WO1995006764A2

WO1995006764A2 - Oligonucleotides with rna cleavage activity

Info

Publication number: WO1995006764A2
Application number: PCT/IB1994/000288
Authority: WO
Inventors: Larry W. Mclaughlin; Dong-Jing Fu; Fritz Benseler; Gerd Kotzorek; Janos Ludwig
Original assignee: Vpi Holdings Ltd.
Priority date: 1993-09-03
Filing date: 1994-09-05
Publication date: 1995-03-09
Also published as: WO1995006764A3; AU7623194A

Abstract

The present invention provides novel oligonucleotides, including oligonucleotides with RNA cleavage activity.

Description

OLIGONUCLEOTIDES WITH RNA CLEAVAGE ACTIVITY

BACKGROUND OF THE INVENTION

The present invention relates to oligonucleotides, including oligonucleotides with RNA cleavage activity. RNA is known to have endoribonuclease activity. Catalytic

RNA molecules called ribozymes have been used to bind to target RNA molecules and catalyze their cleavage, thus blocking the activity of the target RNA. The so-called hammerhead ribozymes have been widely studied. The recognition site and catalytic site of these oligoribonucleotides are well characterized and ribozymes containing a recognition sequence specific for any desired target RNA which contains a specified triplet can be constructed. These compounds can therefore be considered as potential therapeutic agents with possibly higher biological activity than the simple antisense

oligonucleotides. Cotten, M., Trends in Biotechnology 1990, 8, 174- 178.

The structure of a typical hammerhead ribozyme is shown in Figure 1 hereof.

Experiments show that externally supplied ribozymes produce only a transient effect. The catalytic effect is destroyed once the ribozyme has been degraded. The accessibility of the target site also limits ribozyme cleavage activity. Protein bound at the cleavage site has been shown to block ribozyme activity. Further, ribozyme catalytic efficiency has been shown to depend on whether the ribozyme expression occurs in the same cell compartment as that occupied by the target DNA. The hammerhead ribozyme has been shown to have no effect in prokaryotic cells when the ribozyme and target RNA were generated from different genes, whereas it can function at a 1:1 ribozyme:target ratio if co-localization in the same cell is maintained. Studies have shown that a high (1000:1) ribozyrne:substrate ratio is needed for inhibition in vivo in eukaryotic cells. See Cotton, M., supra. This suggests that the catalytic potential of the ribozyme is not being achieved.

Eckstein et al. introduced 2'-amino or 2'-F substituents into the pyrimidine positions or the hammerhead ribozyme. Pieken, W.A.,

Olsen, D.B., Benseler, F., Aurup, A., and Eckstein, F. Science 1991 , 253, 314-317; and Heidenreich, O. and Eckstein, F. J. Biol. Chem. 1992, 267, 1904-1909. The cleavage positions of the ribozymes in cellular extracts were not determined but the all-pyrimidine

substitutions together with phosphorothiolate substitutions at the 3' terminus gave compounds with a markedly inc-eased stability. The influence of these chemical modifications on the catalytic activity or the ribozymes was negligible. Sproat et al. found that ribozymes containing 2'-O-Allyl substituents in all but six positions of the catalytic core are resistant to nuclease attack to some extent and retain their catalytic activity.

Paoella, G., Sproat, B.S., Lammond, A.l. EMBO Journal 1992, 11, 1913-1919. Cedergreen et al. found that mixed DNA / RNA oligomers with 4-7 ribopositions are active in cleaving substrate RNA. These oligomers are three orders or magnitude more stable than the all-RNA ribozymes in incubation with RNase A and yeast extract Yang, J.H., Usman, N., Chartrand, P., Cedergreen, R. Biochemistry 1992, 31,

5005-5009.

Recent results have suggested that the ribonucleotide

backbone is not a strong requirement for catalytic cleavage of mRNA. See, Sproat, et al., supra. When the 2'-OH groups in the

hammerhead structure were systematically replaced with H residues it was observed that the presence of only four 2'-OH groups at defined positions in the catalytic core (the core being nucleotides. 3 to I5.I shown in Figure 1) were necessary to show catalytic activity.

Referring to Figure 1, these positions are G⁵, G⁸, A⁹ and A^15.1. If the

A⁶, A¹⁴ and one nucleotide in helix III (possibly C^15.1) were also left unmodified, catalytic activity was found to reach 1/10th of the all-2'-OH RNA ribozyme. If the substituents were 2'-O-Allyl groups the essential 2'-OH groups needed for catalytic activity were found to be U⁴, G⁵, A⁶, G⁸, G¹², and A^15.1. Those shown in bold are positions identical to the DNA analogue. The kcat/Km for this ribozyme was only 5 times lower than for the all 2'-OH ribozyme.

Other modifications of the 2'-OH groups in the catalytic core of the hammerhead ribozyme have been reported. Williams et at. found only a 15-fold reduction in catalytic activity if the 2'-OH groups at G⁵ and G⁸ are replaced with 2'-NH₂ and the 2'-OH group at G¹² is replaced with a 2'-F group. Olsen et at. noted only a small decrease in catalytic activity if the 2'-OH group at one of the following

adenosines : A⁶, A⁹ or A^15.1, A¹⁴, A¹³ is replaced by 2'-F. Replacement of more 2'-OH-s, however, is not allowed with this substituent.

Williams, D.M., Pieken, W. A., Eckstein, F. P.N.A.S. 1992, 89, 918- 921. Olsen, D.B., Benseler, F., Aurup, H., Pieken, W.A. and Eckstein, F. Biochemistry 1991 , 30, 9735-9741.

Pyle et al. studied the Tetrahymena ribozyme and showed that the essential -OH groups are important for stabilizing base-backbone tertiary interactions by hydrogen bonding. It is not clear, however, whether this is the case, or whether the 2'-OH groups are important for the direct binding of the catalytically important metal ion. Perreault et al. suggest that the 2'-OH groups of A⁹ and G⁵ are directly involved in Mg²⁺ binding. Pyle, A.M., Murphy, F.L, Ceck, T.R. Nature 1992,

358, 123-128. Perreault, J.P., Labuda, D., Usman, N., Yans, J.H., Cedergreen, R. Biochemistry 1991, 30, 4020-4025.

In a recent model McLaughlin et al. proposed a role for the 2'-OH as of G⁵ or G⁸. They interact with H₂O molecules bound in the first co-ordination sphere of the Mg²⁺ cofactor. Fu, D.J. and

McLaughlin, LW. Biochemistry 1992, 31, 10941-10949.

In an effort to determine the minimal sequence requirements for ribozyme activity Jennings et al. observed that the catalytic activity of a hammerhead ribozyme in which the loop of nucleotides 10.1 to 11.1 (see Figure 1) is replaced by four deoxyuridines decreases only by a factor 3 when compared with the original structure. McCall, J.M., Hendry, P., Jennings, PA P.N.A.S. 1992, 89, 5710-5714. This modified .structure has been termed the "miniribozyme structure".

SUMMARY OF THE INVENTION

For exogenous application of preformed synthetic ribozymes several important problems can be identified. It is desirable to increase stability against RNase degradation, increase stability against exonuclease degradation; and facilitate cellular uptake. The present invention provides in a first aspect oligonucleotides selected from the following general sequences of Formulas I through V:

In which - - - X and Y- - - are target-specific RNA recognition sequences

A is adenosine or 2'-deoxyadenosine

C is cytidine or 2'-deoxycytidine

G is guanosine or 2'-deoxyguanosine

U is uridtne or 2'-deoxyuridine

a is either A as defined above or substituted deoxyadenosine according to formula Ia below, and

g is either G as defined above or substituted deoxyguanosine according to formula Ib below,

wherein at least one of groups a or g of the oligonucleotide is other than A or G, respectively,

in which formulas Ia and Ib the C2' stereogenic center is of either S or R configuration, according to the Cahn-lngold-Prelog nomenclature, and preferably is of the R configuration, and the substituent R is selected from -CF₂H, -CF₂, -CCl₂H, -CCI₃, CBr₂H, CBr₃, Cl₂H, Cl₃, - CONH₂, -CONHR', -CONR'R", -NHCOH, -NHCOR", -N(R')COR", -SH, -SR', -NH₂. -NHR', -NHR'R", -COOR', and -NHCOOR', or

combinations thereof;

wherein R' and R" are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or

unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkanoyl, substituted and unsubstituted aminoaikyl, substituted and unsubstituted thioalkyl, and substituted or

unsubstituted alkoxy,

P₁ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₂ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₃ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₄ is a) A-U, b) U-A,c) G-C, or d) C-G,

W is either i) a tetranucleotide loop sequence composed of C, G, A and U residues, for example the tetranucleotide IIa has been used for general sequence IV when P₁ is C-G, P₂ is A-U, P₃ is G-C and

P4 is G-C 5'-C-C-G-A-3' IIa or the nucleotide loop sequence IIb has been used when, P₁ is C-G, P₂ is C-G, P₃ is G-C and P₄ is G-C

5 -G-U-U-A-3' IIb or ii) diol bridges iii connected with phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted phosphoramidate, or methylphosphonate derivative linkages m(Z) iii in which each diol bridge (Z) is of formula iv - [O-(CH₂)_n] - iv where n is 1 - 10,

m diol bridges are connected together, and one or more of the connections may be through phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted

phosphoramidate, or methylphosphonate derivative linkages, and m being 1 - 10. For example, for the general class of sequences of Formula III, when P₁ is C-G and P₂ is G-C, and W is m(Z) with m equal to 6, and n equal to 2, the following material results: .

Alternatively, for the general class of sequences of Formula III, when P₁ is C-G and P₂ is G-C, and W is m(Z) with m equal to 6, and n equal to 2, the following material results, when one of the

connected diol bridges employs a phosphodiester:

For oligonucleotides of Formulas I-V, there is no requirement that the length of the diol be the same in each diol bridge; n may be independently selected for each bridge from 1 to 10. Preferably, n is two or three in each bridge and there are four to six bridges; m is 4 to 6.

In a further aspect of the invention, for an oligonucleotide of Formulas I, II, III, IV or V, more than one a and/or g group of the oligonucleotide is of the formula la or formula lb as defined above, and more preferably each a and g group of the oligonucleotide is other than A or G, i.e. each a and g group of the oligonucleotide is of the formula la or lb as such formulas are defined above.

The present invention provides in another aspect provides oligonucleotides selected from the general sequences Formulas IA- VA:

in which - - - X and Y - - - are target-specific RNA recognition sequences

A is adenosine or 2'-deoxyadenosine

C is cytidine or 2'-deoxycytidine

G is guanosine or 2'-deoxyguanosine

U is uridine or 2'-deoxyuridine

P₁ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₂ is A-U, b) U-A, c) G-C, or d) C-G,

P₃ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₄ is a) A-U, b) U-A, c) G-C, or d) C-G, W is 1) the diol bridges iii connected with phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted phosphoramidate, or methylphosphonate derivative linkages m(Z) iii in which each diol bridge (Z) is of formula iv - [O-(CH2)_n] - iv n is 1 - 10,

m diol bridges are connected together, one or more of the connections may be through phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted

phosphoramidate, or methylphosphonate derivative linkages, and m being 1 - 10, or

W is 2) the tetranucleotide IIa, 5'-C-C-G-A-3' IIa or W is 3) the tetranucleotide IIb 5--G-U-U-A-3' IIb.

It is understood that native hammerhead, as discussed above and depicted in Figure 1 is excluded from the scope of Formula I-V and IA-VA. For oligonucleotides of Formulas IA-VA, there is no

requirement that the length of the diol be the same in each diol bridge; n may be independently selected for each bridge from 1 to 10. Preferably, n is two or three in each bridge and there are four to six bridges; m is 4 to 6.

The invention also provides novel methods, including a method of inhibiting expression of susceptible single-stranded RNA that comprises contacting said susceptible RNA with an expression inhibition effective amount of an oligonucleotide of Formula l-V or IA- VA.

In a still further aspect, the invention provides those

compositions as defined by Formulas l-V and Formulas IA-VA wherein the X and/or Y groups of the sequences are absent

Other aspects of the invention are disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the nucleotide sequence of a hammerhead ribozyme.

Figure 2 shows an example of therapeutic target interaction between the myconcogene mRNA and an oligonucleotide of the invention of the type described in Example 1.

Figure 3 is the ³¹P-NMR spectrum of the 1 ,3-propanediol linker synthesized as the 4,4^,-dimethoxytrityl-β-cyanoethyl-phosphoramidite derivative of Example 8. Figure 4 shows the results of polyacrylamide gel

electrophoresis of the complexes of Example 8.

Figure 5 is a copy of a typical autoradiogram used to monitor the cleavage of the 12-mer substrate in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

Groups X and Y of Formulas I-V and IA-VA above are target specific recognition sequences made up of any deoxyribonucleosides N depending on the target RNA sequence. That is, X and Y each will be deoxyribonucieoside sequences complementary, at least in part, to the sequence of a single-stranded RNA to be cleaved by the particularly oligonucleotide of the invention. X and Y may be of the same or different length. There is no need for the molecule to be symmetrical. Each of X and Y may be 4 to 25 nucteotides long, preferably 6 to 20 nucleotides, containing one or more of A, G, C and U nucleotides. If X and Y are too short, the oligonucleotide loses its specificity. X or Y may include one or more stabilizing modifications, i.e. structural features that inhibit degradation of the oligonucleotide relative to a sequence that lack such features. For example, two or three natural 3'-5' phosphodiester linkages present at least on the 3' end of X may be modified in an attempt to protect the oligonucleotide from attack by 3'-exonucleases. Particularly, 3'-5' phosphodiester linkage may be replaced by phosphorothiate linkages such as thiophosphodiester linkages. Suitable sequences of X and Y for a particular target single-stranded RNA can be readily ascertained by those skilled in the art . For example, the sequence of a target RNA, i.e. an RNA to be cleaved by an oligonucleotide of the present invention, can be determined by known means such as by sequencing the

corresponding DNA, and then the sequences of X and Y are selected to complement at least a substantial number of the nucleotides of the target RNA, e.g. where the nucieotides of X and Y complement at least about 60% of the nucieotides of the target RNA, more preferably at least about 75%, still more preferably at least about 90-96 or 98% and even more preferably all of the nucieotides of X and Y

complement the nucieotides of the target RNA. Of course, conditions of hybridization of an oligonucleotide of the invention and -a target RNA, e.g. salt concentration and temperature, will influence the suitable amounts of complementary bases between the

oligonucleotide and the target RNA.

The 2'-R substituent of oligonucleotides. of Formulas I-V and IA- VA is a non-nucleophilic group which is preferably, but not

necessarily, isoteric and isopoiar with the replaced -OH group. The substituent ideally has both H-bond donor and acceptor abilities. A 2'-COOH substituent, which can chelate with Mg²⁺, is advantageous as -COOH containing amino acid side chains can be important for Mg²⁺ binding in enzymes.

The modifications according to the invention which involve the 2'-R substitution of nucleotides in the catalytic/cleavage region of the oligonucleotide can provide a desirable increase in stability against degradation and can increase catalytic activity due to improved Mg²⁺ binding.

Replacement of the -A-G-C-C- loop sequence or the -G-U-U-A loop sequence with aliphatic diol bridges simplifies the large scale synthesis of these oligonucleotides of the invention and eliminates unwanted intermolecular interactions. Oligonucleotides according to the invention with diol bridges can be made by machine more easily and more cheaply than conventional ribozymes.

Preferred alkyl groups of the oligonucleotides of Formulas l-V include those groups having from 1 to about 12 carbon atoms, more preferably 1 to about 8 carbon atoms, still more preferably 1 to about 6 carbon atoms. Methyl, ethyl, propyl and butyl including isopropyl and branched butyl groups such as sec-butyl and f-butyl are particularly preferred alkyl groups. As used herein, the term alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although of course cyclic groups will comprise at least three carbon ring members. Straight or branched chain noncyclic alkyl groups are generally more preferred than cyclic groups. Preferred alkenyl and alkynyl groups of oligonucleotides of Formulas l-V and IA-VA have one or more unsaturated linkages and from 2 to about 12 carbon atoms, more preferably 2 to about 8 carbon atoms, still more preferably 2 to about 6 carbon atoms, even more preferably 2, 3 or 4 carbon atoms. The terms alkenyl and alkynyl as used herein refer to both cyclic and noncyclic groups, although straight or branched noncyclic groups are generally more preferred. Preferred alkoxy groups of the oligonucleotides of the invention include groups having one or more oxygen linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms, even more preferably 1 , 2, 3 or 4 carbon atoms. Preferred thioalkyl groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Particularly preferred are thioalkyl groups having 1 , 2, 3 or 4 carbon atoms. Preferred

aminoalkyl groups include those groups having one or more primary, secondary and/or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably one to about 8 carbon atoms, still more preferably 1 to about 6 carbon atoms, even more preferably 1 , 2, 3 or 4 carbon atoms. Secondary and tertiary amine groups are generally more preferred than primary amine moieties. Substituted moieties of oligonucleotides of the invention may be substituted at one or more available positions by one or more suitable groups such as, e.g., halogen such as fluoro, chloro, bromo and iodo; cyano; hydroxyl; alkyl groups inctuding those groups having 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms, preferably noncyclic alkyl groups including branched chain groups such as isopropyl and t-butyl; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon or from 2 to about 6 carbon atoms; thioalkyl groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; alkoxy groups having those having one or more oxygen linkages and from 1 to about 12 carbon atoms or 1 to about 6 carbon atoms; and aminoaikyl groups such as groups having one or more N atoms (which can be present as primary, secondary and/or tertiary N groups) and from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms.

The modifications according to the invention which involve substituting diols in the loop region of the oligonucleotide can provide a desirable facilitation of cellular uptake.

The oligonucleotides of the present invention may be used as intermediates for further modification to improve their ease of up-take by the cell (in comparison to unmodified oligonucleotides of the invention and known ribozymes), for example by the attachment of carrier molecules. The embodiments having diol bridges are considered to be particularly useful as intermediates for this purpose. The invention provides methods for inhibiting expression of susceptible mRNA comprising contacting the RNA an expression inhibition effective amount of an oligonucleotide of the invention.

Inhibition effective amounts of particular oligonucleotide can be readily determined, e.g., by the methods of E xample 8 which follows.

The oligonucleotides of the present invention thus are potential antagonists of a wide range of therapeutic targets which involve over-expression of products. By binding to specific targets on mRNA and cleaving the mRNA they can stop translation and hence "switch off a specific gene. In cases where expression of a product (e.g. an enzyme or a protein) by a gene is causative of an illness or

disfunction this could lead to a cure. Alternatively a putative gene as a source of a problem phenomenon could be "switched off' selectively and the therapeutic effect observed. More particularly, the oligonucleotides of the present invention will have utility as anticancer and antiviral agents as well as anti-inflammatory and anti-ulcer agents. The stability of the oligonucleotides of the present invention indicates that they can be used in nanomblar amounts. This offers a significant improvement over known ribozymes which have to be used in relatively large amounts to compensate for their intracellular degradation by nucleases. The oligonucleotides of the invention may achieve true catalytic activity, i.e. they will not be destroyed in the cleavage reaction.

More specifically, oligonucleotides Formulas l-V and IA-VA will be useful as therapeutic for the treatment of mammals, including humans, particularly for the treatment of mammals having cancer cells susceptible to one or more of the oligonucleotides. Thus, the invention provides a method for treatment of susceptible cancer cells, e.g., a solid or disseminated tumor, in mammals including humans, the method comprising administration to the mammal of an antitumor effective amount of one or more oligonucleotides of the invention, once or several times a day or other appropriate schedule, orally, rectally, parenterally, topically, etc.

Other therapeutic uses will be carried out in similar manner. For example, for treatment of a viral infection, a mammal such as a human that has a susceptible viral infection is administered an antiviral effective amount of one or more oligonucleotides of the invention, once or several times a day, or other appropriate schedule, by a suitable route of administration such as orally or parenterally, particularly intravenously. For inflammatory or anti-ulcer applications, an anti-inflammatory or anti-ulcer effective amount of one or more oligonucleotides of the invention to a mammal in need thereof, particularly a human in need thereof, according to an appropriate schedule and route of administration such as orally or parenterally. The oligonucleotides of the present invention may be suitably administered to a subject as a pharmaceutically acceptable salt Such salts can be prepared in a number of ways. For example, where the compound comprises a basic group such as an amino group, salts can be formed from an organic or inorganic acid, e.g. hydrochioride, sulfate, hemisuifate, phosphate, nitrate acetate, oxalate, citrate, maleate, etc. Where the compound comprises a carboxy group, pharmaceutically acceptable salts include those formed form alkali metals, e.g. a sodium salt. For parenteral administration, an oligonucleotide of the invention is typically administered to a subject in aqueous or non-aqueous sterile injection solutions as are known in the art. For oral administration, the therapeutic of the invention may be administered to a subject such as a human in discrete units such as capsules or tablets each containing a predetermined amount of the therapeutic, as a solution or a suspension in an aqueous or non-aqueous liquid, as an oil/water liquid emulsion, in powdered carriers such as lactose or sucrose, etc. Oligonucleotides of Formulas l-V and IA-VA will also be useful in screening methods of the invention, including methods that enable identification of therapeutic agents which inhibit RNA cleavage. Such therapeutics will have significant utility in treatment of subjects, particularly mammals such as humans, that fail to express adequate amounts of particular proteins due to abnormal RNA degradation. Such therapeutics can be identified by a method comprising steps of:

(a) contacting targeted single-stranded RNA molecule(s) with a compound suspected of inhibiting degradation (e.g., cleavage) of said RNA molecule;

(b) adding to the mixture obtained in said step (a) one or more oligonucleotides of Formulas l-V or IA-VA as defined above under conditions suitable for said oligonucleotides to cleave said RNA molecule, and wherein the oligonucleotide can cleave the RNA molecule in the absence of said suspected compound; and

(c) assaying the mixture obtained from step (b), such as by northern blot analysis, to determine cleavage of said RNA molecule, wherein lack of cleavage of said RNA molecule relative to a control indicates that the compound inhibits degradation of the RNA

molecule. Specific conditions for this method will be readily

ascertained by those skilled in the art, and are suitably the same or similar to the procedures of Example 8 which follows with the exception that the test compound suspected of inhibiting RNA degradation will be added to the target single-stranded sequence, i.e. the "substrate" sample, prior to mixing with the oligonucleotide of the invention. As should be clear, the control sample for the above method will be obtained by following the same protocol as for the test compound, but with the omission of adding the test compound to the RNA.

AH documents mentioned herein are incorporated herein by reference. The following examples illustrate the present invention without limitation.

GENERAL COMMENTS

In the following examples the following materials were

employed as specified. The 4,4'-dimethoxytrityl-β-cyanoethylnucleoside phophoramidites of the four common

nucleosides (A, G, C and U) were obtained from Miiligen (New

Bedford, MA). The three linkers, hexaethylene glycol,

triethylenegiyycol and 1,3-propanediol, prepared as the corresponding 4,4'-dimethoxytrityl-β-cyanoethyl phosphoramidite derivatives, were synthesized according to known methods. The ³¹P-NMR resonances of the three phosphoamidite linker derivatives were 148.6, 148.5 and 147.2 ppm, respectively. A typical ³¹P-NMR spectrum is illustrated in Fig. 3 for the propanediol linker. Some of the 1 ,3-propanediol linker was also obtained from Glen Research (Sterling, VA).

Oligonucleotides were synthesized using an Applied Biosystems 381A DNA synthesizer. High performance liquid chromatography (HPLC) was carried out on an ODS-Hypersil column (0.46 × 25 cm, Shandon Southern, England) using a Beckman EPLC system. ¹H NMR spectra were obtained at 300 MHz or 500 MHz on Varian XL-300 or 500 multinuclear spectrometers. ³¹P NMR spectra were obtained at 121 MHZ on the Varian XL-300. Absorption spectra were recorded by a Perkin-Elmer Lambda 3B UV/Vis spectrophotometer. Nuclease S1 is a product of United States Biochemical Corporation (Cleveland, Ohio). RNase T2 was obtained from Sigma (St. Louis, MO).

Example 1

An oligonucleotide having the following general sequence lit

in which

N represents any deoxyribonucleotide recognition sequence specific for the target RNA

N.N represents a thiophosphodiester linkage replacing a natural

3'-5' phosphodiester linkage between two nucieotides

A is deoxyadenosine

C is deoxycytidine

G is deoxyguanosine

U is deoxyuridine

P₁ in this case is C - G and P₂ is A - U,

a is 2'substituted deoxyadenosine according to formula Ia as identified and defined above

g is 2'substituted deoxyguanosine according to formula Ib as identified and defined above.

W is 3'-G-G-A-G-C-C-C-S'

Example 2

An oligonucleotide having the following sequence

in which

N.N represents a thiophosphodiester linkage replacing a natural 3'-5' phosphodiester linkage between two nucieotides

A is deoxyadenosine

C is deoxycytidine

G is deoxyguanosine

U is deσxyuridine

P₁ in this case is C - G and P₂ is C - G,

g is 2'substituted deoxyguanosine according to formula Ib as identified and defined above

W is 3'-C-C-G-U-U-A-G-G-5' Example 3

An oligonucleotide having the following sequence

in which N, N.N, A, C, G, U, a and g are as defined for

Example 1 and the diol bridges are connected with

phosphodiester or substituted neutral phosphotriester derivative linkages.

Example 4

An oligonucleotide having the following sequence

in which N, N.N, A, C, G, U, a and g are as defined for Example 1 and the diol bridges are connected with phosphodiester or substituted neutral phosphotoreceptor derivative linkages.

Example 5

An oligonucleotide having the following sequence

in which N and N.N are as defined for Example I

A is adenosine

C is cytidine

G is guanosine

U is uridine and the diol bridges are connected with phosphodiester or substituted neutral phosphotriester derivative linkages. Example 6

An oligonucleotide having the following sequence

in which N and N.N are as defined for Example I

A is adenosine

C is cytidine

G is guanosine

U is uridine and the diol bridges are connected with phosphodiester or substituted neutral phosphotriester derivative linkages. Example 7

A therapeutic target interaction between mRNA and an oligonucleotide of the invention is shown in Figure 2. The -myc oncogene mRNA secondary structure is shown from nucleotide 1 to nucleotide 900. Translation starts at nucleotide 421 and the triplet cleavage site is positions 433 to 435 (GUU).

Example 8

Native hammerhead has the following structure

Four types of complex (i.e., oligonucleotide) have been prepared, in which loop L as indicated in the above structure is replaced completely or partially with simple synthetic organic linkers. Complexes have been prepared in which loop L comprises a four base-pair stem and a loop comprising linkers based on hexaethylene giycol 8(1), bis(triethylene giycol) phosphate 8(2), tris(propanediol) bisphosphate 8(3), bis(propanediol) phosphate 8(4) and propanediol 8(5), the structures of such complexes specified below. Whilst the base-pair stem of loop L is intact in Examples 8(1) to 8(5), it is reduced to two base pairs in Examples 8(6) and 8(7), one base pair in Examples 8(8), 8(9) and 8(10) and removed from the structure in Examples 8(11) to 8(14). The structures of the Examples follow with the Example number specified within the depicted complexes: Linker Complexes, L = 29

Use of linkers greatly simplifies the preparation of

hammerhead-like catalysts composed of as few as twenty two nucleosides. A series of complexes may be generated that vary in the length of the synthetic linker, which in turn varies the spacing of the tethered nucleoside residues Ag and G₁₀- The linkers differ in the presence or absence of one or more negatively charged

phosphodiesters. The linker building blocks were prepared as

4,4^,-dimethoxytrityl-β-cyanoethylphosphoramidite derivatives and incorporated into the sequence at the desired site under the same conditions used or coupling of the nucleoside phosphoramidites. The linkers were incorporated into the sequences with yields that were comparable with those typically obtained for the four common nucleosides A, G, C and U. Analysis of the sequences by

polyacrylamide gel electrophoresis indicated the presence of a single species and the mobility of the various RNA/linker sequences was directly related to the number of nucleoside residues present. The presence of additional phosphodiester residues in the linkers increased the distance of migration.

Cleavage activity of the oligonucleotide complexes identified above and specified in the below Table was measured under single turnover conditions with a large excess of the ribozyme-like catalyst (i.e., complex) in order to ensure complexation of the substrate sequence as detailed more fully below. First order rate constants characterizing the cleavage reaction were calculated from the halflife of the complexes and were corrected for the extent of cleavage at too as specified below:

Table

Cleavage rates of Ribozyme-like complex containing simple linkers Complex* k_f(min^-1 ) relative cleavage rate %P∞

native 1.1 1.0 88

8(1) 0.81 0.74 89

8(2) 0.71 0.65 91

8(3) 0.12 0.11 86 8(4) 0.0075 0.0068 82

8(5) 0.0034 0.0031 81

8(6) 1.2 1.1 87

8(7) 0.0026 0.0024 69

8(8) 0.11 0.10 81

8(9) 0.050 0.045 75

8(10) 0.032 0.029 80

8(11) 0.0052 0.0047 71

8(12) 0.0055 0.0050 71

8(13) - - -

8(14) 0.0060 0.0055 74 k_f = 0.693/t_1/2.

* Complex are identified as native hammerhead, identified above, or by reference to the particular complex as identified above.

Cleavage activity was identified according to the following protocol. Two 25 ul solutions containing either 1.2 uM catalyst or 0.2 uM substrate (i.e., single stranded RNA) in 50 mM Tris-HCI (pH 7.5) were each heated to 95°C for 1 min and cooled at 25°C. Each solution of catalyst was adjusted to 10 mM MgCI₂ and incubated at 25°C for 15 min. The substrate solution was adjusted to 10 mM MgCI₂. prior to use. The reaction was initiated by mixing the two solutions (final catalyst concentration = 0.6 uM, final substrate concentration = 0.1 uM). Aliquots of 5 - 7 uL were withdrawn, and the reaction was quenched by the addition of an equal volume of 50 mM Na₂EDTA/ 7M urea/ 10% giycerol/ 0.05% xylene cyanol/ 0.05% bromophenol blue. The extent of cleavage were analyzed by PAGE on 20% gels. %P∞ = per cent product at too. The determination of too was at a minimum of 10 x t_½ except, 8(7) was incubated for 38 h (8.5 × t_½). 8(13) exhibited no significant cleavage.

Introduction of the hexaethylene giycol (1) or bis(triethylene giycol) phosphate (2) linkers in place of the GUUA tetranucleotide loop of the native sequence results in activity that is very similar to the 34-mer ribozyme. As the linker replacing the GUUA loop is shortened to 16 atoms, 8(3), 11 atoms, 8(4) and 5 atoms, 8(5), the cleavage rate decreases markedly. Previous work has indicated that a chain length of nine carbon atoms is required to bridge the terminal phosphates of an RNA duplex. Ma, M.Y-X., Reid, L.S., Climie, S.C., Lin, W.C., Kuperman, R., Sumner-Smith, M. Barnett, R.W.

Biochemistry 1993, 32, 1751-1758. While complexes 8(3) and 8(4) provide this chain length, the presence of one or more negatively charged phosphodiesters may result in destabilizing electrostatic repulsive forces.

The number of base pairs in the stem of loop L can be reduced from four to two with the use of the neutral hexaethyleneglycol linker without any change in the cleavage rate (compare 8(6) with 8(1)), but the similar sequence in 8(7) with two negatively charged

phosphodiester residues is less active. Two additional complexes containing the hexaethyleneglycol linker, one with a single base pair 8(8), and one without any base pair 8(11) in the stem are also active catalysts, but the cleavage rates are reduced by 10- and 200-fold, respectively. The reduced cleavage rates with these two complexes suggests that the presence of a base pair, or similar structure, at the base of the stem is critical for formation of the active complex.

Sequences comprising a phosphodiester-containing linker and either a single C-G base pair (8(9) or 8(10)), or no base pair (8(12), 8(13) & 3(14)) in the stem are alt less active than the corresponding

complexes constructed with the neutral hexaethylene giycol linker. All of the linkers employed in complexes 8(8)-8(14) are longer than the nine carbon atom minimum, but the presence or additional charge at some sites may destabilize the active complex. Complex 8(13), lacking any of the base pairs of the stem, and containing a linker with two negatively charged phosphodiesters, did not exhibit any

measurable cleavage activity.

The neutral giycol linker can be employed to replace all of the nucleosides GUUA of loop L and two of the base pairs of the stem without any observable loss of cleavage activity. Some loss of activity is observed as three or all four of the base pairs of the stem are eliminated, but these complexes remain active RNA-cieaving

molecules.

The following procedures were employed in this Example 8 and exemplify suitable procedures for synthesis and analysis of the present oligonucletodies.

Oligonucleotide Synthesis

The oligonucleotides identified above were synthesized from 1 umol of bound nucleoside on wide-pore silica supports using

phosphoramidite chemistry and an Applied Biosystems 381A or 394

DNA synthesizer. After assembly of each sequence, the glass beads were suspended in 4 mL of concentrated ammonium

hydroxide/ethanol (3:1) for 12h at 55°C. The glass beads were removed, the ammonia and ethanol were evaporated to dryness (speed vac), the residue was resuspended in 0.2mL absolute ethanol and evaporated to dryness. To the residue was added 1.1 mL of 1.0M tetrabutyiammonium fluoride in tetrahydrofuran, the suspension was shaken until the residue dissolved, and the reaction was protected from light and kept for 16h at ambient temperature. To the solution was added 0.5mL of sterile 3 M sodium acetate (pH 5.8). After mixing, the solution was extracted (2x) with 1.5mL ethyl acetate. The aqueous phase was transferred to a sterile Eppendorf tube and reduced in volume to 0.4-0.5mL (speed-vac), 1.5-1.6mL of absolute ethanol was added, the solution vortexed and placed in dry ice for 2h.

After spinning at 14k rpm, 15 min at 4°C, the precipitated RNA was separated and dissolved in 0.1 to 0.5mL of sterile water. Purification was carried out in 2mm thick 20% polyacrylamide gels containing 7M urea. After electrophoresis at 23 Watts for approximately 16h, the oligonucleotides were visualized by UV shadowing, excised from the gel and the RNA extracted

electrophoretically using an Elutrap apparatus (Schleicher & Schuell, Germany).

Purity Analysis

The RNA sequences were analyzed by analytical

polyacrylamide gel electrophoresis. Migration distances in a 20% acrylamide 1 % bis acrylamide gel after 250 Volts for 17h for each of the specified oligonucleotides were as follows:

8(1) 12.3 cm

8(2) 12.3 cm 8(3) 13.0 cm

8(4) 13.6 cm

8(5) 13.4 cm

8(6) 14.6 cm

8(7) 14.7 cm

8(8) 15.6 cm

8(9) 15.6 cm

8(10) 15.9 cm

8(11) 16.4 cm

8(12) 16.4 cm

8(13) 16.8 cm

8(14) 16.8 cm

The gel is, illustrated in Fig. 4. The lane numbers and complex numbers are as follows: 1 - 8(2); 2 - 8(1); 3 - 8(3); 4 - 8(4); 5 - 8(5); 6

- 8(6); 7 - 8(7); 8 - 8(9); 9 - 8(8); 10 - 8(10); 11 - 8(12); 12 - 8(11); 13

- 8(13); and 14 8(14). The RNA fragments eluted as single peaks from a reversed-phase column ODS-Hypersil) eluted with 50mM triethyiammonium acetate pH 7.0 and a gradient or acetonitrile.

Nucleoside Analysis

Nucleotide (or nucleoside) composition and the integrity of the nucleoside 3'-5' phosphodiester linkage was determined after S1 nuclease (or S1 nuclease and calf intestinal alkaline pbosphatase) hydrolysis.

A 10uL reaction mixture containing 0.5 A₂₆₀ units oligomer in 200mM sodium chloride/5 mM MgCl₂/0.1 mM ZnSO⁴/25mM sodium acetate, pH 5.5, was incubated for 5 min at room temperature with 267 units of S1 nuclease. A 3 uL aliquot was analyzed by HPLC using a 0.46 × 25 cm column of ODS-Hypersil in 20 mM potassium phosphate, pH 5.5, and a gradient of 0-35 % methanol (60 min). For nucleoside analyses, 5 uL of 0.1 M tris.HCl, pH 8.0, and 1 unit of calf intestinal alkaline phosphatase were added to the remaining 7 uL aliquot of reaction mixture. Following incubation for 60 min at ambient temperature, a 5uL aliquot was analyzed by HPLC.

The presence of the linkers could be confirmed by treatment of the sequence with RNase T2 followed by calf intestinal alkaline phosphatase. Under these conditions the linker remained bound to the nucleoside 5'-hydroxyl through a phosphodiester linkage.

Standards could be prepared by coupling the DMT -phosphoramidrte linker to the appropriate (usually G, but C of sequences 8(7) - 8(10)) followed by ammonia, TBAF and acid deprotection. Standards were generally used without purification.

Radioisotopic Labeling

The 12-mer substrate was 5'-end labeled with [Y-³²P] ATP as follows: A 100 uL reaction mixture containing 2 A^ units of 24-mer (about 0.1 mM), 40mM Tris.HCl, pH 8.0, 10 mM MgCI₂, 10 mM dithiothreitot, 0.2 mM Na₂EDTA, 0.1 mM ATP, 300-600 uCi of [Y-³²P] ATP, and 20 units of T4 potynucleotide kinase was incubated for 60 min at 37°C. The product was isolated by absorption on a C18 Sep-Pak cartridge. The cartridge was washed with water and then with 40-50% aqueous methanol to elute the product. The labeled 12-mer was repurified by electrophoresis in a 20% polyacryiamide/7 M urea gel. The product band was excised, and eiectrophoretically extracted with 0.1 M ammonium acetate, pH 7.0, and desalted with a C18 Sep-Pak cartridge. The specific activity of the 12-mer was typically 0.01 uCi/pmol.

Stoichiometric Cleavage Analysis

Two 25 ul solutions containing either 1.2 uM cataiyst or 0.2uM substrate on 50 mM Tris.HCl (pH 7.5) were each heated to 95°C for 1 min and cooled at 25°C. Each solution of catalyst was adjusted to 10 mM MgCI₂ and incubated at 25°C for 15 min. The substrate solution was adjusted to 10 mM MgCI₂ prior to use. The reaction was initiated by mixing the two solutions (final catalyst concentration = 0.6uM, final substrate concentration = 0.1 uM). Aliquots of 5 - 7 uL were

withdrawn, and the reaction was quenched by the addition of an equal volume of 50 mM Na₂ EDTA/7 M urea/10% glycerol/0.05% xylene cyanol/0.05% brpmophenol blue. The extent of cleavage were analyzed by electrophoresis in 20% polyacrylamide/ 1%

bisacryiamide/7M urea gels (14 × 16 cm) in 89 mM Tris-borate buffer, 2mM Na₃EDTA, pH 8.0. The substrate and product bands were located by autoradiography, for example see Fig. 5 which is a copy of a typical autoradiogram used to monitor cleavage of the 12-mer substrate. The upper band in each case is the 12-mer substrate and the lower band is the 5-mer product. Four radioactive alignment markers are also present After autoradiography, the substrate and product bands were excised, lyophilized to dryness, and the

radioactivity was determined by scintillation counting. The logarithm of the unreacted fraction was plotted against time, and the data points were fitted using a linear least squares analysis. The cleavage half-lives (L_½) were used to obtain first order rate constants (k=0.693/t_½). The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention as set forth in the following claims.

Claims

What is claimed is:

1. An oligonucleotide selected from the following Formulas I, II, III, IV and V:

In which X and Y are target-specific RNA recognition sequences

A is adenosine or 2'-deoxyadenosine

C is cytidine or 2'-deoxycytidine

G is guanosine or 2'-deoxyguanosine

U is uridine or 2'-deoxyguanosine

a is either A as defined above or 2'-substituted deoxyadenosine according to formula la below, and

g is either G as defined above or 2'-substituted

deoxyguanosine according to formula lb below,

wherein at least one of groups a or g of the oligonucletide is other than A or G, respectively,

in which formulas la and lb the C2' stereogenic center is of either S or R configuration, and. the substituent R is selected from -CF₂H, -

CF₃, -CCl₂H, -CCl₃, CBr₂H, CBr₃, Ct₂H, Cl₃, -CONH₂, -CONHR', -

CONR'R", -NHCOH, -NHCOR", -N(R')COR", -SH, -SR', -NH₂, -NHR',

-NHR'R",

-COOR', and -NHCOOR', or combinations thereof; wherein R' and R" are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or

unsubstituted alkoxy,

P₁ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₂ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₃ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₄ is a) A-U, b) U-A,c) G-C, or d) C-G,

W is either

i) a tetranucleotide loop sequence composed of C, G, A and U residues, or

ii) diol bridges iii connected with phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted phosphoramidate, or methylphosphonate derivative linkages m(Z) iii in which each diol bridge (Z) is of formula iv

- [O-(CH₂)_n] - iv where n is 1 - 10,

phosphoramidate, or methylphosphonate derivative linkages, and m being 1 - 10.

2. An oligonucleotide selected from the following Formulas I A, I IAI IIIA, IVA and VA:

in which - - - X and Y- - - are target-specific RNA recognition sequences

A is adenosine or 2'-deoxyadenosine

C is cytidine or 2'-deoxycytidine

G is guanosine or 2'-deoxyguanosine

U is uridine or 2'-deoxyuridine

P₁ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₂ is A-U, b) U-A, c) G-C, or d) C-G,

P₃ is a) A-U, b) U-A, c) G-C, or d) C-G,

P₄ is a) A-U, b) U-A, c) G-C, or d) C-G,

W is the diol bridges iii connected with phosphodiester, substituted neutral phosphotriester, phosphorothioate diester, substituted phosphoramidate, or methylphosphonate derivative linkages m(Z) iii in which each diol bridge (Z) is of formula iv

- [O-(CH₂)_n] - iv n is 1 - 10,

phosphoramidate, or methylphosphonate derivative linkages, and m being 1 - 10, and

or W is the tetranucleotide IIa,

5'-C-C-G-A-3' lla or W is the tetranucleotide IIb 5--G-U-U-A-3' IIb.

3. An oligonucleotide according to claim 1 or 2 in which W is diol bridges III and n is 3 in each diol bridge and m is 2, 3 or 4.

4. An oligonucleotide according to claim 1 or 2 in which W is diol bridges iii and n is 2 in each diol bridge and m is 4, 5 or 6.

5. An oligonucleotide according to claim 1 or 2 in which P₁ is C - G and P₂ is G - C and W is diol bridges iii.

7. An oligonucleotide according to claim 1 or 2 in which X or Y include stabilizing modifications.

8. An oligonucleotide according to claim 1 or 2 in which three natural 3'-5' phosphodiester linkages at the 3'-end of X are replaced by substituted neutral phosphotriester, phosphorothioate diester, substituted phosphoramidate, or methylphoshonate derivative linkages.

9. An oligonucleotide according to claim 1 wherein more than one a or g group of the oligonucleotide is of the formula la or formula lb.

10. An oligonucleotide according to claim 1 wherein each a and g group of the oligonucleotide is of the formula la or formula Ib.

11. A method for preparation of a modified oligonucleotide that exhibits enhanced cellular uptake, comprising attaching a carrier molecule to an oligonucleotide as defined in claim 1 or 2.

12. A method of cleaving single-stranded RNA comprising contacting the RNA with an oligonucletide of claim 1 or 2.

13. A method of inhibiting expression of susceptible single-stranded RNA comprising contacting said susceptible RNA with an expression inhibition effective amount of an oligonucleotide of claim 1 or 2.

14. The method of claim 13 where contact with the oligonucleotide results in cleavage of said RNA.