACAGGGCU CUGAUGAG X CGAA aAGGUGAG 668 1010 AGCCCUGUC CAGUAGCC 168 GGCUACUG CUGAUGAG X CGAA ACAGGGCU 669 1015 UGUCCAGUA GCCCCAAC 169 GUUGGGGC CUGAUGAG X CGAA ACUGGACA 670 1027 CCAACAAUC UGAGCCCA 170 UGGGCUCA CUGAUGAG X CGAA AUUGtJUGG 671 1046 AGGCUGGUC ACAGCCGA 171 UCGGCUGU CUGAUGAG X CGAA ACCAGCCU 672 1092 GCACCAGUA UCUGGGAC 172 GUCCCAGA CUGAUGAG X CGAA ACtJGGUGC 673 1094 ACCAGUAUC UGGGACCC 173 GGGUCCCA CUGAUGAG X CGAA AUACUGGU 674 1119 AACAAAAUU AGGCCUCG 174 CGAGGCCU CtJGAUGAG X CGAA AUUUUGUU 675 1120 ACAAAAUUA GGCCUCGU 175 ACGAGGCC CUGAUGAG X CGAA AAUUUUJGU 676 1126 UUAGGCCUC GUGGACAG 176 CUGUCCAC CUGAUGAG X CGAA AGGCCUAA 677 1141 AGAGAGAUUJ CAAGCUAU 177 AUAGCUUG CUGAUGAG X CGAA AUCUCUCU 678 1142 GAGAGAUUC AAGCUAUU 178 AAUAGCUU CUGAUGAG X CGAA AAUCUCUC 679 1148 UUCAAGCUA UUAUUGGG 179 CCCAAUAA CUGAUGAG X CGAA AGCUUGAA 680 1150 CAAGCUAUTU AUUGGGAA 180 UUCCCAAU CUGAUGAG X CGAA AUAGCUUG 681 1151 AAGCUAUUA UUGGGAAA 181 UUUCCCAA CUGAUGAG X CGAA AAUAGCUU 682 1153 GCUAUUAUU GGGAAAUA 182 UAUUUCCC CUGAUGAG X CGAA AUAAUAGC 683 1161 UGGGAAAUA GAAGCCAG 183 CUGGCUUIC CUGAUGAG X CGAA AUUUCCCA 684 1184 GAUGCUGUC CACUCGGA 184 UCCGAGUG CTJGAUGAG X CGAA ACAGGAUC 685 1189 UGUCCACUC GGAUUGGG 185 CCCAAUCC CUGAUGAG X CGAA AGUGGACA 686 1194 ACUCGGAtJU GGGUCAGG 186 CCUGACCC C!JGAUGAG X CGAA AUCCGAGU 687 1199 GAUUGGGUC AGGCUCUU 187 AAGAGCCU CUGATJGAG X CGAA ACCCAAUC 68 1205 GUCAGGCUC UUUUGGAA 188 UUCCAAAA CUGAUGAG X CGAA AGCCUGAC 689 1207 CAGGCUCUU UUGGAACU 189 AGUUCCAA CUGAUGAG X CGAA AGAGCCUG 690 1208 AGGCUCUUU UGGAACUG 190 CAGUUCCA CUGAUGAG X CGAA AAGAGCCU 691 1209 GGCUCUUTUU GGAACUGU 191 ACAGUUCC CUGAUGAG X CGAA AAAGAGCC 692 1218 GGAACUGtJU UAUAAGGG 192 CCCUUAUA CUGAUGAG X CGAA ACAGUUCC 693 1219 GAACUGUUU AUAAGGGU 193 ACCCUUAU CUGAUGAG X CGAA AACAGUUC 694 1220 AACUGUUUA UAAGGGUA 194 UACCCUUA CUGAUGAG X CGAA AAACAGUU 695 1222 CUGUUUAUA AGGGUAAA 195 UUUACCCU CUGAUGAG X CGAA AUAAACAG 696 1228 AUAAGGGUA AAUGGCAC 196 GUGCCAUU CUGAUGAG X CGAA ACCCUUAU 697 1245 GGAGAUGUU GCAGUAAA 197 tJUUACUGC CUGAUGAG X CGAA ACAUCUCC 698 1251 GUUGCAGUA AAGAUCCU 198 AGGAUCUU CUGAUGAG X CGAA ACUGCAAC 699 1257 GUAAAGAUC CUAAAGGU 199 ACCUUUAG CUGAUGAG X CGAA AUCUTUUAC 700 1260 AAGAUCCUA AAGGUUGU 200 ACAACCUU CUGAUGAG X CGAA AGGAUCUU 701 1266 CUAAAGGUJU GUCGACCC 201 GGGUCGAC CUGAUGAG X CGAA ACCUUUAG 702 1269 AAGGUUGUC GACCCAAC 202 GUTUGGGUC CUGAUGAG X CGAA ACAACCUU 703 1289 AGAGCAAUU CCAGGCCU 203 AGGCCUGG CUGAUGAG X CGAA AUUGCUCU 704 1290 GAGCAAUUC CAGGCCtJU 204 AAGGCCUG CUGAUGAG X CGAA AAUUGCUC 705 1298 CCAGGCCUU CAGGAAUG 205 CAUUCCUG CUGAUGAG X CGAA AGGCCUGG 706 1299 CAGGCCUUC AGGAAUGA 206 UCAUUCCU CUGAUGAG X CGAA AAGGCCUG 7 07 1317 GUGGCUGUU CUGCGCAA 207 UUGCGCAG CUGAUGAG X CGAA ACAGCCAC 708 1318 UGGCUGUUC UGCGCAAA 208 UUUGCGCA CUGAUGAG X CGAA AACAGCCA 709 1344 GUGAACAUU CUGCUUUU 209 AAAAGCAG CUGAUGAG X CGAA AUGUUCAC 710 1345 UGAACAUUC UGCUUUUC 210 GAAAAGCA CUGAUGAG X CGAA AAUGUUJCA 711 1350 AtJUCUGCU UEJCAUGGG 211 CCCAUGAA CUGAUGAG X CGAA AGCAGAAU 712 1351 UUCUGCUUU UCAUGGGG 212 CCCCAUGA CUGAUGAG X CGAA AAGCAGAA 713 1352 UCUGCtJUU CAUGGGGU 213 ACCCCAUG CUGAUGAG X CGAA AAAGCAGA 714 1353 CUGCUU7UUC AUGGGGUA 214 UACCCCAU CUGAUGAG X CGAA AAAAGCAG 715 1361 CAUGGGGUA CAUGACAA 215 UUGUCAUG CUGAUGAG X CGAA ACCCCAUG 716 1386 CUGGCAAUU GUGACCCA 216 UGGGUCAC CUGAUGAG X CGAA AUUGCC-AG 717 1416 AGCAGCCUC UACAAACA 217 UGUUUGUA CUGAUGAG X CGAA AGGCUGCU 718 1418 CAGCCUCUA CAAACACC 218 GGUGUTUUG CUGAUGAG X CGAA AGAGGCUG 719 1434 CUGCAUGUC CAGGAGAC 219 GUCUCCUG CUGAUGAG X CGAA ACAUGCAG 720 1448 GACCAAGUU UCAGAUGU 1220- ACAUCUGA CUGAUGAG X CGAA ACUUGGUC 1721 1449 ACCAAGUUU CAGAUGUU 221 AACAUCUG CUGAUGAG X CGAA AACUUGGU 722 1450 CCAAGUUUC AGAUGUUC 222 GAACAUCU CUGAUGAG X CGAA AAACUUGG 723 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 143 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

1457 UCAGAUGtJU CCAGCUAA 223 UUAGCUGG CUGAUGAG X CGAA ACAUCUGA 724 1458 CAGAUGUUC CAGCUAAU 224 AUtJAGCUG CUGAUGAG X CGAA AACAUCUG 725 1464 UUCCAGCUA AUUGACAU 225 AUGUCAAU CUGAUGAG X CGAA AGCUGGAA 726 1467 CAGCUAAUU GACAUtJGC 226 GCAAUGUC CUGAUGAG X CGAA AUUAGCUG 727 1473 AUUGACAUU GCCCGGCA 227 UGCCGGGC CUGAUGAG X CGAA AUGUCAAU 728 1489 AGACGGCUC AGGGAAUG 228 CAuucccU cUGAUGAG X CGAA AGCCGUCU 729 1502 AAUGGACUA UUUGCAUG 229 CAUGCAAA CUGAUGAG X CGAA AGUCCAUU 730 1504 UGGACUAUTU UGCAUGCA 230 UGCAUGCA CUGAUGAG X CGAA AUAGUCCA 731 1505 GGACUAUUU GCAUGCAA 231 UUGCAUGC CUGAUGAG X CGAA AAUAGUCC 732 1521 AAGAACAUC AUCCAUAG 232 CUAUGGAU CUGAUGAG X CGAA AUGUUCUU 733 1524 AACAUCAUC CAUAGAGA 233 UCUCUAUG CUGAUGAG X CGAA AUGAUGUU 734 1528 UCAUCCAUA GAGACAUG 234 CAUGUCUC CUGAUGAG X CGAA AUGGAUGA 735 1541 CAUGAAAUC CAACAAUA 235 UAXJUGUUG CUGAUGAG X CGAA AUUUCAUG 736 1549 CCAACAAUA UAUUUCUC 236 GAGAAAUA CUGAUGAG X CGAA AUUGUUGG 737 1551 AACAAUAUA UUUCUCCA 237 UGGAGAAA CUGAUGAG X CGAA AUAUUGUU 738 1553 CAAUAUAUU UCUCCAUG 238 CAUGGAGA CUGAUGAG X CGAA AUAUAUUG 739 1554 AAUAUAUUU CUCCAUGA 239 UCAUGGAG CUGAUGAG X CGAA AAUAUAUU 740 1555 AUAUAUTUUC UCCAUGAA 240 UUCAUGGA CUGAUGAG X CGAA AAAUAUAU 741 1557 AUAUTUUCUC CAUGAAGG 241 CCUUCAUG CUGAUGAG X CGAA AGAAAUAU 742 1568 UGAAGGCUU AACAGUGA 242 UCACUGUU CUGAUGAG X CGAA AGCCUUCA 743 1569 GAAGGCUTUA ACAGUGAA 243 UUCACUGU CUGAUGAG X CGAA AAGCCUUC 744 1581 GUGAAAAUU GGAGAUUU 244 AAAUCUCC CUGAUGAG X CGAA AUUUUCAC 745 1588 tJUGGAGAUU UUGGUUUG 245 CAAACCAA CUGAUGAG X CGAA AUCUCCAA 746 1589 UGGAGAUTUU UGGUUUGG 246 CCAAACCA CUGAUGAG X CGAA AAUCUCCA 747 1590 GGAGAUUUU GGUUUTGGC 247 GCCAAACC CUGAUGAG X CGAA AAAUCUCC 748 1594 AUULUGGUU UGGCAACA 248 tJGUUGCCA CUGAUGAG X CGAA ACCAAAAU 749 1595 UUUUJGGUUU GGCAACAG 249 CUGUUGCC CUGAUGAG X CGAA AACCAAAA 750 1605 GCAACAGUA AAGUCACG 250 CGUGACUU CUGAUGAG X CGAA ACUGUUGC 751 1610 AGUAAAGUC ACGCUGGA 251 UCCAGCGU CUGAUGAG X CGAA ACUUUACU 752 1624 GGAGUGGUU CUCAGGAG 252 CUGCUGAG CUGAUGAG X CGAA ACCACUCC 753 1625 GAGUGGUUC UCAGCAGG 253 CCUGCUGA CUGAUGAG X CGAA AACCACtJC 754 1627 GUGGUUCUC AGCAGGUU 254 AACCUGCU CUGAUGAG X CGAA AGAACCAC 755 1635 CAGCAGGUU GAACAACC 255 GGUUJGUUC CUGAUGAG X CGAA ACCUGCUG 756 1645 AACAACCUA CUGGCUCU 256 AGAGCCAG CUGAUGAG X CGAA AGGUUGUU 757 1652 UACUGGCUC UGUCCUCU 257 AGAGGACA CUGAUGAG X CGAA AGCCAGUA 758 1656 GGCUCUGUC CUCUGGAU 258 AUCCAGAG CUGAUGAG X CGAA ACAGAGCC 759 1659 UCUGUCCUC UGGAUGGC 259 GCCAUCCA CUGAUGAG X CGAA AGGACAGA 760 1680 GAGGUGAUC CGAAUGCA 260 UGCAUUCG CUGAUGAG X CGAA AUCACCUC 761 1693 UGCAGGAUA ACAACCCA 261 UGGGUUGU CUGAUGAG X CGAA AUCCUGCA 762 1703 CAACCCAUU CAGUUUCC 262 GGAAACUG CUGAUGAG X CGAA AUGGGUUG 73 1704 AACCCAUUC AGUUUCCA 263 UGGAAACU CUGAUGAG X CGAA AAUGGGUU 764 1708 CAUUCAGUU UCCAGUCG 264 CGACUGGA CUGAUGAG X CGAA ACUGAAUG 765 1709 AUUCAGUUU CCAGUCGG 265 CCGACUGG CUGAUGAG X CGAA AACUGAAU 766 1710 UUCAGUUUC CAGUCGGA 266 UCCGACUG CTJGAUGAG X CGAA AAACUGAA 767 1715 UEJUCCAGUC GGAUGUCU 267 AGACAUCC CUGAUGAG X CGAA ACUGGAAA 768 1722 UCGGAUGUC UACUCCJA 268 UAGGAGuA CUGAUGAG X CGAA ACAUCCGA 769 1724 GGAUGUCUA CUCCUAUG 269 C-AuAGGAG CUGAuGAG X cGAA AGACAUCC 770 1727 UGUCUACUC CUAUGGCA 270 UGCCATJAG CUGAUGAG X CGAA AGUAGACA 771 1730 CUACUCCUA UGGCAUCG 271 CGAUGCCA CtGAUGAG X CGAA AGGAGUAG 772 1737 UAUGGCAUC GUAUUGUA 272 uAC-AAuAc CUGAuGAG X cGAA AUGCCAUA 773 1740 GGCAUCGUA UUGUAUGA 273 UCAUACAA CUGAUGAG X CGAA ACGAUGCC 774 1742 CAUCGUAUU GUAUGAAC 274 GUUCAUAC CUGAUGAG x CGAA AUACGAUG 775 1745 CGUAUUGUA UGAACUGA 275 UCAGUUCA cUGAUGAG X CGAA ACAAUACG 776 1767 GGGGAGCUU CCUUAUUC 276 GAAUAAGG CUGAUGAG X CGAA AGCUCCCC 777 1768 GGGAGCUUC CUUAUUCU 277 AGAAUAAG CUGAUGAG x CGAA AAGCUCCC 778 1771 AGCtUtCCUU AtJUCUCAC 278 GUGAGAAU CUGAUGAG X CGAA AGGAAGCU 779 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 -144ot Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

1772 GCUUCCU)UA UUCUCACA 279 UGUGAGAA CUGAUGAG X CGAA AAGGAAGC 780 1774 UUCCUUAUU CUCACAUC 280 GAUGUGAG CtJGAUGAG X CGAA AUAAGGAA 781 1775 UCCUUAUUTC UCACAUCA 281 UGAUGUGA CUGAUGAG X CGAA AAUAAGGA 782 1777 CUUAUUCUC AC-AucAAC 282 GUEJGAUGU CUGAUGAG X CGAA AGAAUAAG 783 1782 UCUCACAUC AACAACCG 283 CGGUUGUU CUGAUGAG X CGAA AUGUGAGA 784 1795 ACCGAGAUC AGAUCAUC 284 GAUGAUCtJ CUGAUGAG X CGAA ATJCUCGGU 785 1800 GAUCAGAUC AUCUUCAU 285 AUGAAGAU CtJGAUGAG X CGAA AUCUGAUC 786 1803 CAGAUCAUC UUCAUGGU 286 ACCAUGAA CUGAUGAG X CGAA AUGAUCUG 787 1805 GAUCAUCUU CAUGGUGG 287 CCACCAUG CUGAUGAG X CGAA AGAUGAUC 788 1806 AUCAUCUUC AUGGUGGG 288 CCCACCAU CUGAUGAG X CGAA AAGAUGAU 789 1823 CCGAGGAUA UGCCUCCC 289 GGGAGGCA CUGAtJGAG X CGAA AUCCUCGG 790 1829 AUAUGCCUC CCCAGAUC 290 GAUCUGGG CUGAUGAG X CGAA AGGC:AUAU 791 1837 CCCCAGAUC UUAGUAAG 291 CUUACUAA CUGAUGAG X CGAA AUCUGGGG 792 1839 CCAGAUCUU AGUAAGCU 292 AGCUtJACU CUGAUGAG X CGAA AGAUCUGG 793 1840 CAGAUCUUA GUAAGCUA 293 UAGCUUAC CUGAUGAG X CGAA AAGAUCUG 794 1843 AUCUUAGUA AGCUAUAU 294 AUAUAGCU CUGAUGAG X CGAA ACUAAGAU 795 1848 AGUAAGCUA UAUAAGAA 295 UUCUUAUA CUGAUGAG X CGAA AGCUUACU 796 1850 UAAGCUAUA UAAGAACU 296 AGUUCtJUA CUGAUGAG X CGAA AUAGCUUA 797 1852 AGCUAUAUA AGAACUGC 297 GCAGUUCU CUGAUGAG X CGAA AUAUAGCU 798 1884 AGGCUGGUA GCUGACUG 298 CAGUCAGC CUGAUGAG X CGAA ACCAGCCU 799 1905 AAGAAAGUA AAGGAAGA 299 UCUUCCtJU CUGAUGAG X CGAA ACUUUCUU 800 1921 AGAGGCCUC UUUUUCCC 300 GGGAAAAA CUGAUGAG X CGAA AGGCCUCU 801 1923 AGGCCtJCUU UUUCCCCA 301 tGGGGAAA CtJGAUGAG X CGAA AGAGGCCU 802 1924 GGCCUCUUU UUCCCCAG 302 CUGGGGAA CUGAUGAG X CGAA AAGAGGCC 803 1925 GCCUCUUUU UCCCCAGA 303 UCUGGGGA CUGAUGAG X CGAA AAAGAGGC 804 1926 CCUCUUUUU CCCCAGAU 304 AUCUGGGG CUGAUGAG X CGAA AAAAGAGG 805 1927 CUCUUEUUC CCCAGAUC 305 GAUCUGGG CUGAUGAG X CGAA AAAAAGAG 806 1935 CCCCAGAUC CUGUCUUC 306 GAAGACAG CUGAUGAG X CGAA AUCUGGGG 807 1940 GAUCCUGUC UUCCAUUG 307 CAAUGGAA CUGAUGAG X CGAA ACAGGAUC 808 1942 UCCUGUCUU CCAUUGAG 308 CtJCAAUGG CUGAUGAG X CGAA AGACAGGA 809 1943 CCUGUCUUC CAUtJGAGC 309 GCUCAAUG CUGAUGAG X CGAA AAGACAGG 810 1947 UCUTUCCAUU GAGCUGCU 310 AGCAGCUC CUGAUGAG X CGAA AUGGAAGA 811 1956 GAGCUGCUC CAACACUC 311 GAGUGUUG CUGAUGAG X CGAA AGCAGCUC 812 1964 CCAACACUC UCUACCGA 312 UCGGUAGA CUGAUGAG X CGAA AGUGUUGG 813 1966 AACACUCUC UACCGAAG 313 CUUCGGUA CUGAUGAG X CGAA AGAGUGUU 814 1968 CACUCUCUA CCGAAGAU 314 AUCUUCGG CUGAUGAG X CGAA AGAGAGUG 815 1977 CCGAAGAUC AACCGGAG 315 CUCCGGUU CUGAUGAG X CGAA AUCUUCGG 816 1990 GGAGCGCUU CCGAGCCA 316 UGGCUCGG CUGAUGAG X CGAA AGCGCUCC 817 1991 GAGCGCUUC CGAGCCAU 317 AUGGCUCG CUGAUGAG X CGAA AAGCGCUC 818 2000 CGAGCCAUC CUUGCAUC 318 GAUGCAAG CUGAUGAG X CGAA AUGGCUCG 819 2003 GCCAUCCU GC-AUCGGG 319 CCCGAUGC CUGAUGAG X CGAA AGGAUGGC 820 2008 CCUUGCAUC GGGCAGCC 320 GGCUGCCC CUGAUGAG X CGAA AUGCAAGG 821 2029 CUGAGGAUA UCAAUGCU 321 AGCAUEJGA CUGAUGAG X CGAA AUCCUCAG 822 2031 GAGGAUAUC AAUGCUUG 322 CAAGCAUU CUGAUGAG X CGAA AUAUCCUC 823 2038 UCAAUGCJU GCACGCUG 323 CAGCGUGC CUGAUGAG X CGAA AGCAUUGA 824 2054 GACCACGUC CCCGAGGC 324 GCCUCGGG CUGAUGAG X CGAA ACGUGGUC 825 2070 CUGCCUGUC UUCUAGUU 325- AACUAGAA CUGAUGAG X CGAA ACAGGCAG 826 2072 GCCUGUCUU CUAGUUGA 326 uCAACuAG CUGAUGAG X CGAA AGACAGGC 827 2073 CCUGUCUUC UAGUTUGAC 327 GUCAACUA CUGAUGAG X CGAA AAGACAGG 828 2075 UGUCUUCUA GUTUGACUU 328 AAGUCAAC CUGAUGAG X CGAA AGAAGACA 829 2078 CUUCUAGUU GACUUUGC 329 GCAAAGUC CEJGAUGAG X CGAA ACUAGAAG 830 2083 AGUUGACUU UGCACCUG 330 CAGGUGCA CUGAUGAG X CGAA AGUCAACU 831 2084 GUUGACUUU GCACCUGU 331 ACAGGUGC CUGAUGAG X CGAA AAGUCAAC 832 2093 GCACCUGUC UUCAGGCU 332 AGCCUGAA CUGAUGAG X CGAA ACAGGUGC 833 2095 ACCUGUCUU CAGGCUGC 333 GCAGCCUG CUGAUGAG X CGAA AGACAGGU 834 2096 CCUGUCUUC AGGCUGCC 334 GGCAGCCU CUGAUGAG X CGAA AAGACAGG 835 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 145 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

2136 GCACCACUU UUCUGCUC 335 GAGC:AGAA CUGAuGAG X CGAA AGUGGUGC 836 2137 CACCACUtJU UCLJGCUCC 336 GGAGCAGA CUGAUGAG X CGAA AAGUGGUG 837 2138 ACCACUUtJU CUGCUCCC 337 GGGAGCAG CUGAuGAG X CGAA AAAGUGGU 838 2139 CCACUUUUC UGCUCCCU 338 AGGGAGCA CUGAUGAG X CGAA AAAAGUGG 839 2144 UUCUGCUC CCUUtJCUC 339 GAGAAAGG CUGAUGAG X CGAA AGCAGAAA 840 2148 UGCtJCCCUU UCUCCAGA 340 UCUGGAGA CUGAUGAG X CGAA AGGGAGCA 841 2149 GCUCCCUUU CUCCAGAG 341 CUCUGGAG CUGAUGAG X CGAA AAGGGAGC 842 2150 CUCCCUUtUC UCCAGAGG 342 CCUCUGGA CUGAUGAG X CGAA AAAGGGAG 843 2152 CCCUUUCUC CAGAGGCA 343 UGCCUCUG CUGAUGAG X CGAA AGAAAGGG 844 2171 ACACAUGUU UUCAGAGA 344 UCUCUGAA CUGAUGAG X CGAA ACAUGUGU 845 2172 CACAUGUUU UCAGAGAA 345 UUCUCUGA CUGAUGAG X CGAA AACAUGUG 846 2173 ACAUGUUUU CAGAGAAG 346 CUUCUCUG CUGAUGAG X CGAA AAACAUGU 847 2174 CAUGUUUUTC AGAGAAGC 347 GCUUCUCU CUGAUGAG X CGAA AAAACAUG 848 2184 GAGAAGCUC UGCUAAGG 348 CCUUAGCA CUGAUGAG X CGAA AGCUUCUC 849 2189 GCUCUGCUA AGGACCUU 349 AAGGUCCU CUGAUGAG X CGAA AGCAGAGC 850 2197 AAGGACCUU CUAGACUG 350 CAGUCUAG CUGAUGAG X CGAA AGGUCCUU 851 2198 AGGACCUUC UAGACUGC 351. GCAGUCUA CUGAUGAG X CGAA AAGGUCCU 852 2200 GACCUUCUA GACUGCUC 352 GAGCAGUC CUGAUGAG X CGAA AGAAGGUC 853 2208 AGACUGCUC ACAGGGCC 353 GGCCCUGU CUGAUGAG X CGAA AGCAGUCU 854 2218 CAGGGCCUU AACUEJCAU 354 AUGAAGUU CtJGAUGAG X CGAA AGGCCCUG 855 2219 AGGGCCUUA ACUUCAUG 355 CAUGAAGU CUGAUGAG X CGAA AAGGCCCU 856 2223 CCUTUAACUU CAUGUUGC 356 GCAACAUG CUGAUGAG X CGAA AGUUAAGG 857 2224 CUUAACUUC AUGUUGCC 357 GGCAACAU CUGAUGAG X CGAA AAGUUAAG 858 2229 CUUCAUGUU GCCUUCUU 358 AAGAAGGC CUGAUGAG X CGAA ACAUGAAG 859 2234 UGUUGCCUU CUUUUCUA 359 UAGAAAAG CUGAUGAG X CGAA AGGCAACA 860 2235 GUUGCCUUC UUUUCUAU '360 AUAGAAAA CUGAuGAG X CGAA AAGGCAAC 861 2237 UGCCUUCUU UUCUAUCC 361 GGAUAGAA CUGAUGAG X CGAA AGAAGGCA 862 2238 GCCUtJCUUTU UCUAUCCC 362 GGGAUAGA CUGAUGAG X CGAA AAGAAGGC 863 2239 CCUUCUUUU CUAUCCCU 363 AGGGAUAG CUGAUGAG x CGAA AAAGAAGG 864 2240 CUUCUUUULC UAUCCCUUr 364 AAGGGAUA CUGAUGAG X CGAA AAAAGAAG 865 2242 UCUUUUCUA UCCCUUUG 365 CAAAGGGA CUGAUGAG X CGAA AGAAAAGA 866 2244 UUUUCUAUC CCUUTUGGG 366 CCCAAAGG CtGAUGAG X CGAA AUAGAAAA 867 2248 CUAUCCCUU UGGGCCCU 367 AGGGCCCA CUGAUGAG X CGAA AGGGAUAG 868 2249 UAUCCCUUU GGGCCCUG 368 CAGGGCCC CUGAUGAG X CGAA AAGGGAUA 869 2273 GAAGCCAUU UGCAGUGC 369 GCACUGCA CUGAUGAG X CGAA AUGGCUUC 870 2274 AAGCCAUUU GCAGUGCU 370 AGCACUGC CUGAUGAG X CGAA AAUGGCUU 871 2290 UGGUGUGUC CUGCUCCC 371 GGGAGCAG CUGAUGAG X CGAA ACACACCA 872 2296 GUCCUGCUC CCUCCCCA 372 UGGGGAGG CUGAUGAG X CGAA AGCAGGAC 873 2300 UGCUCCCUC CCCACAUU 373 AAUGUGGG CUGAUGAG X CGAA AGGGAGCA 874 2308 CCCCACAUU CCCCAUGC 374 GCAUGGGG CUGAUGAG X CGAA AUGUGGGG 875 2309 CCCACAUUC CCCAUGCU 375 AGCAUGGG CUGAUGAG X CGAA AAUGUGGG 876 2318 CCCAUGCUC AAGGCCCA 376 UGGGCCUU CUGAUGAG X CGAA AGCAUGGG 877 2331 CCCAGCCUU CUGUAGAU 377 AUCUACAG CUGAuGAG x CGAA AGGCUGGG 878 2332 CCAGCCUUC UGUAGAUG 378 CAUCUACA CUGAUGAG x CGAA AAGGCUGG 879 2336 CCUUCUGUA GAUGCGCA 379 UGCGCAUC CUGAUGAG X CGAA ACAGAAGG 880 2354 GUGGAUGUU GAUGGUAG 380 CUACCAUC CUGAUGAG X CGAA ACAUCCAC 881 2361 UUGAUGGUA GUACAAAA 381 UUUUGUAC CTJGAUGAG X CGAA ACCAUCAA 882 2364 AUGGUAGUA CAAAAAGC 382 GCUUUUUG CUGAUGAG X CGAA ACUACCAU 883 2393 CCAGCUGUU GGCUACAU 383 AUGUAGCC CUGAUGAG X CGAA ACAGCUGG 884 2398 UGUUGGCUA CAUGAGUA 384 UACUCAUG CUGAUGAG X CGAA AGCCAACA 885 2406 ACAUGAGUA UUUTAGAGG 385 CCUCUAAA CUGAUGAG X CGAA ACUCAUGU 886 2408 AUGAGUAUU UAGAGGAA 386 UUCCUCUA CUGAUGAG X CGAA AUACUCAU 887 2409 UGAGUAUTUU AGAGGAAG 387 CUUCCUCU CUGAUGAG X CGAA AAUACUCA 888 2410 GAGUAUTUUA GAGGAAGU1 388_ ACUUCCUC CUGAUGAG X CGAA AAAUACU 8 2419 GAGGAAGUA AGGUAGCA 389 UGCUACCU CUGAUGAG X CGAA ACUUCCUC 890 2424 AGUAAGGUA GCAGGCAG 390 CUGCCUGC CUGAUGAG X CGAA ACCUACU 891 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 146 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

2434 CAGGCAGUC CAGCCCUG 391 CAGGGCUG CUGAUGAG X CGAA ACUGCCUG 892 2462 CAUGGGAtJU UUGGAAAU 392 AUUUCCAA CUGAUGAG X CGAA AUCCCAUG 893 2463 AUGGGAUUU UGGAAAUC 393 GAUUUCCA CUGAUGAG X CGAA AAUCCCAU 894 2464 UGGGAUUUU GGAAAUCA 394 UGAUUUCC CUGAUGAG X CGAA AAAUCCCA 895 2471. UtJGGAAAUC AGCUUCUG 395 CAGAAGCU CUGAUGAG X CGAA AUUtJCCAA 896 2476 AAUCAGCUU CUGGAGGA 396 UCCUCCAG CUGAUGAG X CGAA AGCUGAtJU 897 2477 AUCAGCUUC UGGAGGAA 397 UUCCUCCA CUGAUGAG X CGAA AAGCUGAU 898 2493 AUGCAUGUC ACAGGCGG 398 CCGCCUGU CUGAUGAG X CGAA ACAUGCAU 899 2506 GCGGGACUU UCUUCAGA 399 UCUGAAGA CUGAUGAG X CGAA AGUCCCGC 900 2507 CGGGACUUU CUUCAGAG 400 cucuGAAG CUGAUGAG X CGAA AAGUCCCG 901 2508 GGGACUUUC UUCAGAGA 401 UCUCUGAA CUGAUGAG X CGAA AAAGUCCC 902 2510 GACUUUCUU CAGAGAGU 402 ACUCUCUG CTJGAUGAG X CGAA AGAAAGUC 903 2511 ACUUUCUUC AGAGAGUG 403 CACUCUCU CUGAUGAG X- CGAA AAGAAAGU 904 2536 CCAGACAUU UUGCACAU 404 AUGUGCAA CUGAUGAG X CGAA AUGUCUGG 905 2537 CAGACAUUU UGCACAUA 405 UAUGUGCA CUGAUGAG X CGAA AAUGUCUG 906 2538 AGACAUUUU GCACAUAA 406 UUAUGUGC CUGAUGAG X CGAA AAAUGUCU 907 2545 UUGCACAUA AGGCACCA 407 UGGUGCCTJ CUGAUGAG X CGAA AUGUGCAA 908 2577 CCGAGACUC UGGCCGCC 408 GGCGGCCA CtJGAUGAG X CGAA AGUCtJCGG 909 2600 AGCCUGCUU UGGUACUA 409 UAGUACCA CUGAUGAG X CGAA AGCAGGCU 910 2601 GCCUGCUUU GGUACUAU 410 AUAGUACC CUGAUGAG X CGAA AAGCAGGC 911 2605 GCUUUGGUA CUAUGGAA 411 UUCCAUAG CUGAUGAG X CGAA ACCAAAGC 912 2608 UTUGGUACUA UGGAACUU 412 AAGUUCCA CUGAUGAG X CGAA AGUACCAA 913 2616 AUGGAACUU UUCUUAGG 413 CCUAAGAA CUGAUGAG X CGAA AGUUCCAU 914 2617 UGGAACUUU UCUUAGGG 414 CCCUAAGA CUGAUGAG X CGAA AAGtJUCCA 915 2618 GGAACUUUU CUtJAGGGG 415 CCCCUAAG CUGAUGAG X CGAA AAAGU.UCC 916 2619 GAACUUUUC UUAGGGGA 416 UCCCCUAA CUGAUGAG X CGAA AAAAGUUC 917 2621 ACUUUUCUU AGGGGACA 417 UGUCCCCU CUGAUGAG X CGAA AGAAAAGU 918 2622 CUUUUCUUA GGGGACAC 418 GUGUCCCC CUGAUGAG X CGAA AAGAAAAG 919 2633 GGACACGUC CUCCUUUC 419 GAAAGGAG CUGAUGAG X CGAA ACGUGUCC 920 2636 CACGUCCUC CUUUCACA 420 UGUGAAAG CUGAUGAG X CGAA AGGACGUG 921 2639 GUCCUCCUU UCACAGCU 421 AGCUGUGA CUGAUGAG X CGAA AGGAGGAC 922 2640 UCCUCCUUU CACAGCUU 422 AAGCUGUG CtJGAUGAG X CGAA AAGGAGGA 923 2641 CCUCCUUUC ACAGCUtJC 423 GAAGCUGU CUGAUGAG X CGAA AAAGGAGG 924 2648 UCACAGCUU CUAAGGUG 424 CACCUUAG CUGAUGAG X CGAA AGCUGUGA 925 2649 CACAGCUUC UAAGGUGU 425 ACACCUUA CUGAUGAG X CGAA AAGCUGUG 926 2651 CAGCUUCUA AGGUGUCC 426 GGACACCU CUGAUGAG X CGAA A GAAGCUG 927 2658 UAAGGUGUC CAGUGCAU 427 AUGCACUG CUGAUGAG X CGAA ACACCUUA 928 2667 CAGUGCAUU GGGAUGGU 428 ACCAUCCC CUGAUGAG X CGAA AUGCACUG 929 2676 GGGAUGGUU UUCCAGGC 429 GCCUGGAA CUGAUGAG X CGAA ACCAUCCC 930 2677 GGAUGGUUU UCCAGGCA 430 UGCCUG;GA CUGAUGAG x CGAA AACCAUCC 931 2678 GAUGGUUUU CCAGGCAA 431 UUGCCUGG ctJGAuGAG x CGAA AAACCAuC 932 2679 AUGGUUUUC CAGGCAAG 432 cuuGCCUG CUGAUGAG X CGAA AAAACCAU 933 2693 AAGGCACUC GGCCAAUC 433 GAUUGGCC CUGAUGAG X CGAA AGUGCCUU 934 2701 CGGCCAAUC CGCAUCUC 434 GAGAUGCG CUGAUGAG X CGAA AUTJGGCCG 935 2707 AUCCGCAUC UCAGCCCU 435 AGGGCUGA CUGAUGAG X CGAA AUGCGGAU 936 2709 CCGCAUCUC AGCCCUCU 436 AGAGGGCU CUGAUGAG X CGAA AGAUGCGG 937 2716 UCAGCCCUC UCAGGAGC 437 GCUCCUGA CUGAUGAG X CGAA AGGGCUGA 938 272.8 AGCCCUCUC AGGAGCAG 438 CUGCUCCU CUGAUGAG X CGAA AGAGGGCU 939 2728 GGAGCAGUC UUCCAUCA 439 UGAUGGAA CUGAUGAG X CGAA ACUGCUCC 940 2730 AGCAGUCJU CCAUCAUG 440 cAuGAuGG CUGAUGAG X CGAA AGACUGCU 941 2731 GCAGUCUUC CAUCAUGC 441 GCAUGAUG CUGAUGAG X CGAA AAGACUGC 942 2735 UCUCCAUC AUGCUGAA 442 UUCAGcAU CUGAUGAG X CGAA AUGGAAGA .943 2745 UGCUGAAUTU UUGUCUUC 443 GAAGACAA CUGAUGAG X CGAA AUUCAGCA 944 2746 GCUGAAUUU UGUCUUCCl 444__ GGAAGACA CUGAUGAG X CGAA AAUUCAGC 945 2747 CUGAAUUUU GUCUUCCA_. 445 UGGAAGAC CUGAUGAG X CGAA AAAUUCAG 946 2750 AAtJUUUGUC UUCCAGGA 1446 UCCUGGAA CUGAUGAG X CGAA ACAAAAUU 1947 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 147 nt Target SEQ ID. Ribozyme SEQ position Site No. Sequence ID. No.

2752 UUUUGUCUU CCAGGAGC 447 GCUCCUGG CUGAUGAG X CGAA AGACAAAA 948 2753 UtJUGUCtJUC CAGGAGCU 448 AGCUCCUG CUGAUGAG X CGAA AAGACAAA 949 2768 CTJGCCCCUA UGGGGCGG 449 CCGCCCCA CUGAUGAG X CGAA AGGGGCAG 950 2795 CAGCCUGUU UCUCUAAC 450 GUUAGAGA CUGAUGAG X CGAA ACAGGCUG 951 2796 AGCCUGUUU CUCUAACA 451 UGUUAGAG CUGAUGAG X CGAA AACAGGCU 952 2797 GCCUGUUUC UCUAACAA 452 UUGUUAGA CUGAUGAG X CGAA AAACAGGC 9 53_ 2799 CUGUTUUCUC UAACAAAC 453 GUUUGUUA CUGAUGAG X CGAA AGAAACAG 954 2801 GUUCUCUA ACAAACAA 454 UUGUUUGU CUGAUGAG X CGAA AGAGAAAC 955 2825 AACAGCCUU GUUUCUCEJ 455 AGAGAAAc CUGAUGAG X CGAA AGGCUGUU 956 2828 AGCCUTJGUU UCUCtJAGU 456 ACUAGAGA CUJGAUGAG X CGAA ACAAGGCU 957 2829 GCCUUGUUU CUCUAGUC 457 GACUAGAG cuGAUGAG X CGAA AACAAGGC 958 2830 CCUUGUTUUC UCUAGUCA 458 UGACUAGA CUGAUGAG X CGAA AAACAAGG 959 2832 UUGUUUCUC UAGUCACA 459 UGUGACUA CUGAUGAG X CGAA AGAAACAA 960 2834 GUUUCUCUA GUCACAUC 460 GAUGUGAC CUGAUGAG X CGAA AGAGAAAC 961 2837 UCUCUAGUC ACAUCAUG 461 cAUGAUGU CUGAUGAG X CGAA ACUAGAGA 962 2842 AGUCACAUC AUGUGUAU 462 AuAcACAu CUGAUGAG X CGAA AUGUGACU 963 2849 UCAUGUGUA UACAAGGA 463 UCCUUGUA CUGAUGAG X CGAA ACACAUGA 964 2851 AUGUGUAUA CAAGGAAG 464 CUUCCUTUG CUGAUGAG X CGAA AUACACAU 965 2868 CCAGGAAUA CAGGUUTUU 465 AAAACCUG CUGAUGAG X CGAA AUUCCUGG 966 2874 AUACAGGUTU UUCUUGAU 466 AUCAAGAA CUGAUGAG X CGAA ACCUGUAD 967 2875 UACAGGUUU UCUUGAUG 467 CAUCAAGA CUGAUGAG X CGAA AACCUGUA 968 2876 ACAGGUUUU CUUGAUGA 468 UCAUCAAG CUGAUGAG X CGAA AAACCUGU 969 2877 CAGGUUUUC UUGAUGAU 469 AUCAUCAA CUGAUGAG X CGAA AAAACCUG 970 2879 GGUUUUCUU GAUGAUUUJ 470 AAAUCAUC CUGAUGAG X CGAA AGAAAACC 971 2886 UUGAUGAUTU UGGGUUUU 471 AAAACCC:A CUGAUGAG X CGAA AUCAUCAA 972 2887 UGAUGALUU GGGUUUTUA 472 UAAAACCC CUGAUGAG X CGAA AAUCAUCA 973 2892 AUUUGGGUTU UUAAUUUU 473 AAAAUUAA CUGAUGAG X CGAA ACCCAAAU 974 2893 UUUGGGUUU UAAUUUUG 474 CAAAAUUA CUGAUGAG X CGAA AACCCAAA 975 2894 UUGGGUUTUU AAUUUUGU 475 ACAAAAUU CtJGAUGAG X CGAA AAACCCAA 976 2895 UGGGUUUUA AUUUUGUU 476 AACAAAAU CUGAUGAG X CGAA AAAACCCA 977 2898 GUUUUAAUU UUGUUUUU 477 AAAAACAA CUGAUGAG X CGAA AUTUAAAAC 978 2899 -UUUUAAUUU UGUUUUUA 478 UAAAAAC-A CUGAUGAG X CGAA AAUUAAAA 979 2900 UUUAAUUUU GUUUUUAU 479 AUAAAAAC CUGAUGAG X CGAA AAAUUAAA 980 2903 AAUUUUGUU UUUAUUGC 480 GCAAUAAA CUGAUGAG X CGAA ACAAAAUU 981 2904 AUUUUGLTUU UUAUtJGCA 481 UGCAAUAA CUGAUGAG x cGAA AAcAAAAU 982 2905 UUUUGUUtJU UAUUGCAC 482 GUGCAAUA CUGAUGAG X CGAA AAACAAAA 983 2906 UUUGUUUUU AUUGCACC 483 GGUGCAAU CUGAUGAG x CGAA AAAACAAA 984 2907 UUGUUUUUA UUGCACCU 484 AGGUGCAA CUGAUGAG X CGAA AAAAACAA 985 2909 GULULTJUAUU GCACCUGA 485 UCAGGUGC CUGAUGAG X CGAA AUAAAAAC 986 2924 GACAAAAUA CAGUUAUC *486 GAUAACUG CUGAUGAG X CGAA AUUUGUC 987 2929 AAUACAGUU AUCUGAUG 487 CAUCAGAU CUGAUGAG X CGAA ACUGUAUU 988 2930 AUACAGUUA UCUGAUGG 488 CCAUCAGA CUGAUGAG X CGAA AACUGUAU 989 2932 ACAGUUAUC UGAtJGGUC 489 GACCAUCA CUGAUGAG X CGAA AUAACUGU 990 2940 CUGAUGGUC CCUCAAUU 490 AAUUGAGG CUGAUGAG X CGAA ACCAUCAG 991 2944 UGGUCCCUC AAUUAUGU 491 ACAUAAUU CUGAUGAG X CGAA AGGOACCA 992 2948 CCCtJCAAUU AUGUUAUU 492 AAUAACAU CUGAUGAG X CGAA AUUGAGGG 993 2949 CCUCAAUUA UGUTUAUUU 493 AAAUAACA CUGAUGAG x CGAA AAUUGAGG 994 2953 AAUtTAUGUU AUIJUUAAU 494 AUUAAAAU CUGAUGAG X CGAA ACAUAAUU 995 2954 AUUAUGUUA UUUIUAAUA 495 UAUUAAAA CUGAUGAG X CGAA AACAUAAU 996 2956 UAUGUUAUU UUAAUAAA 496 UuAUUAA CUGAUGAG x CGAA AUAACAUA 997 2957 AUGUUALTUU UAAUAAAA 497 LUMUAUUA CUGAUGAG X CGAA AAUAACAU 998 2958 UGUUAUUUU AAUAAAAU 498 AUUUUAUU CUGAUGAG X CGAA AAAUAACA 999 2959 GUUAUUEJUA AUAAAAUA 499 UAUUUUAU CUGAUGAG X CGA.A AAAAUAAC 1000 2962 1AuuuuAAuA AAAuAAAUT 500 AuuuEAuuu cuGAUGAG x CGAA AuuAAAAu 1001 2967 1AAUAAAAUA AAUUAAAU 1501 AUUUAAUU CUGAUGAG X CGAA AUUUUAUU 1002 SUBSTITUTE SHEET (RULE 26) 148 Table XIII: Human C-raf Hairpin Ribozyme and Target Sequence nt. Ribozyme SEQ ID. Target SEQ ID.

Position Sequence No. Sequence No.

12 GCGGGA AGAA GUCA ACCAGAGAAACA X GUACAUUACCUGGUA 1003 UGACC GCC UCCCGC 1078 19 UGAGGG AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1004 CUCCC GCU CCCUCA 1079 31 CCCCGC AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1005 CACCC GCC GCGGGG 1080 61 UUCGGC AGAA GCUU ACCAGAGAAACA X GUACAUUACCUGGUA 1006 AAGCU GCC GCCGAA 1081 64 UCGUUC AGAA GCAG ACCAGAGAAACA X GUACAUUACCUGGUA 1007 CUGCC GCC GAACGA 1082 88 GAGCCA AGAA GCCC ACCAGAGAAACA X GUACAtJUACCUGGUA 1008 GGGCG GCC UGGCUC 1083 205 AGAUGC AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1009 CUCCA GCU GCAUCU 1084 233 UAGCCA AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1010 CAGCA GUU UGGCUA 1085 258 GCCAUC AGAA GAUG ACCAGAGAAACA X GUACAUUACCUGGJA 1011 CAUCA GAU GAUGGC 1086 276 AGAAGG AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1012 UCACA GAU CCUUCU 1087 370 UCAUAA AGAA GUCA ACCAGAGAAACA X GUACAUUACCUGGUA 1013 UGACU GCC UUAUGA 1088 427 GGAGAA AGAA GAAC ACCAGAGAAACA X GUACAUUACCUGGUA 1014 GUUCA GAC UUCUCC 1089 477 CGCAGC AGAA GUAU ACCAGAGAAACA X GUACAUIJACCUGGUA 1015 AUACU GAU GCUGCG 1090 605 CCAUUG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 1016 UUCCU GCU CAAUGG 1091 626 CCACAA AGAA GACA ACCAGAGAAACA X GUACAUUACCUGGUA 1017 UGUCA GAC UUGUGG 1092 655 UGGUGC AGAA GUGC ACCAGAGAAACA X GUACAIJUACCUGGUA 1018 GCACU GUA GCACCA 1093 7B9 CCUGGA AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 1019 AGUCU GUU UCCAGG 1094 859 AUGAGG AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1020 CUCCA GUC CCUCAU 1095 938 UCCACA AGAA GCGU ACCAGAGAAACA X GUACAUUACCUGGUA 1021 ACGCU GCC UGUGGA 1096 990 AGGUGA AGAA GAUU ACCAGAGAAACA X GUACAUUACCUGGUA 1022 AAUCA GCC UCACCO 1097 1002 GGACAG AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1023 CUUCA GCC CUGUCC 1098 1007 CUACUG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1024 GCCCU GUC CAGUAG 1099 1012 UGGGGC AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1025 GUCCA GUA GCCCCA 1100 1049 GUUUUC AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1026 UCACA GCC GAAAAC 1101 1089 CCCAGA AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1027 CACCA GUA UCUGGG 1102 1181 CGAGUG AGAA GCAU ACCAGAGAAACA X GUACAUUACCUGGUA 1028 AUGCU GUC CACUCG 1103 1190 GACCCA AGAA GAGU ACCAGAGAAACA X GUACAUUACCUGGUA 1029 ACUCG GAU UGGGUC 1104 1215 CUUAUA AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1030 GAACU GUU UAUAAG 1105 1314 GCGCAG AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1031 UGGCU GUU CUGCGC 1106 1346 AUGAAA AGAA GAAU ACCAGAGAAACA X GUACAUUACCUGGUA 1032 AUUCU GCU UUUCAU 1107 1411 UGUAGA AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1033 CAGCA GCC UCUACA 1108 1451 UGGAAC AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 1034 UUUCA GAU GUUCCA 1109 1481 UGAGCC AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA -1035 CGGCA GAC GGCUCA 11 cn a m m) -4

M-

m1 nt. Ribozyme SEQ ID. Target SEQ ID.

Position Sequence No. Sequence No.

1485 UCCCUG AGAA GUCU ACCAGAGAAACA X GLACAUUACCUGGUA 1036 AGACG GCU CAGGGA 1111 1653 CCAGAG AGAA GAGC ACCAGAGAAACA X GUACAUUACCUGGUA 1037 GCUCU GUC CUCUGG 1112 1705 ACUGGA AGAA GAAU ACCAGAGAAACA X GUACAUUACCUGGUA 1038 AUUCA GUU UCCAGU 1113 1712 ACAUCC AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 1039 UUCCA GUC GGAUGU 1114 1716 GUAGAC AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 1040 AGUCG GAU GUCUAC 1115 1751 CCCGUC AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1041 GAACU GAU GACGGG 1116 1796 AAGAUG AGAA GAUC ACCAGAGAAACA X GUACAUUACCUGGUA 1042 GAUCA GAU CAUCUU 1117 1833 ACUAAG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1043 CCCCA GAU CUUAGU 1118 1858 CUUUGG AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1044 GAACU GCC CCAAAG 1119 1887 CACACA AGAA GCUA ACCAGAGAAACA X GUACAUUACCUGGUA 1045 UAGCU GAC UGUGUG 1120 1931 GACAGG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1046 CCCCA GAU CCUGUC 1121 1937 AUGGAA AGAA GGAU ACCAGAGAAACA X GUACAUUACCUGGUA 1047 AUCCU GUC UUCCAU 1122 1952 UGUUGG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1048 GAGCU GCU CCAACA 1123 2013 AGUGUG AGAA GCCC ACCAGAGAAACA X GUACAUUACCUGGUA 1049 GGGCA GCC CACACU 1124 2045 GACGUG AGAA GCGU ACCAGAGAAACA X GUACAUUACCUGGUA 1050 ACGCU GAC CACGUC 1125 2063 AAGACA AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1051 AGGCU GCC UGUCUU 1126 2067 CUAGAA AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1052 UGCCU GUC UUCUAG 1127 2090 CCUGAA AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1053 CACCU GUC UUCAGG 1128 2140 AAAGGG AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 1054 UUUCU GCU CCCUEJU 1129 2204 CCUGUG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 1055 AGACU GCU CACAGG 1130_ 2292 GGAGGG AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1056 GLYCCU GCU CCCUCC 1131 2326 ACAGAA AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1057 GCCCA GCC UUCUGU 1132 2333 CGCAUC AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1058 CUUCU GUA GAUGCG 1133 2381 AGCUGG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1059 GCCCA GCC CCAGCU 1134 2387 GCCAAC AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1060 CCCCA GCU GUUGGC 1135 2390 GUAGCC AGAA OCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1061 CAGCU GUU GGCUAC 1136 2431 GGGCUG AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1062 AGGCA GUC CAGCCC 1137 2436 CAUCAG AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1063 GUCCA GCC CUGAUG 1138 2441 CUCCAC AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1064 GCCCU GAU GUGGAG 1139 2472 UCCAGA AGAA GAUU ACCAGAGAAACA X GUACAUUACCUGGUA 1065 AAUCA GCU UCUGGA 1140 2557 GUCCUG AGAA GUUU ACCAGAGAAACA X GUACAUUACCUGGUA 1066 AAACA CCC CAGGAC 1141 2567 AGUCUC AGAA GUCC ACCAGAGAAACA X GUACAUUACCUGGUA 1067 GGACU GCC GAGACU 1142 2582 CCUUCG AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1068 UGGCC GCC CGAAGG 1143 2596 UACCAA AGAA GGCU ACCAGAGAAACA X GUACAUUACCUGGUA 1069 AGCCU GCU UUGGUA 1144 2644 CUUAGA AGAA GUGA ACCAGAGAAACA X GUACAUUACCUCGUA 1070 UCACA CCU UCUAAG 1145 2710 UGAGAG AGAA GAGA ACCAGAGAAACA X GUACAUUACCUGGUA 1071 UCUCA GCC CUCUCA 1146 2725 1AUGGAA AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1072 GAGCA GUC UUCCAU 1147 2761 CAUAGG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1 1073 1GAGCU GCC CCUAUG 1148 2788 GAAACA AGAA GGCC ACCAGAGAAACA X GUACAUUACCUGGUA 1 1074 1GGCCA GCC UGUUUC 1149 I2792 IUAGAGA AAA GGCU ACCAGAGAAACA X GUACAIJUACCUGGUA I 1075 IAGCCU GUll UCUCUA I1150'I 2820 AAACAA AGAA GUUU ACCAGAGAAACA X GUACAUIJACCUGGUA 1076 AAACA GCC UTJGUUU 1151 2933 GGGACC AGAA GAUA ACCAGAGAAACA X GUACAUUACCUGGUA 1 1077 _UAUCU GAU GGUCCC 1 152 Where represents stem IV region of a Hairpin ribozyme. The length of stem IV may be 2 base-pairs.

WO 98/50530 WO 9850530PCTIUS98/09249 151 Table XIV. Hammerhead Ribozyme Sites for A-Raf SEQ ID. SEQ Pos RZ No. Substrate ID. No.

UCCACCCU CUGAUGAG X CGAA AUtJGGGUC 1153 GACCCAAUA AGGGUGGA 1461 28 CUCUGCGG CUGAUGAG X CGAA ACUCAGCC 1154 GGCUGAGUC CCGCAGAG 1462 42 ACUCUCGU CUGAUGAG X CGAA AUtJGGCUC 1155 GAGCCAAUA ACGAGAGU 1463 51 IGCCUCUCG CUGAUGAG X CGAA ACUCUCGU 1156 ACGAGAGUC CGAGAGGC 1464 74 UCCUCACA CUGAUGAG X CGAA AGUCCGCC 1157 GGCGGACUC UGUGAGGA 1465 123 CACGCCGC CUGAUGAG X CGAA ACAGCCGC 1158 GCGGCUGUA GCGGCGUG 1466 166 GAUGGGCU CUGAUGAG X CGAA AGGUGGGG 1159 CCCCACCUC AGCCCAUC 1467 174 UtJUGUCAA CUGAUGAG X CGAA AUGGGCUG 1160 CAGCCCAUC UUTGACAAA 1468 176 AUUTUUGUC CUGAUGAG X CGAA AGAUGGGC 1161 GCCCAUCUU GACAAAAU 1469 185 GAGCCUUA CUGAUGAG X CGAA AUUUUGUC 1162 GACAAAAUC UAAGGCUC 1470 187 UGGAGCCU CUGAUGAG X CGAA AGAUUTJUG 1163 CAAAAUCUA AGGCUCCA 1471 193 GCUCCAUG CUGAUGAG X CGAA AGCCUUAG 1164 CUAAGGCUC CAUGGAGC 1472 238 CUGCCCGG CUGAUGAG X CGAA AUGGCUCG 1165 CGAGCCAUC CCGGGCAG 1473 2 57 UAUACU=t CUGAUGAG X CGAA ACGGUGCC 1166 GGCACCGUC AAAGUAUA 1474 263 GGCAGGUA CUGAUGAG X CGAA ACUUEJGAC 1167 GUCAAAGUA UACCUGCC 1475 2 65 UGGGCAGG CUGAUGAG X CGAA AUACUUJG 1168 CAAAGUAUA CCUGCCCA 1476 299 CCAUCCCG CUGAUGAG X CGAA ACAGUCAC 1169 GUGACUGUC CGGGAUGG 1477 317 GAGUCGUA CUGAUGAG X CGAA ACACUCAU 1170 AUGAGUGUC UACGACUC 1478 319 GAGAGUCG CUGAUGAG X CGAA AGACACUC 1171 GAGUGUCUA CGACUCUC 1479 325 UGUCUAGA CUGAUGAG X CGAA AGUCGUAG 1172 CUACGACUC UCUAGACA 1480 327 CUUGUCUA CUGAUGAG X CGAA AGAGUCGU 1173 ACGACUCUC UAGACAAG 1481 329 GCCUUGUC CUGAUGAG X CGAA AGAGAGUC 1174 GACUCUCUA GACAAGGC 1482 354 CUGAUUUA CUGAUGAG X CGAA ACCCCGCA 1175 UGCGGGGUC UAAAUCAG 1483 356 UCCUGAUU CUGAUGAG X CGAA AGACCCCG 1176 CGGGGUCUA AAUCAGGA 1484 360 GCAGUCCU CUGAUGAG X CGAA. AUUEJAGAC 1177 GUCUAAAUC AGGACUGC 1485 377 AGUCGGUA CUGAUGAG X CGAA ACCACACA 1178 UGUGUGGUC UACCGACU 1486 3 79 1UGAGUCGG CUGAUGAG X CGAA AGACCACA 1179 UGtJGGUCUA CCGACUCA 1487 386 CCCUUGAU CUGAUGAG X CGAA AGUCGGUA 1180 UACCGACUC AUCAAGGG 1488 389 CGUCCCUU CUGAUGAG X CGAA AUGAGUCG 1181 CGACUCAUC AAGGGACG 1489 407 CAGGCAGU CUGAUGAG X CGAA ACCGUCUU 1182 AAGACGGUC ACUGCCUG 1490 428 AGGGGAGC CUGAUGAG X CGAA AUGGCUGU 1183 ACAGCCAUU GCUCCCCU 1491 432 ,AUCCAGGG CUGAUGAG X CGAA AGCAAUGG 1184 CCAUUGCUC CCCUGGAU 1492 452 UCGACAAU CUGAUGAG X CGAA AGCUCCUC 1185 GAGGACC AUEJGUCGA 1493 455 ACCUCGAC CUGAUGAG x CGAA AuGAGCUC 1186 GAGCUCAUU GUCGAGGU 1494 458 AGGACCUC CUGAUGAG X CGAA ACAAUGAG 1187 CUCAUUGUC GAGGUCCU 1495 464 UCUtJCAAG CUGAUGAG x CGAAi ACCUCGAC 1188 GUCGAGGUC CUUGAAGA 1496 467, ACAUCUJC CUGAUGAG X CGAA AGGACCUC 1189 GAGGUCCUU GAAGAUGU 1497 476 GUCAGCGG CUGAUGAG X CGAA ACAUCUUC 1190 GAAGAUGUC CCGCUGAC 1498 495 CCGUACAA CUGAUGAG X CGAA AUtJGUGCA 1191 UGCACAAUU UUGUACGG 1499 496 UCCGUACA CUGAUGAG X CGAA AAUUGUGC 1192 GCACAAUJU UGUACGGA 1500 497 UUCCGUAC CUGAUGAG X CGAA AAAUUGUG 1193 CACAAEJEJUJ GUACGGAA 11501 00 GUCUUCCG CUGAUGAG X CGAA ACAAAAJU 1194 AAXUtGUA CGGAAGAC 1502 511 GGCUGAAG CUGAUGAG X CGAA AGGUCUJC 1195 GAAGACCUU CUUCAGCC 1T503 512 AGGCUGAA CUGAUGAG X CGAA AAGGUCUU 1196 AAGACCUUC UUCAGCCU 1504 514 CCAGGCUG CUGAUGAG X CGAA AGAAGGUC 1197 GACCUtJCUU CAGCCUGG 1505 515 GCCAGGCU CUGAUGAG X CGAA AAGAAGGU 1198 ACCUUCUC AGCCUGGC_ 1506 526 AGUCACAG CUGAUGAG X CGAA ACGCCAGG 1199 CCUGGCGUUE CUGUGACU 1507 527 AAGUCACA CUGAUGAG X CGAA AACGCCA-G 1200 iCUGGCGUJC UGUGACUU 1508 35 1UTAAGGCAG CUGAUGAG X CGAA AGUCACAG. 12011 CUGUGACUU CUGCCUUA 1509 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 152- SEQ ID. SEQ Pos RZ No. Substrate ID. No.

536 UUAAGGCA CUGAUGAG X CGAA AAGUCACA 1202 UGUGACUUC UGCCUUAA 1510 542 AGAAACUU CUGAUGAG X CGAA AGGCAGAA 1203 UUtCUGCCUJ AAGUTUUCU 1511 543 CAGAAACU CUGAUGAG X CGAA AAGGCAGA 1204 UCUGCCUJA AGUUEJCUG 1512 47 GGAACAGA CUGAUGAG X CGAA ACtJUAAGG 1205 CCUUAAGUJ UCUGUUCC 1513 48 UGGAACAG CUGAUGAG X CGAA AACUIJAAG 1206 CUUAAGUUJ CUGUEICCA 1514 549 AUGGAACA CUGAUGAG X CGAA AA.ACUtJAA 1207 UUAAGUUUC UGUUCCAU 1515 553 AGCCAUGG CUGAUGAG X CGAA ACAGAA.AC 1208 GUTUUCUGUU CCAUGGCU 1516 554 AAGCCAUG CUGAUGAG X CGAA AACAGAAA 1209 UUEJCUGUJC CAUGGCUU 15171 562 GGCAACGG CUGAUGAG X CGAA AGCCAUGG 1210 CCAUGGCU-U CCGUUGCC 1518 563 UGGCAACG CUGAUGAG X CGAA AAGCCAUG 1211 CAUGGCUUC CGUtJGCCA 1519 567 GGUUrUGGC CUGAUGAG X CGAA ACGGAAGC 1212 GCUUCCGUJ GCCAAACC 1520 583 GGAACUUG CUGAUGAG X CGAA AGCCACAG 1213 CUGUGGCUA CAAGUUCC 1521 589 GCUGGUGG CUGAUGAG X CGAA ACUUGUAG 1214 CUACAAGUU CCACCAGC 1522 590 UGCUGGUG CUGAUGAG -X CGAA AACUUGUA 1215 UACAAGUUC CACCAGCA 1523 600 GGAGGAAC CUGAUGAG X CGAA AUGCUGGU 1216 ACCAGCAUU GTJUCCUCC 1524 603 CUUGGAGG CUGAUGAG X CGAA ACAAUGCU 1217 AGCAUJGUU CCUCCAAG 1525 604 CCUUGGAG CUGAUGAG X CGAA AACAAUGC 1218 GCAUUGUUC CUCCAAGG 11526 607 GGACCUJG CUGAUGAG X CGAA AGGAACAA 1219 UUGUUCCUC CAAGGUCC 1527 614 ACUGUGGG CUGAUGAG X CGAA ACCUUGGA 1220 UCCAAGGUC CCCACAGU 1528 623 UCAACACA CUGAUGAG X CGAA ACUGUGGG 1221 CCCACAGUC UGUGUA 152 629 CUCAUGUC CUGAUGAG X CGAA ACACAGAC 1222 GUCUGUGUU GACAUGAG 1530 639 GCGGUUGG CUGAUGAG X CGAA ACUCAUGU 1223 ACAUGAGUA CCAACCGC 15311 655 UGUGGUAG CUGAUGAG X CGAA ACUGUJGG 1224 CCAACAGUU CUACCACA 1532 656 CUGUGGUA CUGAUGAG X CGAA AACUGUUG 1225 CAACAGUUC UACCACAG 1533 658 CACUGUGG CUGAUGAG X CGAA AGAACUGU 1226 ACAGUtJCUA CCACAGUG 1534 668 AAAUCCUG CUGAUGAG X CGAA ACACUGUG 1227 CACAGUGUC CAGGAUJU 1535 675 UCCGGACA CUGAUGAG X CGAA AUCCUGGA 1228 UCCAGGAUU UGUCCGGAI 1536 676 CUCCGGAC CUGAUGAG X CGAA AAUCCUGG 1229 CCAGGAUUJ GUCCGGAG 1537 679 AGCCUCCG CUGAUGAG X CGAA ACAAAUCC 1230 GGAUUGUC CGGAGGCU 1538 688 GCUGUCUG CUGAUGAG X CGAA AGCCUCCG 1231 CGGAGGCUC CAGACAGC 1539 705 GUUCGAGG CUGAUGAG X CGAA AGCCUCAU 1232 AUGAGGCUC CCUCGAAC 15401 709 GGCGGUUC CUGAUGAG X CGAA AGGGAGCC 1233 GGCUCCCUC GAACCGCC 1541 730 GGGUUAGC CUGAUGAG X CGAA ACUCAUUC 1234 GAAUGAGUU GCUAACCC 1542 734 UGGGGGGU CUGAUGAG X CGAA AGCAACUC 1235 GAGUEJGCUA ACCCCCCA 1543 747 GGGGCUGG CUGAUGAG X CGAA ACCCUGGG 1236 CCCAGGGUC CCAGCCCC 1544 7 84 GGAAGGGG CUGAUGAG X CGAA AGUGCUCC 1237 GGAGCACUU CCCCUtJCC 15451 785 GGGAAGGG CUGAUGAG X CGAA AAGUGCUC 1238 GAGCACUUC CCCUUCCC 1546 790 GGGCAGGG CUGAUGAG X CGAA AGGGGAAG 1239 CUUCCCCUT CCCUGCCC 1547 791. GGGGCAGG CUGAUGAG X CGAA AAGGGGAA 1240 UTJCCCCUULC CCUGCCCC 1548 815 AUGCGCUG CUGAUGAG X CGAA AGGGGGGC 1241 GCCCCCCUA CAGCGCAU 1549 824 GUGGAGCG CUGAUGAG X CGAA AUGCGCUG 1242 CAGCGCAUC CGCUCCAC 15501 829 UGGACGUG CUGAUGAG X CGAA AGCGGAUG 1243 CAUCCGCUC CACGUCCA 1551 835 UGGGAGUG CUGAUGAG X CGAA ACGUGGAG 1244 CUCCACGUC CACUCCCA 1552 8 40 1GACGUUGG CUGAUGAG X CGAA AGUGGACG 1245 CGUCCACUC CCAACGUC 1553 8 48 AC CAUAUG CUGAUGAG X CGAA ACGUUGGG 1246 CCCAACGUC CAUAUGGU 1554 852 GCUGACCA CUGAUGAG X CGAA AUGGACGU 1247 ACGUCCAUA UGGUCAGC 1555 857 GUGGUGCU CUGAUGAG X CGAA ACCAUAUG 1248 CAUAUGGUC AGCACCAC 1556 880 uGAGGUUG CUGAUGAG x CGAA AGUCCAuG 1249 CAUGGACUC CAACCUCA 1557 887 AGCUGGAU CUGAUGAG X CGAA AGGUJUGGA 1250 UCCAACCUC AUCCAGCU 1558 890 GUGAGCUG CUGAUGAG X CGAA AUGAGGTJU 1251 AACCUCAUC CAGCUCAC 1559 896 UGGCCAGU CUGAUGAG X CGAA AGCUGGAU 1252 AUCCAGCUC ACUGGCCA 1560 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 153 SEQ ID.

SEQ

Pos RZ No. Substrate ID. No.

909 AGUGCUGA CUGAUGAG X CGAA ACUCUGGC 1253 GCCAGAGUU UCAGCACU 1561 910 CAGUGCUG CUGAUGAG X CGAA AACUCUGG 1254 CCAGAGUUU CAGCACUG 1562 911 UCAGUGCU CUGAUGAG X CGAA AAACUCUJG 1255 CAGAGUUJC AGCACUGA 1563 9 30 UCCUCUAC CUGAUGAG X CGAA ACCGGCAG 1256 CUGCCGGUA GUAGAGGA 1564 9 33 ACCUCCUC CUGAUGAG X CGAA ACUACCGG 1257 -CCGGUAGUA GAGGAGGU 1565 942 UCCAUCAC CUGAUGAG X CGAA ACCUCCUC 1258 GAGGAGGUA GUGAUGGA 1566 985 UCCCCGAG CUGAUGAG X CGAA. ACACGCUG 1259 CAGCGUGUC CUCGGGGA :1567 988 UCCUCCCC CUGAUGAG X CGAA AGGACACG 1260 CGUGUCCUC GGGGAGGA 1568 1000 AAUGUGGG CUGAUGAG X CGAA ACUUCCUC 1261 GAGGAAGUC CCCACAUU 1569 1008JUGACUUGG CUGAUGAG X CGAA AUGUGGGG 1262 CCCCACAJU CCAAGUCA 1570 1009 GUGACUUG CUGAUGAG X CGAA AAUGUGGG 1263 CCCACAUUC CAAGUCAC 1571 1015 CUGCUGGU CUGAUGAG X CGAA ACUUGGAA 1264 UUCCAAGUC ACCAGCAG 1572 1042 CGGCCAAG CUGAUGAG X CGAA ACUUCCGC 1265 GCGGAAGUC CUUGGCCG 1573 1045 CAUCGGCC CUGAUGAG X CGAA AGGACUJC 1266 GAAGUCCUU GGCCGAUG 1574 1081, AITJCCCGG CUGAUGAG X CGAA ACCCCAGG 1267 CCUGGGGUA CCGGGANU 1575 10901AAUAGCCU CUGAUGAG X CGAA ANUCCCGG 1268 CCGGGANUC AGGCUAUU 1576 1096 CCCAGUAA CUGAUGAG X CGAA AGCCUGAN 1269 NUCAGGCUA UUACUGGG 1577 1098 CUCCCAGU CUGAUGAG X CGAA AUAGCCUG 1270 CAGGCUAUU ACUGGGAG 1578 1099 CCUCCCAG CUGAUGAG X CGAA AAUAGCCU 1271 AGGCUATUA CUGGGAGG 1579 1109 CUGGGUGG CUGAUGAG X CGAA ACCUCCCA 1272 UGGGAGGUA CCACCCAG 1580 11421CCCGUCCC CUGAUGAG X CGAA AUCCUCUU 1273 AAGAGGAUC GGGACGGG 1581 1153 UGCCAAAC CUGAUGAG X CGAA AGCCCGUC 1274 GACGGGCUC GUUUGGCA 1582 1156 CGGUGCCA CUGAUGAG X CGAA ACGAGCCC 1275 GGGCUCGUU UGGCACCG 1583 1157 ACGGUGCC CUGAUGAG X CGAA AACGAGCC 1276 GGCUCGUUU GGCACCGU 1584, 1168 GCCCUCGA CUGAUGAG X CGAA ACACGGUG 1277 CACCGUGUU UCGAGGGC 1.585 1169,CGCCCUCG CUGAUGAG X CGAA AACACGGU 1278 ACCGUGUUU CGAGGGCG 1586 11701CCGCCCUC CUGAUGAG X CGAA AAACACGG 1279 CCGUGUUUC GAGGGCGG 1587 12081GACACCUU CUGAUGAG X CGAA AGCACCUJ 1280 AAGGUGCUC AAGGUGUC 1588 1216JUGGGCUGG CUGAUGAG X CGAA ACACCUJG 1281 CAAGGUGUC CCAGCCCA .1589 1245 AUUCUtJGA CUGAUGAG X CGAA AGCCUGGG 1282 CCCAGGCUU UCAAGAAU 1590 1246 CAUtJCUUG CUGAUGAG X CGAA AAGCCUGG 1283 CCAGGCUUU CAAGAAUG 1591 1247 UCAUJCUJ CUGAUGAG X CGAA AAAGCCUG 1284 CAGGCUUJC AAGAAUGA 1592 1268 GUCUUCCU CUGAUGAG X CGAA AGCACCUG 1285 CAGGUGCUC AGGAAGAC 1593.

12861AAGAUGUU CUGAUGAG X CGAA ACAUGUCG 1286 CGACAUGUC AACAUCUU 15941 1292 AACAGCAA CUGAUGAG X CGAA AUGUUGAC 1287 GUCAACAUC UJGCUGUU 1595 1294 UAAACAGC CUGAUGAG X CGAA AGAUGUUG 1288 CAACAUCUU GCUGUEJUA 1596 1300 AGCCCAUA CUGAUGAG X CGAA ACAGCAAG 1289 CUUGCUGUJ UAUGGGCU 1597 1301 AAGCCCAU CUGAUGAG X CGAA AACAGCAA 1290 UUGCUGUUU AUGGGCUU 1598 13021GAAGCCCA CUGAUGAG X CGAA AAACAGCA 1291 UGCUGtUUA UGGCUUC 1599 1309 GGGUCAUG CUGAUGAG X CGAA AGCCCAUA 1292 UAUGGGCUU CAUGACCC 1600 1310 CGGGTJCAU CUGAUGAG X CGAA AAGCCCAU 1293 AUGGGCUUC AUGACCCG 1601 1327 UGAUGGCA CUGAUGAG X CGAA AUCCCGGC 1294 GCCGGGAUU UGCCAUCA 1602 1328 AUGAUGGC CUGAUGAG X CGAA AAUCCCGG 1295 CCGGGAUUU GCCAUCAU 1603, 13341UGUGUGAU CUGAUGAG X CGAA AUGGCAAA 1296 LUJUGCCAUC AUCACACA 1604 13371CACUGUGU CUGAUGAG X CGAA AUGAUGGC 1297 GCCAUCAUC ACACAGUG 1605 13571AGAGGCUG CUGAUGAG X CGAA AGCCCUCA 1298 UGAGGGCUC CAGCCUCU 1606 1364JUGAUGGUA CUGAUGAG X CGAA AGGCUGGA 1299 UCCAGCCUC UACCAUCA 1607 13661GGUGAUGG CUGAUGAG X CGAA AGAGGCUG, 1300 CAGCCUCUA CCAUCACC 1608, 1371 AUGCAGGU CUGAUGAG X CGAA AUGGUAGA 1301 _UCUACCAUC ACCUGCAU 1609 1396 CCAUGUCG CUGAUGAG X CGAA AGCGUGUG 1302 CACACGCUU CGACAUGG 1610 1397 ACCAUGUC CUGAUGAG X CGAA AAGCGUGU 1303 1ACACGCUUC GACAUGGU 1611 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 154- SEQ ID. SEQ Pos RZ No. Substrate ID. No.

1406 AUGAGCUG CUGAUGAG X CGAA ACCAUGUC 1304 GACAUGGUC CAGCUCAU 1612 1412 ACGUCGAU CUGAUGAG X CGAA AGCUGGAC 1305 GUCCAGCUC AUCGACGU 1613 1415 GCCACGUC CUGAUGAG X CGAA AUGAGCUG 1306 CAGCUCAUC GACGUGGC 1614 14501CAUGGAGG CUGAUGAG X CGAPA AGUCCAUG 1307 CAUGGACUA CCUCCAUJG 1615 1454 UEJGGCAUG CUGAUGAG X CGAA AGGUAGUC 1308 GACUACCUC CAUGCCAA 1616 1469 CGGUGGAU CUGAUGAG X CGAA AUGUUCUU 1309 AAGAACAUC AUCCACCG 1617 1472 UCUCGGUG CUGAUGAG X CGAA AUGAUGUJ 1310 AACAUCAUC CACCGAGA 1618 1482 AGACUUGA CUGAUGAG X CGAA AUCUCGGU 1311 ACCGAGAUC UCAAGUCU 1619 14841UUAGACUU CUGAUGAG X CGAA AGAUCUCG 1312 CGAGAUCUC AAGUCUAA 1620 1489 UGUUGUEJA CUGAUGAG X CGAA ACUUGAGA 1313 UCUCAAGUC UAACAACA 1621 1491 GAUGUUGU CUGAUGAG X CGAA AGACUGA 1314 UCAAGUCUA ACAACAUC 1622 1499 UGUAGGAA CUGAUGAG X CGAA AUGTJUGUU 1315 AACAACAUC UEJCCUACA 1623 1501 CAUGUAGG CUGAUGAG X CGAA AGAUGUUG 1316 CAACAUCUU CCUACAUG 1624 1502 UCAUGUAG CUGAUGAG X CGAA AAGAUGUU 1317 AACAUCUUC CUACAUGA 1625 1505 CCCTJCAUG CUGAUGAG X CGAA AGGAAGAU 1318 AUCUCCUA CAUGAGGG 1626 1517 UUCACCGU CUGAUGAG X CGAA AGCCCCUC 1319 GAGGGGCUC ACGGUGAA 1627" 1529 AAGUCACC CUGAUGAG X CGAA AUCUtJCAC 1320 GUGAAGAUC GGUGACUJ 1628 1537 CCAAGCCA CUGAUGAG X CGAA AGUCACCG 1321 CGGUGACIJU UGGCUUGG 1629 1538 GCCAAGCC CUGAUGAG X CGAA AAGUCACC 1322 GGUGACUJU GGCUEJGGC 1630 1543 CUGUGGCC CUGAUGAG X CGAA AGCCAAAG 1323 CUUEJGGCUU GGCCACAG 1631 1560 GCUCCAUC CUGAUGAG X CGAA AGUCUEJCA 1324 UGAAGACUC GAUGGAGC 1632 1582 GCUGCUCC CUGAUGAG X CGAA AGGGCUGG 1325 CCAGCCCUU GGAGCAGC 1633 1594 CAGAUCCU CUGAUGAG X CGAA AGGGCUGC 1326 GCAGCCCUC AGGAUCUG 1634 1600,ACAGCACA CUGAUGAG X CGAA AtJCCUGAG 1327 CUCAGGAUC UGUGCUGU 1635 1628 UGCAUACG CUGAUGAG X CGAA AUCACCUC 1328 GAGGUGAUC CGUAUGCA 1636 1632 GUCCUGCA CUGAUGAG X CGAA ACGGAUCA 1329 UGAUCCGUA UGCAGGAC 1637 1651 GGAAGCUG CUGAUGAG X CGAA AGGGGUJC 1330 GAACCCCUA CAGCUUCC 1638 1657 CUGACUGG CUGAUGAG X CGAA AGCUGUAG 1331 CUACAGCUUJ CCAGUCAG 1639 1658 UCUGACUG CUGAUGAG X CGAA AAGCUGUA 1332 UACAGCUUC CAGUCAGA 1640 1663 AGACGUCU CUGAUGAG X CGAA ACUGGAAG 1333 CUUCCAGUC AGACGUCU 1641 1670 UAGGCAUA CUGAUGAG X CGAA ACGUCUGA 1334 UCAGACGUC UAUGCCUA 1642 1672 CGUAGGCA CUGAUGAG X CGAA AGACGUCU 1335 AGACGUCUA UGCCUACG 1643 1678 CAACCCCG CUGAUGAG X CGAA AGGCAUAG 1336 CUAUGCCUA CGGGGUUG 1644 16851UAGAGCAC CUGAUGAG X CGAA ACCCCGUA 1337 UACGGGGUTU GUGCUCUA 1645 1691 AGCUCGUA CUGAUGAG X CGAA AGCACAAC 1338 GUUGUGCUC UACGAGCU 1646 1693 UAAGCUCG CUGAUGAG X CGAA AGAGCACA 1339 UGUGCUCUA CGAGCUJA 1647 1700 CCAGUCAU CUGAUGAG X CGAA AGCUCGUA 1340 UACGAGCUJ AUGACUGG 1648 1701 GCCAGUCA CUGAUGAG X CGAA AAGCUCGU 1341 ACGAGCUUA UGACUGGC 1649 17111AAGGCAGU CUGAUGAG X CGAA AGCCAGUC 1342 GACUGGCUC ACUGCCUTU 1650 1719 GUGGCUGU CUGAUGAG X CGAA AGGCAGUG 1343 CACUGCCUJ ACAGCCAC 1651 1720 UGUGGCUG CUGAUGAG X CGAA AAGGCAGU 1344 ACUGCCUUA CAGCCACA 1652 1730 CGGCAGCC CUGAUGAG X CGAA AUGUGGCU 1345 AGCCACAUU GGCUGCCG 1653 1748AUAAGA CUAUGA X GAAAUCGGUC134 GACAGUD AC~tUAU 165 1749 AUAAAGAU CUGAUGAG X CGAA AUCUGGUC 1347 GACCAGAUU AUCUAU 16551 1751 ACCAUAAA CUGAUGAG X CGAA AUAUCUG 1348 CAGAUUAUC ULJUAUGGU 1656 1753 CCACCAUA CUGAUGAG X CGAA AGAUAAUC 1349 GAUUAUCUU UAUGGUGG 1657 1754 CCCACCAU CUGAUGAG X CGAA AAGAUAAU 1350 AUUAUCUUU AUGGUGGG 1658 1755 GCCCACCA CUGAUGAG X CGAA AAAGAUAA 1351 UUAUCUtJUA UGGUGGGC 1659, 1771 GGGAAGA CUGAUGAG X CGAA AGCCACGG 1352 CCGUGGCUA UCUGUCCC 16601 1773 CGGGGACA CUGAUGAG X CGAA AUAGCCAC 1353 GUGGCUAUC UGUCCCCG 1661 1777 GGUCCGGG CUGAUGAG X CGAA ACAGAUAG 1354 CUAUCUGUC CCCGGACC 16621 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 155 SEQ ID. SEQ Pos RZ No. Substrate ID. No.

1787 AUUUUGCU CUGAUGAG X CGAA AGGUCCGG 1355 CCGGACCUC AGCAAAAU 1663 1796 UUGCUGGA CUGAUGAG x CGAA AUULTJGCU 1356 AGCAAAAUC UCCAGCAA 1664 1798, AGUtJGCUG CUGAUGAG X CGAA AGAUUtJUG 1357 CAP.AAUCUC CAGCAACU 1665 18341GGCAGUCA CUGAUGAG X CGAA ACAGCAGG 1358 CCUGCUGUC UGACUGCC 1666 1844 UGGAACUU CUGAUGAG X CGAA AGGCAGUC 1359 GACUGCCUC AAGUtJCCA 1667 1849 CCCGCUGG CUGAUGAG X CGAA ACUUGAGG 1360 CCUCAAGUJ CCAGCGGG 1668 1850 UCCCGCUG CUGAUGAG X CGAA AACUUGAG 1361 CUCAAGUJC CAGCGGGA 1669 1871 UGGGGGAA CUGAUGAG X CGAA AGGGGCCG 1362 CGGCCCCJC UTJCCCCCA 1670 1873,UCUGGGGG CUGAUGAG X CGAA AGAGGGGC 1363 GCCCCUCUU CCCCCAGA 1671 18741AUCUGGGG CUGAUGAG X CGAA AAGAGGGG 1364 CCCCUCUJC CCCCAGAU 1672 18831GUGGCCAG CUGAUGAG X CGAA AUCUGGGG 1365 CCCCAGAUC CUGGCCAC 1673 18951AGCAGCUC CUGAUGAG X CGAA AUUEGUGGC 1366 GCCACAAUU GAGCUGCU 1674 1912JUGGGGAGU CUGAUGAG X CGAA ACCGUEJGC 1367 GCAACGGUC ACUCCCCA 1675 19161AUCUGGG CUGAUGAG X CGAA AGUGACCG 1368 CGGUCACUC CCCAAGAU 1676 19251CUCCGCUC CUGAUGAG X CGAA AUCUUGGG 1369 CCCAAGAUJ GAGCGGAG 1677 19391AGGGUUJCC CUGAUGAG X CGAA AGGCACUC 1370 GAGUGCCUC GGAACCCU 1678 1948IGGUGCAAG CUGAUGAG X CGAA AGGGUUCC 1371 GGAACCCUC CUUGCACC 1679 1951JUGCGGUGC CUGAUGAG X CGAA AGGAGGGU 1372 ACCCUCCUU GCACCGCA 1680 19751AGGCAGGC CUGAUGAG X CGAA ACUCAUCG 1373 CGAUJGAGUU GCCUGCCU 1681 19881GCGCUGAG CUGAUGAG X CGAA AGGCAGGC 1374 GCCUGCCUA CUCAGCGC 1682 19911GCUGCGCU CUGAUGAG X CGAA AGUAGGCA 1375 UGCCUACUC AGCGCAGC 11683 2006 UAAGGCAC CUGAUGAG X CGAA AGGCGGGC 1376 GCCCGCCUU GUGCCUUA 1684 2013 CGGGGCCU CUGAUGAG X CGAA AGGCACAA 1377 UJUGUGCCUJ AGGCCCCG 1685 2014 GCGGGGCC CUGAUGAG X CGAA AAGGCACA 1378 UGUGCCUJA GGCCCCGC 1686 2044 AGGGCUGA CUGAUGAG X CGAA AUUGGCUC 1379 GAGCCAAUC UCAGCCCU 1687 2046 GGAGGGCU CUGAUGAG X CGAA AGAtJUGGC 1380 GCCAAUCUC AGCCCUCC 1688 20531UUGGCGUG CUGAUGAG X CGAA AGGGCUGA 1381 UCAGCCCUC CACGCCAA 1689 2069JUGGUGGGC CU-GAUGAG X CGAA AGGCUCCU 1382 AGGAGCCUJ GCCCACCA 1690 2084 CGAACAUJ CUGAUGAG X CGAA AUUGGCUG 1383 CAGCCAAUC AAUGUUCG 1691 2090 CAGAGACG CUGAUGAG X CGAA ACAUUGAU 1384 AUCAAUGUU CGUCUCUG 1692 2091 GCAGAGAC CUGAUGAG X CGAA AACAUJGA 1385 UCAAUGUUC GUCUCUGC 1693 2094 AGGGCAGA CUGAUGAG X CGAA ACGAACAU 1386 AUGUEJCGUC UCUGCCCU 1694 20961UCAGGGCA CUGAUGAG X CGAA AGACGAAC 1387 GUUCGUCUC UGCCCUGA 1695 2113 GGGAUCCU CUGAUGAG X CGAA AGGCAGCA 1388 UGCUGCCUC AGGAUCCC 1696 2119 GAAUGGGG CUGAUGAG X CGAA AUCCUGAG 1389 CUCAGGAUC CCCCAUUC 1697 2126 GGGUGGGG CUGAUGAG X CGAA AUGGGGGA 1390 UCCCCCAUU CCCCACCC 1698 2127 AGGGUGGG CUGAUGAG X CGAA AAUGGGGG 1391 CCCCCAUUC CCCACCCU 1699 2151 CACAUGGG CUGAUGAG X CGAA ACCCCCUC 1392 GAGGGGGUC CCCAUGUG 1700 2162 AACUGGAA CUGAUGAG X CGAA AGCACAUG 1393 CAUGUGCUU UUCCAUU 1701 2163 GAACUGGA CUGAUGAG X- CGAA AAGCACAU 1394 AUGUGCUUJ UCCAGUTUC 1702 2164 AGAACUGG CUGAUGAG X CGAA AAAGCACA 1395 UGUGCUUUU CCAGUUCU 1703 2165 AAGAACUG CUGAUGAG x CGAA AAAAGCAC 1396 GUGCUUUUC CAGUUCUU 1704 2170[UCCAGAAG CUGAUGAG X CGAA ACUGGAAA 1397 UUUJCCAGUU UCUG 1705 2171 UtJCCAGAA CUGAUGAG x CGAA AACUGGAA 1398 UEJCCAGUUC TUUCUGGAAL 1706 2173 AAUUCCAG CUGAUGAG X CGAA AGAACUGG 1399 CCAGUtJCUU CUGGAAUU 1707 2174 CAAUUCCA CUGAUGAG X CGAA AAGAACUG 1400 CAGUtJCUUC UGGAAEJUG 1708 2181 GGUCCCCC CUGAUGAG X CGAA ATJUCCAGA 1401 UCUGGAAtJU GGGGGACC 1709, 2214,AUGGAGGA CUGAUGAG X CGAA ACAGGGGG 1402 CCCCCUGUC UCCUCCAU 17101 2216 UGAUGGAG CUGAUGAG X CGAA AGACAGGG 1403 CCCUGUCUC CUCCAUCA 17111 2219 AAAUGAUG CUGAUGAG X CGAA AGGAGACAI 1404 UGUJCUCCUC CAUCAUUJ 17121 2223 AACCAAAU CUGAUGAG X CGAA AUGGAGGAI 1405 1UCCUCCAUC AUUUGGUUJ11 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 156- SEQ ID. SEQ Pos RZ No. Substrate ID. No.

2226 GGAAACCA CUGAUGAG X CGAA AUGAUGGA 1406 UCCAUCAUU UGGUTUUCC 2.714 2227 AGGAAACC CUGAUGAG X CGAA AAUGAIJGG 1407 CCAUCAUUU GGUUUCCU 1715 2231,CAAGAGGA CUGAUGAG X CGAA ACCAAAUG 1408 CAUUUGGUU UCCUCUUG 1716 2232 CCAAGAGG CUGAUGAG X CGAA AACCAAAU 1409 ALUUGGUUJ CCUCUUGG 1717 2233 GCCAAGAG CUGAUGAG X CGAA AA.ACCAAA 1410 UUUGGUJUC CUCUJEGGC 1718 2236 AAAGCCAA CUGAUGAG X CGAA AGGAAACC 1411 GGUrUECCUC UEJGGCUUUJ 1719 2238 CCAAAGCC CUGAUGAG X CGAA AGAGGAAA 1412 UEJUCCUCUU GGCUUUGG 1720 2243,UAUCCCCA CUGAUGAG X CGAA AGCCAAGA 1413 UCUEJGGCUU UGGGGAUA 1721 2244 GUAUCCCC CUGAUGAG X CGAA AAGCCAAG 1414 CUUGGCUUJ GGGGAUAC 1722 2251 UTUUAGAAG CU-GAUGAG X CGAA AUCCCCAA 1415 UUGGGGAUA CUUCUAAA 1723 2254 AAAUUtJAG CUGAUGAG X CGAA AGUAUCCC 1416 GGGAUACUU CUAAAUUU[ 1724 2255 AAAAUUtA CUGAUGAG X CGAA AAGUAUCC 1417 GGAUACUUC UAAAUUUU 1725 2257,CCAA.AAUU CUGAUGAG X CGAA AGAA GUAU 1418 AUACUEJCUA AAUUUtJGG 1726 2261 GCUCCCAA CUGAUGAG X CGAA. AUUtJAGAA 1419 UUCUAAAUJ UUGGGAGC 1727 2262 AGCUCCCA CUGAUGAG X CGAA AATJUUAGA 1420 UCUAAAUUJ UGGGAGCU 1728 2263 GAGCUCCC CUGAUGAG X CGAA AAAUUUAG 1421 CUAAAUJUtJ GGGAGCUC 1729 2271 AGAUGGAG CUGAUGAG X CGAA AGCUCCCA 1422 UGGGAGCUC CUCCAUCU 1730 2274 UGGAGAUG CUGAUGAG X CGAA AGGAGCUC 1423 GAGCUCCUC CAUCUCCA 1731 2278 CCAUUGGA CUGAUGAG X CGAA AUGGAGGA 1424 UCCUCCAUC UCCAAUGG 1732 2280 AGCCAUUG CUGAUGAG X CGAA AGAUGGAG 1425 CUCCAUCUC CAAUGGCU 1733 2294 CUGCCACA CUGAUGAG X CGAA AUCCCAGC 1426 GCUGGGAUU UGUGGCAG 1734 2295 CCUGCCAC CUGAUGAG X CGAA AAUCCCAG 1427 CUGGGAUUJ GUGGCAGG 1735 2307 CUGAGUGG CUGAUGAG X CGAA AUCCCUGC 1428 GCAGGGAUU CCACUCAG 1736 2308 UCUGAGUG CUGAUGAG X CGAA AAUCCCUG 1429 CAGGGAUUC CACUCAA 1737 2313 GAGGUtCU CUGAUGAG X CGAA AGUGGAAU 1430 AUUCCACUC AGAACCUC 1738, 2321 AUUCCAGA CUGAUGAG X CGAA AGGUUCUG 1431 CAGAACCUC UCUGGAAJ 1739 2323 AAAUUCCA CUGAUGAG X CGAA AGAGGUUC 1432 GAACCUCUC UGGAAUUJ 1740 2330 CAGGCACA CUGAUGAG X CGAA AUUCCAGA 1433 UCUJGGAAUU UGUGCCUG 1741 2331 UCAGGCAC CUGAUGAG X CGAA AAUUCCAG 1434 CUGGAAUUU GUGCCUGA 1742 2347 UCCAGUGG CUGAUGAG X CGAA AGGCACAU 1435 AUGUGCCUU CCACUGGA 1743 2348 AUCCAGUG CUGAUGAG X CGAA AAGGCACA 1436 UGUGCCUUC CACUGGAU 1744 2357 AACCCCAA CUGAUGAG X CGAA AUCCAGUG 1437 CACUGGAUU UUGGGGUU 1745 2358 GAACCCCA CUGAUGAG X CGAA AAUCCAGU 1438 ACUGGAUUU UGGGGUJC 1746 2359 GGAACCCC CUGAUGAG X CGAA AAAUCCAG 1439 CUGGAUTUUU GGGGUUCC 1747 2365 GUGCUGGG CUGAUGAG X CGAA ACCCCAAA 1440 UUtJGGGGUU CCCAGCAC 1748 2366 GGUGCUGG CUGAUGAG X CGAA AACCCCAA 1441 UEJGGGGUUC CCAGCACC 1749 2385 CCCCCCAA CUGAUGAG X CGAA. AUCCACAU 1442 _AUGUGGAUU UEJGGGGGG 1750 2386 ACCCCCCA CUGAUGAG X CGAA AAUCCACA 1443 UGUGGALTJ UGGGGGGU 11751 2387 GACCCCCC CUGAUGAG X CGAA AAAUCCAC 1444 GUGGAUUUU GGGGGGTJC 175 2395 ACAAAAGG CUGAUGAG X CGAA ACCCCCCA 1445 UGGGGGGUC CCUUUEJGU 1753 2399 AGACACAA CUGAUGAG X CGAA AGGGACCC 1446 GGGUCCCUJ UUGUGUCU 1754 2400 GAGACACA CUGAUGAG x CGAA AAGGGACC 1447 GGUCCCUUU UGUGUCUC 1755 2401,GGAGACAC CUGAUGAG X CGAA AAAGGGAC 1448 GUCCCVUUUJ GUGUCUCC 1756 2406 GCGGGGGA CUGAUGAG X CGAA ACACAA.AA 1449 UUJEUGUGUC UCCCCCGC 1757 2408 UGGCGGGG CUGAUGAG X CGAA AGACACAA 1450 UETJGUGUCUC CCCCGCCA 1758 2418 AGUCCUUG CUGAUGAG X CGAA AUGGCGGG 1451 CCCGCCAUU CAAGGACU 1759 2419 GAGUCCUU CUGAUGAG X CGAA AAUGGCGG 1452 CCGCCAUUC AAGGACUC 1760 24271AAAGAGAG CUGAUGAG X CGAA AGUCCUG 1453 CAAGGACUC CUCUCUUU 1761 2430 AAGAAAGA CUGAUGAG X CGAA AGGAGUCC 1454 GGACUCCUC UCUUUCUU 1762 2432 UGAAGAAA CUGAUGAG X CGAA AGAGGAGU 1455 ACUCCUCUC UU-UCUUCA_ 17631 2434 GGUGAAGA CUGAUGAG X CGAA AGAGAGGA 1456 UCCUCUCUU UTCUEJCACC 1764: SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 157- SEQ ID. SEQ POS UGGGZ No. Substrate ID. No.

2435 UGGA CUGAUGAG X CGAA AAGAGAGG 1457- CCUCUCUTUU CUtJCACCA 1765 2436 UEIGGUGAA CUGAUGAG X CGAA AAAGAGAG 1458 CUCIJCUtJUC UEJCACCAA 1766 2438 UCUUGGUG CUGAUGAG X CGAA AGAAAGAG 1459 CUCUUtJCUU CACCAAGA 1767 24391UUCUUGGU CUGAUGAG X CGAA AAGAAAGA 1460 UCUCCACAGA 16 Where represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Res. 20: 3252). The length of stem II may be 2 base-pairs.

Acids SUBSTITUTE SHEET (RULE 26) 158 Table XV: Human A-raf Hairpin Ribozyme and Target Sequence nt. Position Ribozyme Sequence SEQ ID. No. Target Sequence SEQ ID. No.

69 CACAGA AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1769 AGGCG GAC UCUGUG 1841 117 CGCUAC AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 1770 CGGCG GCU GUAGCG 1842 120 CGCCGC AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 1771 CGGCU GUA GCGGCG 1843 151 GGGCUG AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 1772 CACCU GCC CAGCCC 1844 156 AGGUGG AGAA GGGC ACCAGAGAAACA X GtJACAUUACCUGGUA 1773 GCCCA GCC CCACCU 1845 167 AAGAUG AGAA GAGG ACCAGAGAAACA X GUACAUUACCUGGUA 1774 CCUCA GCC CAUCUU 1846 268 UUGUUG AGAA GGUA ACCAGAGAAACA X GUACAUUACCUGGUA 1775 UACCU GCC CAACAA 1847 296 AUCCCG AGAA GUCA ACCAGAGAAACA X GUACAUUACCUGGUA 1776 UGACU GUC CGGGAU 1848 366 CCACAC AGAA GUCC ACCAGAGAAACA X GUACAUUACCUGGUA 1777 GGACU GCU GUGUGG 1849 381 UGAUGA AGAA GUAG ACCAGAGAAACA X GUACAUUACCUGGUA 1778 CUACC GAC UCAUCA 1850 410 GUCCCA AGAA GUGA ACCAGAGAAACA X GUACAUUACCUGGUA 1779 UCACU GCC UGGGAC 1851 478 AUGGUC AGAA GGAC ACCAGAGAAACA X GUACAUUACCUGGUA 1780 GUCCC GCU GACCAU 1852 481 UGCAUG AGAA GCGG ACCAGAGAAACA X GUACAUUACCUGGUA 1781 CCGCU GAC CAUGCA 1853 516 ACGCCA AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1782 CUUCA GCC UGGCGU 1854 537 ACUUAA AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1783 CUUCU GCC UUAAGU 1855 550 CCAUGG AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 1784 UUUCU GUU CCAUGG3 1856 564 UUUGGC AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 1785 CUUCC GUU GCCAAA 1857 620 AACACA AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 1786 CCACA GUC UGUGUU 1858 652 UGGUAG AGAA GUUG ACCAGAGAAACA X GUACAUUACCUGGUA 1787 CAACA GUU CUACCA 1859 714 UCAGGG AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 1788 GAACC GCC CCCUGA 1860 750 UGCGGG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 1789 UCOCA GOC CCCGCA 1861 794 GGCUGG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 1790 UCCCU GOC CCAGCC 1862 825 ACGUGG AGAA GAUG ACCAGAGAAACA X GUACAUUACCUGGUA 1791 CAUCC GCU CCACGU 1863 866 CAUGGG AGAA GUGG ACCAGAGAAACA X GUACAUTUACCUGGUA 1792 CCACG GOC CCCAUG 1864 892 CCAGUG AGAA GGAU ACCAGAGAAACA X GUACAUUACCUGGUA 1793 AUCCA GCU CACUGG 1865 917 GGCAGC AGAA GUGC ACCAGAGAAACA X GUACAUUACCUGGUA 1794 GCACU GAU GOUGOC 1866 923 ACUACO AGAA GCAU ACCAGAGAAACA X GUACAUUACCUGGUA 1795 AUGCU GCC GGUAGU 1867 927 CUCUAC AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1796 UGCCG GUA GUAGAG 1868 969 UGGCUG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1797 CCOA GCC CAGCCA 1869 1049 CUUGUC AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1798 UGGCC GAU GACAAG 1870 1126 UUCAGC AGAA GCAC ACCAGAGAAACA X GUACAUUACCUGGUA 1799 GUGCA GCU GCUGAA 1871 1129 CUCUUC AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 1800 CAGCU GCU GAAGAG 1872 1219 GCUGUG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 1801 UCCCA GCC CACAGC 1873 1226 CUGOUC AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 1802 CCACA GCU GAGOAG 1874 0

U'

00

(A

0 th w, nt. Position Ribozyme Sequence SEQ ID. No. Target Sequence SEQ ID. No.

1297 CCCAUA AGAA GCAA ACCAGAGAAACA X GUACAUUACCUGGUA 1803 UUGCU GUJ UAUGGG 1875 1318 AAUCCC AGAA GGGU ACCAGAGAAACA X GUACAUUACCUGGUA 1804 ACCCG GCC GGGAUU 1876 1359 GGUAGA AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 1805 CUCCA GCC UCUACC 1877 1408 UCGAUG AGAA GGAC ACCAGAGAAACA X GUACAUtJACCUGGUA 1806 GUCCA GCU CAUCGA 1878 1429 UGGGCA AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 1807 CGGCA GAC UGCCCA 1879 1433 GCCCUG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 1808 AGACU GCC CAGGGC 1880 1576 UCCAAG AGAA GGGC ACCAGAGAAACA X GUACAtJUACCUGGUA 1809 GCCCA GCC CUUGGA 1881 1588 CCUGAG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1810 GAGCA GCC CUCAGG 1882 1616 CACCUC AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1811 UGGCA GCU GAGGUG 1883 1629 CCUGCA AGAA GAUC ACCAGAGAAACA X GUACAUUACCUGGUA 1812 GAUCC GUA UGCAGG 1884 1653 ACUGGA AGAA GUAG ACCAGAGAAACA X GUACAtJUACCUGGUA 1813 CUACA GCU UCCAGU 1885 1664 AUAGAC AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 1814 AGUCA GAC GUCUAU 1886 1714 CUGUAA AGAA GUGA ACCAGAGAAACA X GUACAtJUACCUGGUA 1815 UCACU GCC UUACAG 1887 1734 GGUCAC AGAA GCCA ACCAGAGAAACA X GUACAUUACCUGGUA 1816 UGGCU GCC GUGACC 1888 1744 AAGAUA AGAA GGUC ACCAGAGAAACA X GUACAUUACCUGGUA 1817 GACCA GAU UAUCUU 1889 1774 UCCGGG AGAA GAUA ACCAGAGAAACA X GUACAUUACCUGGUA 1818 UAUCU GUC CCCGGA 1890 1781 GCUGAG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1819 CCCCG GAC CUCAGC 1891 1806 CCUUGG AGAA GUUG ACCAGAGAAACA X GUACAUUACCUGGUA 1820 CAACU GCC CCAAGG 1892 1828 UCAGAC AGAA GGCG ACCAGAGAAACA X GUACAUUACCUGGUA 1821 CGCCU GCU GUCUGA 1893 1831 CAGUCA AGAA GCAG ACCAGAGAAACA X GUACAUUACCUGGUA 1822 CUGCU GUC UGACUG 1894 1835 GAGGCA AGAA GACA ACCAGAGAAACA X GUACAUIJACCUGGUA 1823 UGUCU GAC UGCCUC 1895 1839 ACUUGA AGAA GUCA ACCAGAGAAACA X GUACAUUtACCUGGUA 1824 UGACU GCC UCAAGU 1896 1864 AAGAGG AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1825 GAGCG GCC CCUCUU 1897 1879 GCCAGG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1826 CCCCA GAU CCUGGC 1898 1900 CGUUGC AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 1827 GAGCU GCU GCAACG 1899 1967 CAACUC AGAA GCCU ACCAGAGAAACA X GUACAUUACCUGGUA 1828 AGGCC GAU GAGUUG 1900 1979 UAGGCA AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1829 UGCCU GCC UGCCUA 1901 1983 UGAGUA AGAA GGCA ACCAGAGAAACA X GUACAUUACCUGGUA 1830 UGCCU GCC UACUCA 1902 1997 AAGGCG AGAA GCGC ACCAGAGAAACA X GUACAUUACCUGGUA 1831 GCGCA GCC CGCCUU 1903 2001 GCACAA AGAA GGCU ACCAGAGAAACA X GUACAUUACCUGGUA 1832 AGCCC GCC UUGUGC 1904 2020 GGCUUG AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1833 GCCCC GCC CAAGCC 1905 2047 GUGGAG AGAA GAGA ACCAGAGAAACA X GUACAUUACCUGGUA 1834 UCUCA GCC CUCCAC 1906 2097 CAUCAG AGAA GAGA ACCAGAGAAACA X GUACAUUACCUGGUA 1835 UCUCU GCC CUGAUG 1907 2102 GGCAGC AGAA GGGC ACCAGAGAAACA X GUACAUUACCUGGUA 1836 GCCCU GAU GCUGCC 1908 2108 UCCUGA AGAA GCAU ACCAGAGAAACA X GUACAUUACCUGGUA 1837 AUGCU GCC UCAGGA 1909 2167 CAGAAG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 1838 UUCCA GUU CUUCUG 19 2211 GGAGGA AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 1839 CCCCU GUC UCCUCC 1911 160 0 nt. Position Ribozyme Sequence ISEQ ID. No. J Target Sequence ISEQ ID. No. '0 2337 AGGCAC AGAA GGCA ACCAGAGAAACA X GUACAUtJACCUGGUA 1840 UGCCU GAO GUGCCU 1912 a 0z Where represents stem IV region of a Hairpin ribozyme. The length of stem IV may be 2 base-pairs.

CO)

M

11 t- 00 WO 98/50530 WO 9850530PCTIUS98/09249 161 Table XVI: Hammerhead Ribozyme Sites for B-raf nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

17 CGGGCGGG CUGAUGAG X CGAA AGGGGGCC 1913 GGCCCCCUC CCCGCCCG 2354 38 CGGGGCCC CUGAUGAG X CGAA AGCGGCCG 1914 CGGCCGCUC GGGCCCCG 2355 AUAACCGA CUGAUGAG X CGAA AGCCGGGG 1915 CCCCGGCUC UCGGUUAU 2356 52 UtJAUAACC CUGAUGAG X CGAA AGAGCCGG 1916 CCGGCUCUC GGUtJAUAA 235-7 56 CAUCUUAU CUGAUGAG X CGAA ACCGAGAG 1917 CUCUCGGUJ AUAAGAUG 2358 57 CCAUCUUA CUGAUGAG X CGAA AACCGAGA 1918 UCUCGGUUA UAAGAUGG 2359 59 CGCCAUCU CUGAUGAG X CGAA AUAACCGA 1919 UCGGtJUAUA AGAUGGCG 2360 113 GUUGAACA CUGAUGAG X CGAA AGCCUGGC 1920 GCCAGGCUC UGUUCAAC 2361 117 CCCCGUUG CUGAUGAG X CGAA ACAGAGCC 1921 GGCUCtJGUU CAACGGGG 2362 118 UCCCCGUU CUGAUGAG X CGAA AACAGAGC 1922 GCUCUGUUC AACGGGGA 2363 165 CAGCCGAA CUGAUGAG X CGAA AGGCCGCG 1923 CGCGGCCUC UUCGGCUG 2364 167 CGCAGCCG CUGAUGAG X CGAA AGAGGCCG 1924 CGGCCUCUU CGGCUGCG 2365 168 CCGCAGCC CUGAUGAG X CGAA AAGAGGCC 1925 GGCCUCUJC GGCUGCGG 2366 187 UCCUCCGG CUGAUGAG X CGAA AUGGCAGG 1926 CCUGCCAUJ CCGGAGGA 2367 188 CUCCUCCG CUGAUGAG X CGAA AAUGGCAG 1927 CUGCCAUUC CGGAGGAG 2368 206 UUGUtJGA CUGAUGAG X CGAA AUUCCACA 1928 UGUGGAAUA UCAAACAA 2369 208 AUTUUGUUU CUGAUGAG X CGAA AUAUtJCCA 1929 UGGAAUAUC AAACAAAU 2370 220 GUCALACUJ CUGAUGAG X CGAA AUCAUUJG 1930 CAAAUGAUU AAGtJUGAC 2371 221 UGUCAACU CUGAUGAG X CGAA AAUCAUTUU 1931 AAAUGAUUA AGUEJGACA 2372 225 CCUGUGUC CUGAUGAG X CGAA ACUUAAUC 1932 GAUUAAGUJ GACACAGG 2373 239 GGCCUCUA CUGAUGAG X CGAA AUGUEJCC-U 1933 AGGAACAUA UAGAGGCC 2374 241 AGGGCCUC CUGAUGAG X CGAA AUAUGUUC 1934 GAACAUAUA GAGGCCCU 2375 250 UtGUCCAA CUGAUGAG X CGAA AGGGCCUC 1935 GAGGCCCUA UUGGACAA 2376 252 AUUJUGUCC CUGAUGAG X CGAA AUAGGGCC 1936 GGCCCUAUU GGACAAAU 2377 261 CCCCACCA CUGAUGAG X CGAA AUUUGUCC 1937 GGACAAAUJ UGGUGGGG 2378 262 UCCCCACC CUGAUGAG X CGAA AAUUEJGUC 19 38 GACAAAUUU GGUGGGGA 2379 275 UGGUGGAU CUGAUGAG X CGAA AUGCUCCC 1939 GGGAGCAUA AUCCACCA 2380 278 UGAUGGUG CUGAUGAG X CGAA AUEJAUGCU 1940 AGCAtJAAUC CACCAUCA 2381 285 GAUAUAUJ CUGAUGAG X CGAA AUGGUGGA 1941 UCCACCAUC AAUAUAUC 2382 289 UCCAGAUA CUGAUGAG X CGAA AUEJGAUGG 1942 CCAUCAAUA UAUCUGGA 2383 291 CCUCCAGA CUGAUGAG X CGAA AUAUUGAU 1943 AUCAAUAUA UCUGGAGG 2384 293 GGCCUCCA CUGAUGAG X CGAA AUAUAUUG 1944 CAAUAUAUC UGGAGGCC 2385 303 AUUCUUCA CUGAUGAG X CGAA AGGCCUCC 1945 GGAGGCCUA UGAAGAAU 2386 312 UGCUGGUG CUGAUGAG X CGAA AUtJCUUCA 1946 UGAAGAAUA CACCAGCA 2387 325 AGUGCAUC CUGAUGAG X CGAA AGCUtJGCU 1947 AGCAAGCUA GAUGCACU 2388 334 CUUJUGUUG CUGAUGAG X CGAA AGUGCAUC 1948 GAUGCACUC CAACAAAG 2389 354 AUEJCCAAU CUGAUGAG X CGAA ACUGUUGU 1949 ACAACAGUEJ AUUGGAAU 2390 355 GAUUCCAA CUGAUGAG X CGAA AACUGUEJG 1950 CAACAGUtJA UUGGAAUC 2391 357 GAGAUUCC CUGAUGAG X CGAA AUAACUGU 1951 ACAGUtJAUU GGAAUCUC 2392 363 IUCCCCAGA CUGAUGAG X CGAA ALUCCAAU 1952 AUTUGGAAUC UCUGGGGA 2393 365 GUEJCCCCA CUGAUGAG X CGAA AGAUUCCA 1953 UGGAAUCUC UGGGGAAC 2394 383 AACAGAAA CUGAUGAG X CGAA AUCAGUUC 1954 GAACUGAUEJ UUUCUGUU 2395 384 AAACAGAA CUGAUGAG x CGAA AAUCAGUU 1955 AACUGAUUU UUCUGUUU 2396 385 GAAACAGA CUGAUGAG X CGAA AAAUCAGU 1956 ACUGATJU UCUGUUUC 2397 386 AGAA.ACAG CUGAUGAG x CGAA AAAAUCAG 1957 CUGAUUUtUU CUGUUUCU 2398 387 UAGAAACA CUGAUGAG X CGAA AAAAAUCA 1958 UGAUUEUEJC UGUUUCUA 2399 391 GAGCUAGA CUGAUGAG X CGAA ACAGAAAA 1959 ULTUUCUGUU UCUAGCUC 2400 392 AGAGCUAG CUGAUGAG X CGAA AACAGAAA 1960 UT.UCUGUUU CUAGCUCU 2401 39 AGGU CUGAUGAG X CGAA AAACAGAA 1961 UUCUGUTUUC UAGCUCUG, 2402 SUBSITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 162 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

395 UGCAGAGC CUGAUGAG X CGAA AGAAACAG 1962 CUGLUUCUA GCUCUGCA 2403 399 UUGAUGCA CUGAUGAG X CGAA AGCUAGAA 1963 UUCUAGCUC UGCAUCAA 2404 405 UAUCCAUTU CUGAUGAG X CGAA AUGCAGAG 1964 CUCUGCAUC AAUGGAUA 2405 413 UGUAACGG CUGAUGAG X CGAA AUCCAUUG 1965 CAAUGGAUA CCGUJUACA 2406 418 GAAGAUGU CUGAUGAG X CGAA ACGGUAUC 1966 GAUACCGUJ ACAUCUC 2407 419 AGAAGAUG CUGAUGAG X CGAA AACGGUAU 1967 AUACCGUJA CAUCUUCJ 2408 423 AGGAAGAA CUGAUGAG X CGAA AUGUAACG 1968 CGUUACAUC ULTCUEJCCU 2409 425 AGAGGAAG CUGAUGAG X CGAA AGAUGUAA 3.969 TJACAUCU CUUCCUCU 2410 426 AAGAGGAA CUGAUGAG X CGAA AAGAUGUA 1970 UACAUCUJC UTJCCUCU 2411 428 AGAAGAGG CUGAUGAG X CGAA AGAAGAUG 1971. CAUCUtJCUU CCUCUUCU 2412 429 UAGAAGAG CUGAUGAG X CGAA AAGAAGAU 1972 AUCUEJCUUC CUCUUCUA 2413 432 GGCUAGAA CUGAUGAG X CGAA AGGAAGAA 1973 UUCUUCCUC UUCUAGCC 2414 434 AAGGCUAG CUGAUGAG X CGAA AGAGGAAG 1974 CUtJCCUCUJ CUAGCCUU 2415 435 AAAGGCUA CUGAUGAG X CGAA AAGAGGAA 1975 UUCCUCUUC UAGCCUUJ 2416 437 UGAAAGGC CUGAUGAG X CGAA AGAAGAGG 1976 CCUCUtJCUA GCC~TJCA 2417 442 AGCACUGA CUGAUGAG X CGAA AGGCUAGA 1977 UCUAGCCUU UCAGUGCU 2418 443 UAGCACUG CUGAUGAG X CGAA AAGGCUAG 1978 CEJAGCCUUEJ CAGUGCUA 2419 444 GUAGCACU.CUGAUGAG X CGAA AAAGGCUA 1979 UAGCCUUUC AGUGCUAC 2420 451 GAUGAAGG CUGAUGAG X CGAA AGCACUGA 1980 UCAGUGCUA CCUEJCAUC 2421 455 AAGAGAUG CUGAUGAG X CGAA AGGUAGCA 1981 UGCUACCUU CAUCUCUU 2422 456 AAAGAGAU CUGAUGAG X CGAA AAGGUAGC 1982 GCUACCUUC AUCUCUUJE 2423 459 CUGAAAGA CUGAUGAG X CGAA AUGAAGGU 1983 ACCUUCAUC UCUUUCAG 2424 461 AACUGAAA CUGAUGAG X CGAA AGAUGAAG 1984 CtJUCAUCUC UUUCAGUU 2425 463 AAAACUGA CUGAUGAG X CGAA AGAGAUGA 1985 UCAUCUCUJ UCAGUUUU 2426 464 AAAAACUG CUGAUGAG X CGAA AAGAGAUG 1986 CAUCUCUUU CAGUUUUE 2427 465 GAAAAACU CUGAUGAG X CGAA AAAGAGAU 1987 AUCUCUUUC AGU=tUEC 2428 469 UULUJGAAA CUGAUGAG X CGAA ACUGAAAG 1988 CUUUCAGUJ UUE3CAAAA 2429 470 AUUUUGAA CUGAUGAG X CGAA AACUGAAA 1989 LUUCAGUnUUTECAAAAU 2430 471 GAUUUUGA CUGAUGAG X CGAA AAACUGAA 1990 LTUCAGUTUUU UCAAAAUC 2431 472 GGAUUUUG CUGAUGAG X CGAA AAAACUGA 1991 UCAGUUUUJ CAAAAUCC 2432 473 GGGAUUU CUGAUGAG X CGAA AAAAACUG 1992 CAGUUUEJC AAAAUCCC 2433 479 AUCUGUGG CUGAUGAG X CGAA AUUEJUGAA 1993 UUCAAAAUC CCACAGAU 2434 510 UTUUGUGGU CUGAUGAG X CGAA ACUEJGGGG 1994 CCCCAAGUC ACCACAAA 2435 524 UCUAACGA CUGAUGAG X CGAA AGGUUUUJ 1995 AAAAACCUA UCGUUAGA 2436 526 ACUCUAAC CUGAUGAG X CGAA AUAGGUUE 1996 AAACCUAUC GUJUAGAGU 2437 529 AAGACUCU CUGAUGAG X CGAA ACGAUAGG 1997 CCUAUCGUU AGAGUCUU 2438 530 GAAGACUC CUGAUGAG X CGAA AACGAUAG 1998 CUAUCGUJA GAGUCUUC 2439 535 GGCAGGAA CUGAUGAG X CGAA ACUCUAAC 1999 GUUAGAGUC UUCCUGCC 2440 537 UGGGCAGG CUGAUGAG X CGAA AGACUCUA 2000 UAGAGUCUU CCUGCCCA 2441 538 UtJGGGCAG CUGAUGAG X CGAA AAGACUCU 2001 AGAGUCUUC CUGCCCAA 2442 565 ICUtJGCAGG CUGAUGAG X CGAA ACCACUGU 2002 ACAGUGGUA CCUGCAAG 2443 583 CGGACUGU CUGAUGAG X CGAA ACUCCACA 2003 UGUGGAGUU ACAGUCCG 2444 584 UCGGACUG CUGAUGAG X CGAA AACUCCAC 2004 GUGGAGUUA CAGUCCGA 2445 589 CUGUCUCG CUGAUGAG X CGAA ACUGUAAC 2005 GUUACAGUC CGAGACAG 2446 599 UUUCUUUA CUGAUGAG X CGAA ACUGUCUC 2006 GAGACAGUC UAAAGAAA 2447 601 GCUJUCUUE CUGAUGAG X CGAA AGACUGUC 2007 GACAGUCUA AAGAAAGC. 2448 626 UGGGAUtJA CUGAUGAG X CGAA ACCUCUCA 2 0 08 UGAGAGGUC UAAUCCCA 2449 628 UCUGGGAU CUGAUGAG X CGAA AGACCUCU 2 0 09 AGAGGUCUA AUCCCAGA 2450 631 CACUCUGG CUGAUGAG X CGAA AUUAGACC 2010 GGUCUAAUC CCAGAGUG 2451 649 AUUJCUGUA CtJGAUGAG X CGAA ACAGCACA,2011 UGUGCUGUU UACAGAAU 2452 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 163 nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

650 AAUUCUGU CUGAUGAG X CGAA AACAGCAC 2012 GUGCUGUUU ACAGAAUU 2453 651 GAAUUCUG CUGAUGAG X CGAA AAACAGCA 2013 UGCUGUUUA CAGAAUUC 2454 658 ICCAUCCUG CUGAUGAG X CGAA AUtJCUGUA 2014 UACAGAAUU CAGGAUGG 2455 659 UCCAUCCU CUGAUGAG X CGAA AAUtJCUGU 2015 ACAGAAUUC AGGAUGGA 2456 682 UCCCAACC CUGAUGAG X CGAA AUUGGUUU 2016 AAACCAAUU GGUUGGGA 2457 686 AGUGUCCC CUGAUGAG X CGAA~ ACCA-AUEJG 2017 CAAUUGGUU GGGACACU 2458 698 CcAGGAAA CUGAUGAG X CGAA AUCAGUGU 202.8 ACACUGAUA UEUtCCUGG 2459 700 AGCCAGGA CUGAUGAG X CGAA AUAUCAGU 20219 ACUGAUAUU UCCUGGCU 2460 701 AAGCCAGG CUGAUGAG X CGAA AAUAUCAG 2020 CUGAUAUUU CCUGGCUU 2461 702 UAAGCCAG CUGAUGAG X CGAA AAAUAUCA 2021 UGAUAUUUC CUGGCUUA 2462 709 UCUCCAGU CUGAUGAG X CGAA AGCCAGGA 2022 tCCUGGCTUU ACUGGAGA 2463 710 UtJCUCCAG CUGAUGAG X CGAA AAGCCAGG 2023 CCUGGCUUA CUGGAGAA 2464 723 CCACAUGC CUGAUGAG X CGAA AUUCUUCU 2024 AGAAGAAUU GCAUGUGG 2465 738 CAUtJCUCC CUGAUGAG X CGAA ACACUUCC 2025 GGAAGUGUU GGAGAAUG 2466 748 GUAAGUGG CUGAUGAG X CGAA ACAUtJCUC 2026 GAGAAUGtJU CCACUUAC 2467 749 UGUAAGUG CUGAUGAG X CGAA AACAUUCU 2027 AGAAUGUUC CACUtJACA 2468 754 UGUGUUGU CUGAUGAG, X CGAA AGUGGAAC 2028 GUtJCCACUJ ACAACACA 2469 755 GUGUGUJG CUGAUGAG X CGA1A AAGUGGAA 2029 UUCCACUUA CAACACAC 2470 768 UUCGUACA CUGAUGAG X CGAA AGUUGUGU 2030 ACACAACUU UGUACGAA 2472.

769 UTUUCGUAC CUGAUGAG X CGAA AAGUtJGUG 2031 CACAACUUU GUACGAAA 2472 772 GUTUUUUCG CUGAUGAG X CGAA ACAAAGUU 2032 AACUUUGUA CGAA-AAAC 2473 783 AGGUGAAA CUGAUGAG X CGAA ACGUUUUU 2033 AAAAACGUU UUUCACCU 2474 784 AAGGUGAA CUGAUGAG X CGAA AACGUUU 2034 AAAACGUUU UUCACCUU 2475 785 UAAGGUGA CUGAUGAG X CGAA AAACGUUU 2035 AAACGUUUU UCACCUUA 2476 786 CUAAGGUG CUGAUGAG X CGAA AAAACGUU 2036 AACGUTUJ CACCUUAG 2477 787 GCUAAGGU CUGAUGAG X CGAA AAAAACGU 2037 ACGUtJUUUC ACCUUAGC 2478 792 AAAAUGCU CUGAUGAG X CGAA AGGUGAAA 2038 UUUCACCUU AGCAUUUU 2479 793 CAAAAUGC CUGAUGAG X CGAA AAGGUGAA 2039_ UEJCACCUUA GCAUtJUUG 2480 798 AGUCACAA CUGAUGAG X CGAA AUGCUAAG 2040 CUUEAGCAUJ UEJGUGACU 2481 799 AAGUCACA CUGAUGAG X CGAA AAUGCUAA 2041 UJEAGCAUUU UGUGACUU 2482 800 AAAGUCAC CUGAUGAG X CGAA AAAUGCUA 2042 UAGCAUUnt GUGACUUU 2483 807 UUCGACAA CUGAUGAG X CGAA AGUCACAA 2043 UUGUGACUU LUGUCGAA 2484 808 UUUCGACA CUGAUGAG X CGAA AAGUCACA 2044 UGUGACUUTU UGUCGAAA 2485 809 CUUtJCGAC CUGAUGAG X CGAA AAAGUCAC 2045 GUGACUUUU GUCGAAAG 2486 812 CAGCUUUC CUGAUGAG X CGAA ACAAAAGU 2046 ACUUUtJGUC GAAAGCUG 2487 823 CCCUGGAA CUGAUGAG X CGAA AGCAGCUU 2047 AAGCUGCUTJ UUCCAGGG 2488 824 ACCCUGGA CUGAUGAG X CGAA AAGCAGCU 2048 AGCUGCUUJ UCCAGGGU 2489 825 AACCCUGG CUGAUGAG X CGAA AAAGCAGC 2049 GCUGCUEJUU CCAGGGUU 2490 826 AAACCCUG CUGAUGAG X CGAA AAAAGCAG 2050 CUGCUUUUC CAGGGUTUU 2491 833 ACAGCGGA CUGAUGAG X CGAA. ACCCUGGA 2051 UCCAGGGUU UCCGCUGU 2492 834 GACAGCGG CUGAUGAG X CGAA AACCCUGG 2052 CCAGGGUUJ CCGCUGUC 2493 835 UGACAGCG CUGAUGAG X CGAA AAACCCUG 2053 CAGGGUUUC CGCUGUCA 2494 842 ACAUGUUJ CUGAUGAG X CGAA ACAGCGGA 2054 UCCGCUGUC AAACAUGU 2495 854 AAAUUUAU CUGAUGAG X CGAA ACCACAUG 2055 CAUGUGGUU AUAAAUUU 2496 855 GAAAUUUA CUGAUGAG X CGAA AACCACAU 2056 AUGUGGUUA UAAAUUUC 2497 857 GUGAAAUU CUGAUGAG X CGAA AUAACCAC 2057 GUGGUUAUA AAUUUCAC 2498 861 GCUGGUGA CUGAUGAG X CGAA AUUUAUAA 2058 UUAUAAAUJ UCACCAGC 2499 862 CGCUGGUG CUGAUGAG X CGAA AAUUUAUA 2059 UAUAAAUUU CACCAGCG 2500 863 ACGCUGGU CUGAUGAG X CGAA AAAUUUAU 2060 AUAAAUUUC ACCAGCGU 2501 872 lUGUACUAC CUGAUGAG X CGAA ACGCUGGU 2 061 ACCAGCGUTU GUAGUACA 2502 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 -164nt. SEQ E Position Ribozyme ID. No. Substrate ID. No.

875 UtJCUGUAC CUGAUGAG X CGAA ACAACGCU 2062 AGCGUtJGUA GUACAGAA 2503 878 AACUUCUG CUGAUGAG X CGAA ACUACAAC 2063 GtJUGUAGUA CAGAAGUIJ 2504 886 IAUCAGUGG CUGAUGAG X CGAA ACUUCUGU 2064 ACAGAAGUU CCACUGAU 2505 887 CAUCAGUG CUGAUGAG X CGAA AACUUCUG 2065 CAGAAGUUC CACUGAUG 2506 901 UCAUAAUU CUGAUGAG X CGAA ACACACAU 2066 AUGUGUGJU AAUUAUGA 2507 902 GUCAUAAU CUGAUGAG X CGAA AACACACA 2067 UGUGUGUUA AUEJAUGAC 2508 905 UUGGUCAU CUGAUGAG X CGAA AUUAACAC 2068 GUGUUAAUU AUGACCAA 2509 906 GUUGGUCA CUGAUGAG X CGAA AAUUAACA 2069 UGUtJAAUUA UGACCAAC 2510 916 AGCAAAUC CUGAUGAG X CGAA AGUEIGGUC 2070 GACCAACUJ GAUUEJGCU 2511 920 AAACAGCA CUGAUGAG X CGAA AUCAAGUU 2071 AACUtJGAUU UGCUGUUU 2512 921 CAAACAGC CUGAUGAG X CGAA AAUCAAGU 2072 ACUUGAUUU GCUGUUUG 2513 927 UGGAGACA CUGAUGAG X CGAA ACAGCAAA 2073 UEJUGCUGUU UGUCUCCA 2514 928 UtJGGAGAC CUGAUGAG X CGAA AACAGCAA 2074 UUGCUGU~nJ GUCUCCAA 2515 931 AACUUGGA CUGAUGAG X CGAA ACAAACAG 2075 CUGUUUJGUC UCCAAGULU 2516 933 AGAACUUG CUGAUGAG X CGAA AGACAAAC 2076 GUUrUGUCUC. CAAGUEJCU 2517 939 GUUCAAAG CUGAUGAG X CGAA ACUUGGAG 2077 CUCCAAGUJ CUtJUGAAC 2518 940 UGUtJCAAA CUGAUGAG X CGAA AACUUGGA 2078 UCCAAGUJC UUJEGAACA 2519 942 GGUGUUCA CUGAUGAG X CGAA AGAACUEJG 2079 CAAGUUCUEJ UGAACACC 2520 943 UGGUGUtJC CUGAUGAG X CGAA AAGAACUU 2080 AAGUtJCUUU GAACACCA 2521 958 UCCUGUGG CUGAUGAG X CGAA AUUGGGUG 2081 CACCCAAUA CCACAGGA 2522 975 CUGCUAAG CUGAUGAG X CGAA ACGCCUCU 2082 AGAGGCGUC CUUAGCAG 2523 978 UCUCUGCU CtJGAUGAG X CGAA AGGACGCC 2083 GGCGUCCUU AGCAGAGA 2524 979 GUCUCUGC CUGAUGAG X CGAA AAGGACGC 2084 GCGUCCUUA GCAGAGAC 2525 994 CCAGAUGU CUGAUGAG X CGAA AGGGCAGU 2085 ACUGCCCUA ACAUCUGG 2526 999 AUGAUCCA CUGAUGAG X CGAA AUGUUAGG 2086 CCUAACAUC UGGAUCAU 2527 1005 AAGGGGAU CUGAUGAG X CGAA AUCCAGAU 2087 AUCUGGAUC AUCCCCUU 2528 1008 CGGAAGGG CUGAUGAG X CGAA AUGAUCCA 2088 UGGAUCAUC CCCUUCCG 2529 1013 GGGUGCGG CUGAUGAG X CGAA AGGGGAUG 2089 CAUCCCCUU CCGCACCC 2530 1014 CGGGUGCG CUGAUGAG X CGAA AAGGGGAU 2090 AUCCCCUUC CGCACCCG 2531 1026 IUAGAGUCC CUGAUGAG X CGAA AGGCGGGU 2091 ACCCGCCUC GGACUCUA 2532 1032 GCCCAAUA CUGAUGAG X CGAA AGUCCGAG 2092 CUCGGACUC UAUUGGGC 2533 1034 GGGCCCAA CUGAUGAG X CGAA AGAGUCCG 2093 CGGACUCUA UEJGGGCCC 2534 1036 UGGGGCCC CtJGAUGAG X CGAA AUAGAGUC 2094 GACUCUAUU GGGCCCCA 2535 1048 CUGGUGAG CUGAUGAG X CGAA AUUUGGGG 2095 CCCCAAAUU CUCACCAG 2536 1049 ACUGGUGA CUGAUGAG X CGAA AAUUUGGG 2096 CCCAAAUUC UCACCAGU 2537 1051 GGACUGGU CUGAUGAG X CGAA AGAAUUUG 2097 CAAAUUCUC ACCAGUCC 2538 1058 AGGAGACG CUJGAUGAG X CGAA ACUGGUGA 2098 UCACCAGUC CGUCUCCU 2539 1062 UtJGAAGGA CUGAUGAG X CGAA ACGGACUG 2099 CAGUCCGUC UCCUUCAA 2540 1064 LJEJEJGAAG CUGAUGAG X CGAA AGACGGAC 2100 GUCCGUCUC CUUCAAAA 2541 1067 GGAUUUUtG CUGAUGAG X CGAA AGGAGACG 2101 CGUCUCCUU CAAAAUCC 2542 1068 UGGAUUUJ CUGAUGAG X CGAA AAGGAGAC 2102 GUCUCCUUC AAAAUCCA 2543 1074 UUGGAAUG CUGAUGAG X CGAA AUUUUGAA 2103 UUCAAAAUC CAUUCCAA 2544 1078 GGAAUtJGG CUGAUGAG X CGAA AUGGAUUU 2104 AAAUCCAUU CCAAUUCC 2545 1079 UGGAAUJG CUGAUGAG X CGAA AAUGGAUU 2105 AAUCCAUUC CAAUtJCCA 2546 1084 GGCUGUGG CUGAUGAG X CGAA AUJUGGAAU 2106 AUrJCCAAUU CCACAGCC 2547 1085 GGGCUGUG CUGAUGAG X CGAA AAUUGGAA 2107 UtJCCAAUUC CACAGCCC 2548 1095 CUGGUCGG CUGAUGAG X CGAA AGGGCUGU 2108 ACAGCCCUU CCGACCAG 2549 1096 GCUGGUCG CUGAUGAG X CGAA AAGGGCUG 2109 CAGCCCUUC CGACCAGC 2550 1115 AUUUCGAU CUGAUGAG X CGAA AUCUEJCAU 12110 AUGAAGAUC AUCGAAAU 2551 1118 tJUGAUUUC CUGAUGAG X CGAA AUGAUCUUI 2111 JAAGAUCAUC GAAAUCAA 2552 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 165 nt. -SEQ

SEQ

Position Ribozyme ID. No. Substrate ID. No.

1124 cccAAAUtJ CUGAUGAG X CGAA AtJUUCGAU 2112 AUCGAAAUC AAUUUGGG 2553 1128 GUUGCCCA CUGAUGAG X CGAA AUtJGAUUJ 21.13 AAAUCAAUU UGGGCAAC 2554 12.29 CGTJEGCCC CUGAUGAG X CGAA AAUUGAUU 2114 AAUCAA3UUU GGGCAACG 2555 1146 CUGAUGAG CUGAUGAG X CGAA AUCGGUCU 2115 AGACCGAUC CUCAUCAG 2556 1149 GAGCUGAU CUGAUGAG X CGAA AGGAUCGG 2116 CCGAUCCUC AUCAGCUC 2557 1152 UGGGAGCU CUGAUGAG X CGAA AUGAGGAU 2117 AUJCCUCAUC AGCUCCCA 2558 1157 CACAUUGG CUGAUGAG X CGAA AGCUGAUG 2118 CAUCAGCUC CCAAUGUG 2559 1169 UGUGUUUA CUGAUGAG X CGAA AUGCACAU 2119 AUGUGCAUA UAAACACA 2560 1171 AUUGUGUU CUGAUGAG X CGAA AUAUGCAC 2120 GUGCAUAUA AACACAAU 2561 1180 ACAGGUUC CUGAUGAG X CGAA AUUGUGUU 22.21 AACACAAUA GAACCUGU 2562 1189 UCAAUAUU CUGAUGAG X CGAA ACAGGUJC 2122 GAACCUGUC AAUAUUGA 2563 1193 GUCAUCAA CUGAUGAG X CGAA AUJtGACAG 21.23 CUGUCAAUA UUGAUGAC 2564 1195 AAGUCAUC CUGAUGAG X CGAA AUAUtJGAC 2124 GUCAAUAUU GAUGACUIJ 2565 1203 CUCUAAUC CUGAUGAG X CGAA AGUCAUCA 2125 UGAUGACUU GAUUJAGAG 2566 1207 UGGUCUCU CUGAUGAG X CGAA AUCAAGUC 2126 GACUUGAUU AGAGACCA 2567 1208 UUGGUCUC CUGAUGAG X CGAk AAUCAAGU 2127 ACUUrGAUUA GAGACCAA 2568 1221 CACCACGA CUGAUGAG X CGAA AUCCUUGG 2128 CCAAGGAUU UCGUGGUG 2569 1222 UCACCACG CUGAUGAG X CGAA AAUCCUUG 2129 CAAGGAUUUE CGUGGUGA 2570 1223 AUCACCAC CUGAUGAG X CGAA AAAUCCUU 2130 AAGGAUUUC GUGGUGAU 2571 1239 CUGUGGUJ CUGAUGAG X CGAA AUCCUCCA 2131 UGGAGGAUC AACCACAG 2572 1250 AGCAGACA CUGAUGAG X CGAA ACCUGUGG 2132 CCACAGGUU UGUCUGCU 2573 1251 UAGCAGAC CUGAUGAG X CGAA AACCUGUG 2133 CACAGGUUU GUCUGCUA 2574 1254 GGGUAGCA CUGAUGAG X CGAA ACAAACCU 2134 AGGUUUGUC UGCUACCC 2575 1259 AGGGGGGG CUGAUGAG X CGAA AGCAGACA 2135 UGUCUGCUA CCCCCCCU 2576 1272 CAGGUAAU CUGAUGAG X CGAA AGGCAGGG 2136 CCCUGCCUC AUtJACCUG 2577 1275 AGCCAGGU CUGAUGAG X CGAA AUGAGGCA 2137 UGCCUJCAUU ACCUGGCU 2578 1276 GAGCCAGG CUGAUGAG X CGAA AAUGAGGC 2138 GCCUCAUUA CCUGGCUC 2579 1284 UAGUUAGU CUGAUGAG X CGAA AGCCAGGU 2139 ACCUGGCUC ACUAACUA 2580 1288 ACGUIJAGU CUGAUGAG X CGAA AGUGAGCC 2140 GGCUCACUA ACUAACGU 2581 1292 UUtJCACGU CUGAUGAG X CGAA AGUUAGUG 2141 CACUAACUA ACGUGAAA 2582 1305 AUUtJCUGU CUGAUGAG X CGAA AGGCUUJC 2142 GAAAGCCUU ACAGAAAU 2583 1306 GAUUUCUG CUGAUGAG X CGAA AAGGCUUU 22.43 AAAGCCUUA CAGAAAUC 2584 1314 GUCCUGGA CUGAUGAG X CGAA AUJUUCUGU 2144 ACAGAAAUC UCCAGGAC 2585 1316 AGGUCCUG CUGAUGAG X CGAA AGAUUtJCU 2145 AGAAAUCUC CAGGACCU 2586 1325 UUCUCGCU CUGAUGAG X CGAA AGGUCCUG 2146 CAGGACCUC AGCGAGAA 2587 1341 AUGAAGAU CUGAUGAG X CGAA ACUUCCUU 2147 AAGGAAGUC AUCUUCAU 2588 1344 AGGAUGAA CUGAUGAG X CGAA AUGACUUC 2148 GAAGUCAUC UUCAUCCU 2589 1346 UGAGGAUG CUGAUGAG X CGAA AGAUGACU 2149 AGUCAUCUU CAUCCUCA 2590 1347 CUGAGGAU CUGAUGAG X CGAA AAGAUGAC 22.50 GUCAUCUUC AUCCUCAG 2591 1350 CUUCUGAG CUGAUGAG X CGAA AUGAAGAU 2151 AUCUtJCAUC CUCAGAAG 2592 1353 UGUCUUCU CUGAUGAG X CGAA AGGAUGAA 22.52 UTJCAUCCUC AGAAGACA 2593 1367 UUUCAUUC CUGAUGAG X CGAA AUUCCUGU 2153 ACAGGAAJC GAAUGAAA 2594 1381 CGUCUACC CUGAUGAG X CGAA AGUGUtJUU 2154 AAAACACUU GGUAGACG 2595 1385 GUCCCGUC CUGAUGAG X CGAA ACCAAGUG 2155 CACUUGGUA GACGGGAC 2596 1395 CAUCACUC CUGAUGAG X CGAA AGUCCCGU 2156 ACGGGACUC GAGUGAUG 2597 1406 AAUCUCCC CUGAUGAG X CGAA AUJCAUCAC 2157 GUGAUGAUTU GGGAGAUTU 2598 1414 CCAUCAGG CUGAUGAG X CGPJA AUCUCCCA 2158 UGGGAGAUU CCUGAUGG 2599 1415 CCCAUCAG CUGAUGAG X CGAA AAUCUCCC 2159 GGGAGAUUC CUGAUGGG 2600 1429 CCCACUGU CUGAUGAG X CGAA AUCUGCCC 2160 GGGCAGAUUT ACAGUGGG 2601 1430 UCCCACUG CUGAUGAG X CGAA AAUCUGCC, 2161 GGCAGAUUTA CAGUGGGA 2602 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 166 nt. SEQ

SEQ

Position Ribozyme ID. No. Substrate ID. No.

1447 CCAGAUCC CUGAUGAG X CGAA AUtJCUUUG 2162 CAAAGAAUEJ GGAUCUGG 2603 1452 AUGAUCCA CUGAUGAG X CGAA AUCCAAUU 2163 AAUtJGGAUC UGGAUCAU 2604 1458 IUUCCAAAU CUGAUGAG X CGAA AUCCAGAU 2164 AUCUGGAUC AUUUGGAA 2605 1461 CUGUEICCA CUGAUGAG X CGAA AUGAUCCA 2165 UGGAUCAUU UGGAACAG 2606 1462 ACUGUUCC CUGAUGAG X CGAA AAUGAUCC 2166 IGGAUCAUUU GGAACAGU 2607 1471 CCCUtJGUA CUGAUGAG X CGAA ACUGUUCC 2167 GGAACAGUC tACAAGGG 2608 1473 UUCCCUJG CUGAUGAG X CGAA AGACUGUU 2168 AACAGUCUA CAAGGGAA 2609 1512 UCACAUEJC CUGAUGAG X CGAA ACAUUUJC 2169 GAAAAUGUU GAAUGUGA 2610 1529 CUGAGGUG CUGAUGAG X CGAA AGGUGCUG 2170 CAGCACCUA CACCUCAG 2611 1535 UAACUGCU CUGAUGAG X CGAA AGGUGUAG 2171 CUACACCUC AGCAGUUA 2612 1542 AGGCUGU CUGAUGAG X CGAA ACUGCUGA 2172 JUCAGCAGUU ACAAGCCU 2613 1543 AAGGCUUG CUGAUGAG X CGAA AACUGCUG 2173 ICAGCAGU3A CAAGCCUU 2614 1551 CAUUUUUG CUGAUGAG X CGAA AGGCUUGU 2174 ACAAGCCUU CAAAAAUG 2615 1552 UCAUUUUU CUGAUGAG X CGAA AAGGCUUG 2175 CAAGCCTUC AAAAAUGA 2616 1564 AGUACUCC CUGAUGAG X CGAA ACUIJCAUU 2176 AAUGAAGUA GGAGUACU 2617 1570 UUCCUGAG CUGAUGAG X CGAA ACUCCUAC 2177 GUAGGAGUA CUCAGGAA 2618 1573 GUUUUCCU CUGAUGAG X CGAA AGUACUCC 2178 GGAGUACUC AGGAAAAC 2619 1595 GAGUAGGA CUGAUGAG X CGAA AUUCACAU 2179 AUGUGAAUA UCCUACUC 2620 1597 AAGAGUAG CUGAUGAG X CGAA AUAUtJCAC 2180 GUGAAUAUC CUACUCUU 2621 1600 AUGAAGAG CUGAUGAG X CGAA AGGAUAUJ 2181 AAUAUCCUA CUCUUCAU 2622 1603 CCCAUGAA CUGAUGAG X CGAA AGUAGGAU 2182 AUCCUACUC UUCAUGGG 2623 1605 AGCCCAUG CUGAUGAG X CGAA AGAGUAGG 2183 CCUACUCUJ CAUGGGCU 2624 1606 UAGCCCAU CUGAUGAG X CGAA AAGAGUAG 2184 CUACUCUUC AUGGGCUA 2625 1614 UUGUGGAA CUGAUGAG X CGAA AGCCCAUG 2185 CAUGGGCUA UUCCACAA 2626 1616 CUUEGUGG CUGAUGAG X CGAA AUAGCCCA 2186 UGGGCUAUU CCACAAAG 2627 1617 GCUUUGUG CUGAUGAG X CGAA AAUAGCCC 2187 GGGCUAUUC CACAAAGC 2628 1637 GGUAACAA CUGAUGAG X CGAA AGCCAGUU 2188 AACUGGCUA UUGUUACC 2629 1639 UGGGUAAC CUGAUGAG X CGAAL AUAGCCAG 2189 CUGGCUAUU GUIJACCCA 2630_ 1642 CACUGGGU CUGAUGAG X CGAA ACAIWAGC 2190 GCUAUUGUU ACCCAGUG 2631 1643 CCACUGGG CUGAUGAG X CGAA AACAAUAG 2191 CUAUUGUEJA CCCAGUGG 2632 1662 ACAAGCUG CUGAUGAG X CGAA AGCCCUCA 2192 UGAGGGCUC CAGCUtIGU 2633 1668 GGUGAUAC CUGAUGAG X CGAA AGCUGGAG 2193 CUCCAGCUU GUAUCACC 2634 1671 GAUGGUGA CUGAUGAG X CGAA ACAAGCUG 2194 CAGCUtJGUA UCACCAUC 2635 1673 GAGAUGGU CUGAUGAG X CGAA AUACAAGC 2195 GCUUGUAUC ACCAUCUC 2636 1679 GAUAUGGA CUGAUGAG X CGAA AUGGUGAU 2196 AUCACCAUC UCCAUAUC 2637 1681 AUGAUAUG CUGAUGAG X CGAA AGAUGGUG 2197 CACCAUCUC CAUAUCAU 2638 1685 CUCAAUGA CUGAUGAG X CGAA AUGGAGAU 2198 AUCUCCAUA UCAUUGAG 2639 1687 GUCUCAAU CUGAUGAG -X CGAA AUAUGGAG 2199 CUCCAUAUC AUUGAGAC 2640 1690 UTJGGUCUC CUGAUGAG X CGAA AUGAUAUG 2200 CAUAUCAUJ GAGACCAA 2641 1701 UCAUCUCA CUGAUGAG X CGAA AUUUGGUC 2201 GACCAAAUU UGAGAUGA 2642 1702 AUCAUCUC CUGAUGAG X CGAA AAUUJEGGU 2202 ACCAAAUUU GAGAUGAU 2643 1711 AUAAGUUU CUGAUGAG X CGAA AUCAUCUC 2203 GAGAUGAUC AAACUUAU 2644 1717 AUAUCUAU CUGAUGAG X CGAA AGUUEJGAU, 2204 AUJCAAACUU AUAGAUAU 2645 1718 AAUAUCUA CUGAUGAG X CGAA AAGUUtJGA 2205 uCAAACUUA UAGAUAUU 2646 1720 GCAAUAUC CUGAUGAG X CGAA AUAAGUUU 2206 AAACUUAUA GAUAUJUGC 2647 1724 UCGUGCAA CUGAUGAG X CGAA AUCUAUAA 2207 UUAUAGAUA UUGCACGA 2648 1726 UGUCGUGC CUGAUGAG X CGAA AUAUCUAU 22-08 AUAGAUAUU GCACGACA 2649 1754 GUGUAAGU CUGAUGAG X CGAA AUCCAUGC 2209 GCAUGGA-UU ACUUACAC 2650 1755 CGUGUAAG CUGAUGAG X CGAA AAUCCAUG 2210 CAUGGAUUA CUUACACG 2651 1758 IUGGCGUGU CUGAUGAG X CGAA AGUAAUCCI 2211 GGAUUJACUU ACACGCCA 26521 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 167 nt. SEQ

SEQ

Position Ribozyme ID. No. Substrate ID. No.

1759 UUGGCGUG CUGAUGAG X CGAA AAGUAAUC 2212 GAUEJACUUA CACGCCAA 2653 1770 GGAUGAUU CUGAUGAG X CGAA ACUEGGCG 2213 CGCCAAGUC AAUCAUCC 2654 1774 CUGUGGAU CUGAUGAG X CGAA AUUGACUJ 2214 AAGUCAAUC AUCCACAG 2655 1777 UCUCUGUG CUGAUGAG X CGAA AUGAUUGA 2215 UCAAUCAUC CACAGAGA 2656 1789 UUACUCUU CUGAUGAG X CGAA AGGUCUCU 2216 AGAGACCUC AAGAGUAA 2657 1796 UAUAUUAU CUGAUGAG X CGAA ACUCUGA 2217 UCAAGAGUA AUAAUAUA 2658 1799 AAAUAUAU CUGAUGAG X CGAA AUUACUCU 2218 AGAGUAAUA AUAUAUUU 2659 1802 AAGAAAUA CUGAUGAG X CGAA AUUAUUAC 2219 GUAAUAAUA UAUUJCUJ 2660 1804 UGAAGAAA CUGAUGAG X CGAA AUAUUAUU 2220 AAUAAUAUA UUtJCUUCA 2661 1806 CAUGAAGA CUGAUGAG X CGAA AUAUAIJUA 2221 UAAIJAUAUU UCUUCAUJG 2662 1807 UCAUGAAG CUGAUGAG X CGAA AAUAUAUU 2222 AAUAUAUUU CUUCAUGA 2663 1808 UUCAUGAA CUGAUGAG X CGAA AAAUAUAU 2223 AUAUAUUUC UUCAUGAA 2664 1810 UCUUCAUG CUGAUGAG X CGAA AGAAAUAU 2224 AUAUUUCUU CAUGAAGA 2665 1811 GUCUUCAU CUGAUGAG X CGAA A.AGAAAUA 2225 UAUUUCUUC AUGAAGAC 2666 1822 UUUACUGU CUGAUGAG X CGAA AGGUCUUC 2226 GAAGACCUC ACAGUAAA 2667 1828 CCUAUUUU CUGAUGAG X CGAA ACUGUGAG 2227 ICUCACAGUA AAAAUAGG 2668 1834 AAAUCACC CUGAUGAG X CGAA AUEJUUUAC 2228 GUAAAAAUA GGUGAUUU 2669 1841 UAGACCAA CUGAUGAG X CGAA AUCACCUA 2229 UAGGUGAUU UUGGUCUA 2670 1842 CUAGACCA CUGAUGAG X CGAA AAUCACCU 2230 AGGUGAUUU UGGUCUAG 2671 1843 GCUAGACC CUGAUGAG X CGAA AAAUCACC 2231 GGUGAUUUU GGUCUAGC 2672 1847 UGUAGCUA CUGAUGAG X CGAA ACCAAAAU 2232 AUUUJGGTJC UAGCUACA 2673 1849 ACUGUAGC CUGAUGAG X CGAA AGACCAAA 2233 UUUGGUCUA GCUACAGU 2674 1853 UUUCACUG CUGAUGAG X CGAA AGCUAGAC 2234 GUCUAGCUA CAGUGAAA 2675 1863 UCCAUCGA CUGAUGAG X CGAA AUUUCACU 2235 AGUGAAAUC UCGAUGGA 2676 1865 ACUCCAUC CUGAUGAG X CGAA AGAUEJUCA 2236 UGAALAUCUC GAUGGAGU 2677 1878 ACUGAUGG CUGAUGAG X CGAA ACCCACUC 2237 GAGUGGGUC CCAUCAGU 2678 1883 UUCAAACU CUGAUGAG X CGAA AUGGGACC 2238 GGUCCCAUC AGUUUGAAL 2679 1887 ACUGUUCA CUGAUGAG X CGAA ACUGAUGG 2239 CCAUCAGUU UGAACAGU 2680 1888 AACUGUJC CUGAUGAG X CGAA AACUGAUG 2240 CAUCAGUJU GAACAGUU .26 81 1896 AUCCAGAC CUGAUJGAG X CGAA ACUGUtJCA 2241 UGAACAGUU GUCUGGAU 2682 1899 UGGAUCCA CUGAUGAG X CGAA ACAACUGU 2242 ACAGUIJGUC UGGAUCCA 2683 1905 ACAAAAUG CUGAUGAG X CGAA AUCCAGAC 2243 GUCUGGAUC CAUUUUGU 2684 1909 AUCCACAA CUGAUGAG X CGAA AUGGAUCC 2244 GGAUCCAUJ UUGUGGAU- 2685 1910 CAUCCACA CUGAUGAG X CGAA AAUGGAUC 2245 GAUCCAUJU UGUGGAUG 2686 1911 CCAUCCAC CUGAUGAG X CGAA AAAUGGAU 2246 AUCCAUUUJ GUGGAUGG 2687 1930 AUEJCUGAU CUGAUGAG X CGAA ACUUCUGG 2247 CCAGAAGUC AUCAGAAU 2688 1933 UGCAUUCU CUGAUGAG X CGAA AUGACUJC 2248 GAAGUCAUC AGAAUGCA 2689 1946 UGGAUUUJ CUGAUGAG X CGAA AUCUUGCA 2249 UGCAAGAUA AAAAUCCA 2690 1952 GCUGUAUG CUGAUGAG X CGAA AUUTJUUAU 2250 AUAAAAAUC CAUACAGC 2691 1956 GAAAGCUG CUGAUGAG X CGAA AUGGAUTUU 2251 AAAUCCAUA CAGCULTJC 2692 1962 CUGACUGA CUGAUGAG X CGAA AGCUGUAU 2252 AUACAGCUU UCAGUCAG 2693 1963 UCUGACUG CUGAUGAG X CGAA AAGCUGUA 2253 UACAGCUUU CAGUCAGA 2694 1964 AUCUGACU CUGAUGAG X CGAA AAAGCUGU 2254 ACAGCUUJC AGUCAGAU 2695 1968 AUACAUCU CUGAUGAG X CGAA ACUGAAAG 2255 CUUUCAGUC AGAUGUAU 2696 1975 AAUGCAUA CUGAUGAG X CGAA ACAUCUGA 2256 UCAGAUGUA UAUGCAUU 2697 1977 CAAAUGCA CUGAUGAG X CGAA AUACAUCU 2257 AGAUGUAUA UGCAtJUUG 2698 1983 CAAUCCCA CUGAUGAG X CGAA AUGCAUAU 2258 AUAUGCAUU UGGGAUUG 2699 1984 ACAAUCCC CUGAUGAG X CGAA AAUGCAUA 2259 UAUGCAUUU GGGAUUGU 2700 1990 UACAGAAC CUGAUGAG X CGAA AUCCCAAA,2260 UVUGGGAUU GUEJCUGUA 2701 1993 JUCAUACAG CUGAUGAG X CGAA ACAAUCCCI 2261 GGGAUUJGUU CUGUAUGA 2702 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCT/US98/09249 168 -nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

1994 UUCAUACA CUGAUGAG X CGAA AACAAUCC 2262 GGAIJUGUUC UGUAUGAA 2703 1998 UCAAUUCA CUGAUGAG X CGAA ACAGAACA 2263 UGUUCUGUA UGAAUUGA 2704 2004 CAGUCAUC CUGAUGAG X CGAA AUUCAUAC 2264 GUAUGAAUU GAUGACUG 2705 2019 AAUAAGGU CUGAUGAG X CGAA ACUGUCCA 2265 JUGGACAGUU ACCUUAUU 2706 2020 GAAUAAGG CUGAUGAG X CGAA AACUGUCC 2266 GGACAGUUA CCUUAUJC 2707 2024 GUUIJGAAU CUGAUGAG X CGAA AGGUAACU 2267 AGUUACCUU AUUCAAAC 2708 2025 UGUUtJGAA CUGAUGAG X CGAA AAGGUAAC 2268 GUUACCUUA UUCAAACA 2709 2027 GAUGEJUUG CUGAUGAG X CGAA AUAAGGtJA 2269 UACCUUAUJ CAAACAUC 2710 2028 UGAUGUUU CUGAUGAG X CGAA AAUAAGGU 2270 IACCUJAUUC AAACAUCA 2711 2035 CUGUUGUU CUGAUGAG X CGAA AUGUUTUGA 2271 UCAAACAUC AACAACAG 2712 2053 AUAAAAAU CUGAUGAG X CGAA AUCUGGUC 2272 GACCAGAUA AUUUUUAU 2713 2056 ACCAUAAA CUGAUGAG X CGAA AUEJAUCUG 2273 CAGAUAAUU UUtJAUGGU 2714 2057 CACCAUAA CUGAUGAG X CGAA AAUUAUCU 2274 AGAUAAUUU UUAUGGUG 2715 2058 CCACCAUA CUGAUGAG X CGAA AAAUEJAUC 2275 GAUAAUUUU UAUGGUGG 2716 2059 CCCACCAU CUGAUGAG X CGAA AAAAtJUAU 2276 AUAAUUUUU AUGGUGGG 2717 2060 UCCCACCA CUGAUGAG X CGAA AAAAAUUA 2277 UAAUUUTItJA UGGUGGGA 2718 2076 GAGACAGG CUGAUGAG X CGAA AUCCUCGU 2278 ACGAGGAUA CCUGUCUC 2719 2082 GAUCUGGA CUGAUGAG X CGAA ACAGGUAU 2279 AUACCUGUC UCCAGAUC 2720 2084 GAGAUCUG CUGAUGAG X CGAA AGACAGGU 2280 ACCUGUCUC CAGAUCUC 2721 2090 CUUACUGA CUGAUGAG X CGAA AUCUGGAG 2281 CUCCAGAUC UCAGUAAG 2722 2092 ACCUUACU CUGAUGAG X CGAA AGAUCUGG 2282 CCAGAUCUC AGUAAGGU 2723 2096 CCGUACCU CUGAUGAG X CGAA ACUGAGAU 2283 AUCUCAGUA AGGUACGG 2724 2101 UUACUCCG CUGAUGAG X CGAA ACCUUACU 2284 AGUAAGGUA CGGAGUAA 2725 2108 UGGACAGU CUGAUGAG X CGAA ACUCCGUA 2285 UACGGAGUA ACUGUCCA 2726 2114 GGCUUUJEG CUGAUGAG X CGAA ACAGUUAC 2286 GUAACUGUC CAAAAGCC 2727 2133 CUGCCAUJ CUGAUGAG X CGAA AUCUCUUC 2287 GAAGAGAUJU AAUGGCAG 2728 2134 UCUGCCAU CUGAUGAG X CGAA AAUCUCUJ 2288 AAGAGAUUA AUGGCAGA 2729 2149 UUCUJUTJ CUGAUGAG X CGAA AGGCACUC 2289 GAGUGCCUC AAAAAGAA 2730 2176 UGGGGAAA CUGAUGAG X CGAA AGUGGUCU 2290 AGACCACUC UUUCCCCA 2731 2178 UUtJGGGGA CUGAUGAG X CGAA AGAGUGGU 2291 ACCACUCUU UCCCCAAA 2732 2179 AUUUGGGG CUGAUGAG X CGAA AAGAGUGG 2292 CCACUCUUU CCCCAAAU 2733 2180 AAL=tGGG CUGAUGAG X CGAA AAAGAGUG 2293 CACUCUUJC CCCAAAUJ 2734 2188 GAGGCGAG CUGAUGAG X CGAA AUUUGGGG 2294 CCCCAAAUJ CUCGCCUC 2735 2189 AGAGGCGA CUGAUGAG X CGAA AAUUEJGGG 2295 CCCAAAUUC UCGCCUCU 2736 2191 AUAGAGGC CUGAUGAG X CGAA AGAAUUUG 2296 CAAAUUCUC GCCUCUAU 2737 2196 GCUCAAUA CUGAUGAG X CGAA AGGCGAGA 2297 UCUCGCCUC UAUUGAGC 2738 2198 CAGCUCAA CUGAUGAG X CGAA AGAGGCGA 2298 UCGCCUCUA UtJGAGCUG 2739 2200 AGCAGCUC CUGAUGAG X CGAA AUAGAGGC 2299 GCCUCUAUJU GAGCUGCU 2740 2217 UUGGCAAUJCUGAUGAG X CGAA AGCGGGCC 2300 GGCCCGCUC AUUGCCAA 2741 2220 UUUJEGGC CUGAUGAG X CGAA AUGAGCGG 2301 CCGCUCAUU GCCAAAAA 2742 2230 CUGCGGUG CUGAUGAG X CGAA AUUUtJUGG 2302 CCAAAAAUU CACCGCAG 2743 2231 ACUGCGGU CUGAUGAG X CGAA AAUtIUUUG 2303 CAAAAAUUC ACCGCAGU 2744 2244 AGGGUUCJ CUGAUGAG X CGAA AUGCACUG 2304 CAGUGCAUC AGAACCCU 2745 2253 GAUUCAAG CUGAUGAG X CGAA AGGGUJCU 2305 AGAACCCUC CUUGAAUC 2746 2256 CCCGAUUC CUGAUGAG X CGAA AGGAGGGU 2306 ACCCUCCUU GAAUCGGG 2747 2261 ACCAGCCC CUGAUGAG X CGAA AUUCAAGG 2307 CCUUGAAUC GGGCUGGU 2748 2270 UGLUUGGA CUGAUGAG X CGAA ACCAGCCC 2308 GGGCUGGUU UCCAAACA 2749 2271 CUGUUUGG CUGAUGAG X CGAA AACCAGCC 2309 GGCUGGUUU CCAAACAG 2750 2272 UCUGUUtG CUGAUGAG X CGAA AAACCAGCI 2310 1GCUGGUUUC CAAACAGA, 2751 L2285 JUAGACUAA CUGAUGAG X CGAA AUCCUCUGI 2311 ICAGAGGAUU UUAGUCUA12752 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 169nt. SEQ SEQ Position Ribozyme ID. No. Substrate ID. No.

2286 AUAGACUA CUGAUGAG X CGAA AAUCCUCU 2312 AGAGGAUUU UAGUCUAU 2753 2287 UAUAGACU CUGAUGAG X CGAA AAAUCCUC 2313 GAGGAUUUU AGUCUAUA 2754 2288 AUAUAGAC CUGAUGAG X CGAA AAAAUCCU 2314 AGGAUUUUA GUCUAUAU 2755 2291 AGCAUAUA CUGAUGAG X CGAA ACUAAAAU 2315 AUtUUAGUC UAUAUGCU 2756 2293 CAAGCAUA CUGALIGAG X CGAA AGACUAAA 2316 UUUAGUCUA UAUGCUJG 2757 2295 CACAAGCA CUGAUGAG X CGAA AUAGACUA 2317 tJAGUCUAUA UGCUUGUG 2758 2300 AGAAGCAC CUGAUGAG X CGAA AGCAUAUA 2318 tJAUAUGCUU GUGCUUCU 2759 2306 UUUEJGGAG CUGAUGAG X CGAA AGCACAAG 2319 CUUGUGCJU CUCCAAAA 2760 2307 UUUUEJGGA CUGAUGAG X CGAA AAGCACAA 2320 UUGUGCUUC UCCAAAAA 2761 2309 UGUUUUUG CUGAUGAG X CGAA AGAAGCAC 2321 GUGCUUCUC CAAAAACA 2762 2323 CCUGCCUG CUGAUGAG X CGAA AUGGGUGU 2322 ACACCCAUC CAGGCAGG 2763 2337 ACGCACCA CUGAUGAG X CGAA AUCCCCCU 2323 AGGGGGAUA UGGUGCGU 2764 2346 GGACAGGA CUGAUGAG X CGAA ACGCACCA 2324 UGGUGCGUJU UCCUGUCC 2765 2347 UGGACAGG CUGAUGAG X CGAA AACGCACC 2325 GGUGCGUUJ CCUGUCCA 2766 2348 GUGGACAG CUGAUGAG X CGAA AAACGCAC 2326 GUGCGUUUC CUGUCCAC 2767 2353 U!JUCAGUG CUGAUGAG X CGAA ACAGGAAA 2327 UUUCCUGUC CACUGAAA 2768 2379 CUCUCCUG CUGAUGAG X CGAA ACUCUCUC 2328 GAGAGAGUU CAGGAGAG 2769 2380 ACUCUCCU CUGAUGAG X CGAA AACUCUCU 2329 AGAGAGU3C AGGAGAGU 2770 2389 UUUGUUGC CUGAUGAG X CGAA ACUCUCCU 2330 AGGAGAGUA GCAACAAA 2771 2406 UGUUCAEIJ CUGAUGAG X CGAA AUUUUCCU 2331 AGGAAAAUA AAUGAACA 2772 2416 AGCAAACA CUGAUGAG X CGAA AUGUUtCAU 2332 AUGAACAUA UGUUtJGCU 2773 2420 UAUAAGCA CUGAUGAG X CGAA ACAUAUGU 2333 ACAUAUGUJ UGCUUAUA 2774 2421 AUAUAAGC CUGAUGAG X CGAA AACAUAUG 2334 CAUAUGUUt GCUUAUAU 2775 2425 UAACAUAU CUGAUGAG X CGAA AGCAAACA 2335 UGUUtJGCUU AUAUGUUA 2776 2426 UTJAACAUA CUGAUGAG X CGAA AAGCAAAC 2336 GUUtJGCUUA UAUGUUAA 2777 2428 AtJEUAACA CUGAUGAG X CGAA AUAAGCAA 2337 UUGCUUAUA UGUUAAAU 2778 2432 UUCAAUUU CUGAUGAG X CGAA ACAUAUAA 2338 UUAUAUGUU AAAUUGAA 2779 2433 AUUCAAUU CUGAUGAG X CGAA AACAUAUA 2339 UAUAUGUJA AAUUGAAU 2780 2437 UUUJUAUUC CUGAUGAG X CGAA ALUUtAACA 2340 UGUtJAAAUU GAAUAAAA 2781 2442 GAGUAUUU CUGAUGAG X CGAA AUUCAAUU 2341 AAUtJGAAUA AAAUACUC 2782 2447 AAAGAGAG CUGAUGAG X CGAA AUUUUAUU 2342 AAIJAAAAUA CUCUCUU 2783 2450 AAAAAAGA CUGAUGAG X CGAA AGUAUUUU 2343 AAAAUACUC UCUUUUU 2784 2452 AAAAAAAA CUGAUGAG X CGAA AGAGUAU-U 2344 AAUACUCUC UUUUUUUJ 2785 2454 UAAAAAAA CUGAUGAG X CGAA AGAGAGUA 2345 UACUCUCJU UtUUUUUA 2786 2455 LJUAAAAAA CUGAUGAG X CGAA A.AGAGAGU 2346 ACUCUCUUU UUUUUUAA 2787 2456 CUUAAAAA CUGAUGAG X CGAA AAAGAGAG 2347 CUCUCUJUE UEJUUAAG 2788 2457 CCUUAAAA CUGAUGAG X CGAA AAAAGAGA 2348 UCUCUUUUU UUJAAGG 2789 2458 ACCUUAAA CUGAUGAG X CGAA AAAAAGAG 2349 CUCUUUUUU UEJUAAGGU 2790 2459 CACCUAA CUGAUGAG X CGAA AAAAAAGA 2350 UCUUUUUU UUAAGGUG 2791 2460 CCACCUJA CUGAUGAG X CGAA AAAAAAG 2351 CUUTUjUUUU UAAGGUGG 2792 2461 UCCACCUU CUGAUGAG X CGAA AA AAAAA 2352 tJTUUTJTJUUU AAGGUGGA 2793 2462 UtJCCACCU CUGAUGAG X CGAA AAAAAAAA1 2353 UTJEU~UtJUA AGGUGGAA 2794 Where represents stem HI region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20: 3252). The length of stem II may be 3 2 base-pairs.

SUBSTITUTE SHEET (RULE 26) 170 Table XVII: Human B3-raf Hairpin Ribozyme and Target Sequence nt. Position Rihozyrne Sequence SEQ ID. No. Target Sequence SEQ ID. No.

9 GGAGGG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 2795 UCCCG GCC CCCUCC 2846 CUGUCG AGAA GGGA ACCAGAGAAACA X GUACAUUACCUGGUA 2796 UCCCC GCC CGACAG 2847 31 CCGAGC AGAA GCUG ACCAGAGAAACA X GtACAUUACCUGGUA 2797 CAGCG GCC GCUCGG 2848 34 GGCCCG AGAA GCCG ACCAGAGAAACA X GUACAUUACCUGGUA 2798 CGGCC GCU CGGGCC 2849 46 ACCGAG AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 2799 CCCCG GCU CUCGGU 2850 114 CCGUUG AGAA GAGC ACCAGAGAAACA X GUACAUUACCUGGUA 2800 GCUCU GUU CAACGG 2851 149 CGGGCC AGAA GGCG ACCAGAGAAACA X GUACAUUACCUGGUA 2801 CGCCG GCC GGCCCG 2852 153 GCCGCG AGAA GGCC ACCAGAGAAACA X GUACAUUACCUGGUA 2802 GGCCG GCC CGCGGC 2853 160 CGAAGA AGAA GCGG ACCAGAGAAACA X GUACAUUACCUGGUA 2803 CCGCG GCC UCUUCG 2854 169 GUCCGC AGAA GAAG ACCAGAGAAACA X GLJACAUUACCUGGUA 2804 CUUCG GCU GCGGAC 2855 175 GGCAGG AGAA GCAG ACCAGAGAAACA X GUACAUUACCUGGUA 2805 CUGCG GAC CCUGCC 2856 379 AGAAAA AGAA GtJUC ACCAGAGAAACA X GUACAtJUACCUGGUA 2806 GAACU GAU UUUUCU 2857 388 GCUAGA AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 2807 UUUCU GUll UCUAGC 2858 466 UUGAAA AGAA GAAA ACCAGAGAAACA X GUACAUUACCUGGUA 2808 UUUCA GUU UUUCAA 2859 484 UGCCAC AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGtJA 2809 CCACA GAU GUGGCA 2860 540 UUGUUG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 2810 UUCCU GCC CAACAA 2861 586 GUCUCG AGAA GUAA ACCAGAGAAACA X GUACAUUACCUGGUA 2811 UUACA GUC CGAGAC 2862 596 UCUUUA AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 2812 AGACA GUC UAAAGA 2863_ 612 CUCAUC AGAA GUGC ACCAGAGAAACA X GUACAUUACCUGGUA 2813 GCACU GAU GAUGAG 2864 646 UCUGUA AGAA GCAC ACCAGAGAAACA X GUACAUUACCUGGUA 2814 GUGCU GUU UACAGA 2865 819 UGGAAA AGAA GCUU ACCAGAGAAACA X GUACAUUACCUGGUA 2815 AAGCU GCU UUUCCA 2866 836 UUUJGAC AGAA GAAA ACCAGAGAAACA X GUACAUTJACCUGGUA 2816 UUUCC GCU GUCAAA 2867 891 ACACAC AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 2817 CCACU GAU GUGUGU 2868 924 GAGACA AGAA GCAA ACCAGAGAAACA X GUACAUUACCUGGUA 2818 UUGCU GUl UGUCUC 2869 988 UGUUAG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 2819 AGACU GCC CUAACA 2870 1021 GUCCGA AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 2820 CACCC GCC UCGGAC 2871 1027 AAUAGA AGAA GAGG ACCAGAGAAACA X GUACAUUACCUGGUA 2821 CCUCG GAC UCUAUU 2872 1055 GAGACG AGAA GGUG ACCAGAGAAACA X GUACAUUACCUGGUA 2822 CACCA GUC CGUCUC 2873 1059 GAAGGA AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 2823 AGUCC GUC UCCUUC 2874 1089 CGGAAG AGAA GUGG ACCAGAGAAACA X GUACAUUACCUGGUA 2824 CCACA GCC CUUCCG 2875 1097 CUGCUG AGAA GAAG ACCAGAGAAACA X GUACAUUACCUGGUA 2825 CUUCC GAC CAGCAG 2876 1105 AUCUUC AGAA GCUG ACCAGAGAAACA X GUACAUUACCUGGUA 2826 CAGCA GAUGGA 287 1142 AUGAGG AGAA GUCU ACCAGAGAAACA X GUACAUUACCUGGUA 2827 AGACC GAU CCUCAU 2878 1153 AUUGGG AGAA GAUG ACCAGAGAAACA X GUACAUUACCUGGUA 2828 CAUCA GCU CCCAAU 2879 nt. Position Ribozyme Sequence SEQ ID. No. Target Sequence SEQ ID. No.

1267 UAAUGA AGAA GGGG ACCAGAGAAACA X GUACAUUACCUGGUA 2829 CCCCU GCC UCAUUA 2880 1417 CUGCCC AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 2830 UUCCU GAU GGGCAG 2881 1425 ACUGUA AGAA GCCC ACCAGAGAAACA X GUACAtJUACCUGGUA 2831 GGGCA GAU UACAGU 2882 1468 CUUGUA AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 2832 GAACA GUC UACAAG 2883 1664 GAUACA AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 2833 CUCCA GCU UGUAUC 2884 1734 UGUGCA AGAA GUCG ACCAGAGAAACA X GUACAUUACCUGGUA 2834 CGACA GAC UGCACA 2885 1884 UGUUCA AGAA GAUG ACCAGAGAAACA X GUACAUJIACCUGGJA 2835 CAUCA GUU UGAACA 2886 1893 CCAGAC AGAA GUUC ACCAGAGAAACA X GUACAUUACCUGGUA 2836 GAACA GUU GUCUGG 2887 1958 ACUGAA AGAA GUAU ACCAGAGAAACA X GUACAUUACCUGGUA 2837 AUACA GCU UUCAGU 2888 1969 AUAUAC AGAA GACU ACCAGAGAAACA X GUACAUUACCUGGUA 2838 AGUCA GAU GUAUAU 2889 1995 AAUUCA AGAA GAAC ACCAGAGAAACA X GUACAUUACCUGGUA 2839 GUUCU GUA UGAAUU 2890 2079 UCUGGA AGAA GGUA ACCAGAGAAACA X GUACAtJUACCUGGUA 2840 UACCU GUC UCCAGA 2891 2086 ACUGAG AGAA GGAG ACCAGAGAAACA X GUACAUUACCUGGUA 2841 CUCCA GAU CUCAGU 2892 2111 CUUUUG AGAA GUUA ACCAGAGAAACA X GUACAtJUACCUGGUA 2842 UAACU GUC CAAAAG 2893 2205 CGGGCC AGAA GCUC ACCAGAGAAACA X GUACAUUACCUGGUA 2843 GAGCU GCU GGCCCG 2894 2213 GCAAUG AGAA GGCC ACCAGAGAAACA X GUACAUUACCUGGUA 2844 GGCCC GCU CAUUGC 2895 2350 UCAGUG AGAA GGAA ACCAGAGAAACA X GUACAUUACCUGGUA 2845 UUCCU GUC CACUGA 2896 Where represents stem IV region of a Hairpin ribozyme. The length of stem IV may be 2 base-pairs.

WO 98/50530 PCT/US98/09249 -172- Table XVII. Hammerhead (HH) Ribozyme target with c-raf and A-raf sequence homology between nt. Position Target Seq I.D. No.

452 AGGAGCU C AUUGUCG 2897 527 UGGCGUJ C UGUGACU 2898 583 UGUGGCU A CAAGUUC 2899 668 ACAGUGU C CAGGALTU 2900 857 AUAUGGU C AGCACCA 2901 1096 UCAGGCU A UJACJGG 2902 1098 AGGCUAU U ACUGGGA 2903 1246 CAGGCU U CAAGAAU 2904 1247 AGGCULTU C AAGAAUG 2905 1309 AUGGGCU U CAUGACC 2906 1327 CCGGGA U UGCCAUC 2907 1357 GAGGGCU C CAGCCUC 2908 1412 UCCAGCU C AUCGACG 2909 1469 AGAACAU C AUCCACC 2910 1628 AGGUGAU C CGUAUGC 2911 1658 ACAGCUU C CAGUCAG 2912 1663 UUCCAGU C AGACGUC 2913 1748 ACCAGAU U AUCUUUA 2914 1749 CCAGAULT A UCUUUAU 2915 1751 AGAUUAU C UUUAUGG 2916 1753 AUUAUCU U UAUGGUG 2917 1754 UUAUCULT U AUGGUGG 2918 1871 GGCCCCU C UUCCCCC 2919 1874 CCCUCUU C CCCCAGA 2920 1951 CCCUCCU U GCACCGC 2921 2046 CCAAUCU C AGCCCUC 2922 2127 CCCCAUU C CCCACCC 2923 2174 AGUUCUU C UGGAAJU 2924 2251 UGGGGAU A CUUCUAA 2925 2400 GUCCCUU U UGUGTCU 2926 2432 CUCCUCU C UUUCtUC 2927 SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCTIUS98/09249 173- Table XIX. Hammerhead Ribozyme Target with and B-raf sequence homology between c-raf nt. Position Target Sequence Seq. I. D. No.

17 GCCCCCU C CCCGCCC 2928 405 UCUGCAU C AAUGGAU 2929 426 ACAUCUU C UUCCUCU 2930 479 UCAAAAU C CCACAGA 2931 702 GAUAUUU C CUGGCUU 2932 754 UUCCACU U ACAACAC 2933 861 UAUAAAU U UCACCAG 2934 931 UGUUUGU C UCCAAGU 2935 1034 GGACUCU A UUGGGCC 2936 1259 GUCUGCU A CCCCCCC 2937 1344 AAGUCAU C UUCAUCC 2938 1603 UCCUACU C UUCAUGG 2939 1662 GAGGGCU C CAGCUUG 2940 1802 UAAUAAU A UAUUUCU 2941 1804 AUAAUAU A UtUUCUUC 2942 1806 AAUAUAU U UCUUCAU 2943 1807 AUAUAUU U CUUCAUG 2944 1808 UAUAUUU C UUCAUGA 2945 1810 UAUUUCU U CAUGAAG 2946 1834 UAAAAAU A GGUGAUU 2947 1842 GGUGAUU U UGGUCUA 2948 1847 UUUUGGU C UAGCUAC 2949 1956 AAUCCAU A CAGCUIU 2950 2035 CAAACAU C AACAACA 2951 2059 UAAUUUU U AUGGUGG 2952 2090 UCCAGAU C UCAGUAA 2953 2092 CAGAUCU C AGUAAGG 2954 2200 CCUCUAU U GAGCUGC 2955 2256 CCCUCCU U GAAUCGG 2956 SUBSTITUTE SHEET (RULE 26) WO 98/50530 WO 9850530PCTIUS98/09249 174- Table XX.

Experimental Group RPI.4610 RPI.461 I RPI.4733 RPI .473 4 Saline Ribozyme Activity/T'arget Active/fit-] Inactive/fit-I Active/flk-J Inactive/fik-J

NA

Dose (mg/kg/day) 1, 3, 10, 30, 100 1, 3, 10, 30, 100 1, 3,10, 30, 100 1, 3, 10, 30, 100 12 R1/day Sample Size per dose SUBSTITE SHEET (RULE 26)

Claims (2)

175- Claims 1. A method for identification of a nucleic acid molecule capable of modulating a process in a biological system comprising the steps of: a) introducing a random library of a nucleic acid catalyst with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence, into said biological system under conditions suitable for modulating said process; and b) determining the nucleotide sequence of at least a portion of the substrate binding domain of said nucleic acid catalyst from a said biological system in which the process has been modulated. 2. A method for identifying one or more nucleic acid molecules involved in a process in a biological system comprising the steps of: a) providing a library of a nucleic acid catalyst, with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises a random sequence, to said biological system under conditions suitable for said process to be altered; b) identifying any said nucleic acid catalyst present in said biological system where said process has been altered by said any said nucleic acid catalyst; and c) determining the nucleotide sequence of at least a portion of the binding arm of said any said nucleic acid catalyst to allow said identification of said nucleic acid molecule involved in said process in said biological system. 3. A method for identification of a nucleic acid catalyst capable of modulating a process in a biological system comprising the steps of: a) introducing a random library of a nucleic acid catalyst with a substrate binding domain and a catalytic domain, wherein said substrate binding domain comprises SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -176- a random sequence, into said biological system under conditions suitable for modulating said process; and b) identifying said nucleic acid catalyst from said biological system in which the process has been modulated. 4. The method of any of claims 1-3, wherein said biological system is a bacterial cell. The method of any of claims 1-3, wherein said biological system is of plant origin. 6. The method of any of claims 1-3, wherein said biological system is of mammalian origin. 7. The method of any of claims 1-3, wherein said biological system is of yeast origin. 8. The method of any of claims 1-3, wherein said biological system is Drosophila. 9. The method of any of claims 1-3, wherein said nucleic acid catalyst is in a hammerhead motif. The method of any of claims 1-3, wherein said nucleic acid catalyst is in a hairpin motif. 11. The method of any of claims 1-3, wherein said nucleic acid catalyst is in a hepatitis delta virus ribozyme motif. 12. The method of any of claims 1-3, wherein said nucleic acid catalyst is in group I intron, group II intron, VS ribozyme or RNase P ribozyme motif. 13. The method of any of claims 1-3, wherein said process is selected from the group consisting of growth, proliferation, apoptosis, morphology, angiogenesis, differentiation, migration, viral multiplication, drug resistance, signal SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249 -177- transduction, cell cycle regulation, temperature sensitivity and chemical sensitivity. 14. The method of any of claims 1-3, wherein said random library of nucleic acid catalysts is encoded by an expression vector in a manner which allows expression of said nucleic acid catalysts. The method of claim 14, wherein said expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said nucleic acid catalyst; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst. 16. The method of claim 14, wherein said expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said nucleic acid catalyst, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst. 17. The method of claim 14, wherein said expression vector comprises: a) a transcription initiation region; SUBSTITUTE SHEET (RULE 26) WO 98/50530 PCT/US98/09249

178- b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid catalyst; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst. 18. The method of claim 14, wherein said expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid catalyst, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid catalyst. 19. The method of claim 14, wherein said expression vector is derived from a retrovirus. The method of claim 14, wherein said expression vector is derived from an adenovirus. 21. The method of claim 14, wherein said expression vector is derived from an adeno-associated virus. SUBSTITUTE SHEET (RULE 26) 179 The claims defining the invention are as follows: 1. A nucleic acid molecule with an endonuclease activity having the formula III: L M-3' 7 -A-G-C 4 -U 3 wherein, N is independently a nucleotide or a non-nucleotide linker, which may be s same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single- stranded and/or double-stranded region; represents a chemical linkage; and A, C, U and G represent adenosine, cytidine, uridine and guanosine nucleotides, respectively. 2. A nucleic acid molecule with catalytic activity having the formula IV: 3 5 A Z3 A Z4 G A A G GZ 7 SC A C G (N)o (N)n L wherein, N is independently a nucleotide or a non-nucleotide linker, which may be 15 same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single- stranded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is 2'-C-allyl uridine; Z7 is 6-methyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively. 3. A nucleic acid molecule with catalytic activity having the formula V: [R:\LIBz]02943.doc:aak 180 3 m PQ 5 A Z3 A Z4 G A A G GZ 7 A C 0 G (N)o (N)n \L/ wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single- stranded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is o0 2'-methylthiomethyl cytidine; Z7 is 6-methyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively. 4. A nucleic acid molecule with catalytic activity having the formula VI: 3' Q A Z3 A Z4 A A Z7 G G A C 0 G (N)o (N)n \L wherein, N is independently a nucleotide or a non-nucleotide linker, which may be same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but AU when present, is a nucleotide and/or a non-nucleotide linker, which may be a single- nded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is [R:\LIBz]02943.doc:aak 181 2'-methylthiomethyl cytidine; Z7 is 2'-C-allyl uridine; represents a chemical linkage; and A, and G represent adenosine and guanosine nucleotides, respectively. A nucleic acid molecule with catalytic activity having the formula VII: A Z3 A z4 G A A z7 G G A C G (N)o (N)n \L wherein, N is independently a nucleotide or a non-nucleotide linker, which may be *same or different; M and Q are independently oligonucleotides of length sufficient to stably interact with a target nucleic acid molecule; o and n are integers greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; L is a linker which may be present or absent, but 10 when present, is a nucleotide and/or a non-nucleotide linker, which may be a single- stranded and/or double-stranded region; Z3 is 2'-methylthiomethyl uridine; Z4 is 2'-methylthiomethyl cytidine; Z7 is pyridine-4-one; and represents a chemical 9 linkage; and A, and G represent adenosine and guanosine nucleotides, respectively. 6. The nucleic acid molecule of any one of claims 1-5, wherein said (N)o and 15 (N)n are nucleotides and said o and n are integers greater than or equal to 3. 7. The nucleic acid molecule of any one of claims 1-5, wherein said L is a nucleotide linker. 8. The nucleic acid molecule of any one of claims 1-7, wherein said nucleic acid cleaves a separate nucleic acid molecule. 9. The nucleic acid molecule of claim 8, wherein said separate nucleic acid molecule is RNA. The nucleic acid molecule of claim 8, wherein said nucleic acid comprises between 12 and 100 bases complementary to said separate nucleic acid molecule. 11. The nucleic acid molecule of claim 8, wherein said nucleic acid comprises between 14 and 24 bases complementary to said separate nucleic acid molecule. 12. A nucleic acid molecule with an endonuclease activity, substantially as 6 ereinbefore described with reference to any one of the examples. [R:\LIBzj02943.doc:aak 182 13. A cell including the nucleic acid molecule of any one of claims 1-12. 14. The cell of claim 13, wherein said cell is a mammalian cell. The cell of claim 14, wherein said cell is a human cell. 16. An expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of any one of claims 1-12, in a manner which allows expression of that nucleic acid molecule. 17. A cell including the expression vector of claim 16. 18. The cell of claim 17, wherein said cell is a mammalian cell. 19. The cell of claim 18, wherein said cell is a human cell. 20. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-12. 21. A method for modulating expression ofa gene in a plant cell'by administering 44**g to said cell the nucleic acid molecule of any one of claims 1-12. :I 22. A method for modulating expression of gene in a mammalian cell by administering to said cell the nucleic acid molecule of any one of claims 1-12. 23. Use of a nucleic acid of any one of claims 1-12 for the manufacture of a medicament for modulating expression of a gene in a mammalian cell. 24. A method of cleaving a separate nucleic acid comprising contacting the *o nucleic acid molecule of any one of claims 1-12 with said separate nucleic acid molecule 20o under conditions suitable for the cleavage of said separate nucleic acid molecule. 4* *4 25. The method of claim 24, wherein said cleavage is carried out in the presence So. of a divalent cation. 2+ .4 26. The method of claim 25, wherein said divalent cation is Mg 2 27. The nucleic acid molecule of any one of claims 1-12, wherein said nucleic acid is chemically synthesized. 28. The expression vector of claim 16, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. 29. The expression vector of claim 16, wherein said vector comprises: a) a transcription initiation region; I b) a transcription termination region; [R:\LIBz]02943.doc:aak 183 c) an open reading frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The expression vector of claim 16, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and ,wherein said gene is operably linked to said initiation region, said intron and said 9termination region, in a manner which allows expression and/or delivery of said nucleic e* o acid molecule. is 31. The expression vector of claim 16, wherein said vector comprises: a) a transcription initiation region; S°b) a transcription termination region; c) an intron; d) an open reading frame; 20 e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. 32. A method for identifying variants of a nucleic acid catalyst comprising the steps of: a) selecting at least three positions within said nucleic acid catalyst to be varied with a predetermined group of different nucleotides; b) synthesizing a first class of different pools of said nucleic acid catalyst, wherein the number of pools synthesized is equal to the number of nucleotides in the predetermined group of different nucleotides, wherein at least one of the positions to be varied in each pool comprises a defined nucleotide selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random /U mixture of nucleotides selected from the predetermined group of different nucleotides; [R:\LIBz]02943.doc:aak 184 c) testing the different pools of said nucleic acid catalyst under conditions suitable for said pools to show a desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant in subsequent steps; d) synthesizing a second class of different pools of nucleic acid catalyst, wherein at least one of the positions to be varied in each of the second class of different pools comprises a defined nucleotide selected from the predetermined group of different nucleotides and the remaining positions to be varied comprise a random mixture of nucleotides selected from the predetermined group of different nucleotides; e) testing the second class of different pools of said nucleic acid catalyst under conditions suitable for showing desired attribute and identifying the pool with said desired attribute and wherein the position with the defined nucleotide in the pool with the desired attribute is made constant in subsequent steps; and f) repeating the process similar to steps d and e until every position S. 1is selected in said nucleic acid catalyst to be varied is made constant. Dated 8 March, 2002 Ribozyme Pharmaceuticals, Incorporated 0 S •Patent Attorneys for the Applicant/Nominated Person •SPRUSON FERGUSON 2 **O [R:\LIBz]02943.doc:aak