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WO1994006811A1 - Analogues d'oligonucleotides a modification siloxy - Google Patents

Analogues d'oligonucleotides a modification siloxy Download PDF

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Publication number
WO1994006811A1
WO1994006811A1 PCT/US1993/008980 US9308980W WO9406811A1 WO 1994006811 A1 WO1994006811 A1 WO 1994006811A1 US 9308980 W US9308980 W US 9308980W WO 9406811 A1 WO9406811 A1 WO 9406811A1
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Prior art keywords
siloxy
nucleic acid
group
moieties
oligonucleotides
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PCT/US1993/008980
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English (en)
Inventor
Joseph A. Walder
Zigun Li
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Integrated Dna Technologies, Inc.
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Priority to AU51629/93A priority Critical patent/AU5162993A/en
Publication of WO1994006811A1 publication Critical patent/WO1994006811A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12

Definitions

  • oligonucleotide analogs containing one or more stable internucleotide siloxy linkages is presented here.
  • Such oligonucleotides may be single or double stranded.
  • oligonucleotides is very easy, requiring no specially modified nucleosides, and is flexible enough to allow for the production of a diverse family of compounds.
  • oligonucleotides containing siloxy linkages are essentially identical to unmodified oligonucleotides containing only phosphodiester linkages.
  • this easily produced oligonucleotide class is ideal for use in therapeutic administration of oligonucleotides that can be targeted toward the treatment of a large number of deleterious processes and disorders, ranging from viral infection to malignant growth.
  • the siloxy oligonucleotide analogs may be used in any case where the expression of a specific gene is to be modulated, and may alternatively be used in diagnostic procedures where resistance to nucleases, such as is the case with in situ hybridizations, is required.
  • oligonucleotides could down-regulate the expression of specific cellular genes. Since then, due to the great potential this technique holds for the treatment of a wide range of disorders and deleterious processes, the design of strategies and methods to modulate the expression of cellular or viral genes through the introduction of exogenous oligonucleotides has been a focus of research. By selectively blocking the expression of particular genes, oligonucleotides may be used therapeutically, for example, to specifically inhibit viral or
  • Oligonucleotide inhibition of gene expression may be post-transcriptional.
  • oligodeoxyribonucleotides (referred to with respect to this process as "antisense oligonucleotides") having a nucleotide sequence complementary to a portion of a specified mRNA bind to that mRNA, causing the expression of the corresponding gene to be blocked. It has been shown that the predominant mechanism for such expression inhibition is the degradation of the mRNA in such an RNA/DNA heteroduplex by the enzyme RNase H (Walder, R.Y. and Walder, J.A., 1988, Proc. Natl. Acad. Sci. USA 85: 5011-5015).
  • RNase H The use of oligodeoxyribonucleotides in conjunction with RNase H has the potential advantage of acting as a catalytic process. This is due to the fact that many copies of a
  • transcript may be degraded for every oligodeoxyribonucleotide introduced, since RNase H only cleaves the RNA, not the DNA in a heteroduplex, meaning that once an mRNA is degraded, the
  • oligodeoxyribonucleotide is freed and can hybridize to another transcript. Additionally, oligonucleotides can function to post-transcriptionally inhibit gene expression in an RNase H-independent manner by
  • oligonucleotide inhibition of gene expression may also occur at the
  • an oligonucleotide can interfere with transcription of a specific mRNA, through the formation of a triple helix with the endogenous double stranded DNA via Hoogsteer., as opposed to the usual Watson-Crick, base pairing.
  • a potential advantage to this approach is that only one molecule of oligonucleotide is required for each copy present of the gene to be inhibited.
  • Bielinska et al. Bielinska, A. et al., 1990, Science 250:997-1000.
  • the Bielinska group employed double-stranded oligonucleotide analogs to inhibit gene expression by using them to compete inside the nucleus with endogenous promoter sequences for the binding of specific transcription factors.
  • RNA oligonucleotides termed
  • ribozymes may also provide a means by which to inhibit specific gene expression. This method takes advantage of the fact that mRNA splicing occurs via autocatalytic RNAs, which cleave RNA through the enzymatic use of the 2'-OH of a specific seguence (Cech, T.R., 1986, Cell 44:207-210). Ribozymes are designed to hybridize to a specific sequence of RNA and cleave this target RNA by transesterification. By targeting a single mRNA species, gene expression may be controlled in a specific manner. Catalytic RNA cleavage by ribozymes occurs independent of any protein. Once cleavage is completed, the ribozyme is freed to bind to a new target RNA.
  • oligonucleotide-based therapeutics Blackwell, T.K. and Weintraub, 1990, Science 250:1104-1110; Blackwell, T.K. and Weintraub, 1990, Science 250:1149-1151;
  • oligonucleotides Pools of these oligonucleotides, containing upwards of 10 13 different sequences, are produced. Such oligonucleotides may be DNA or RNA, and either single or double stranded. Next, sequences are selected that, by chance, have the correct three-dimensional structure to bind a target molecule.
  • the target molecule may range from a small organic compound
  • oligonucleotides can be selected that bind with high affinity to any molecule whose inhibition may be of therapeutic interest. For example, any molecule whose inhibition may be of therapeutic interest. For example, any molecule whose inhibition may be of therapeutic interest. For example, any molecule whose inhibition may be of therapeutic interest.
  • extracellular molecule may be targeted, circumventing the need for cell permeation, described below, that is faced with traditional applications of oligonucleotide therapies.
  • a nucleic acid probe often an oligonucleotide, is used to detect the presence of a complementary nucleic acid (DNA or RNA seguence).
  • the probe hybridizes to its complementary sequence if it is present within the sample.
  • the target sequence may be analyzed in solution or, as is frequently the case, it may first be immobilized on a solid support, such as nitrocellulose or a nylon membrane.
  • the probe carries a label, e.g., a radioactive, fluorescent, or enzyme marker, to permit their detection.
  • a label e.g., a radioactive, fluorescent, or enzyme marker
  • oligonucleotides As potential therapeutic agents is the rapid enzymatic nuclease degradation that the oligonucleotides undergo in the bloodstream and within cells. Unmodified oligonucleotides are degraded sufficiently rapidly in blood, and even more quickly in cells, that their effect as drugs becomes
  • hybridizations where probes are hybridized directly to tissue samples.
  • Nucleases are enzymes that hydrolyze the
  • Exonucleases are further divided into those that cleave from the 5' end of the nucleic acid molecule inward (5' ⁇ 3'), and those that cleave from the 3' end of the nucleic acid molecule inward (3' ⁇ 5'). It has been demonstrated that the predominant mechanism responsible for the rapid degradation of oligonucleotides is a 3' ⁇ 5' mechanism (Walder, J.A. et al., 1989, WO 89/05358), although there is a minor effect observed from 5' ⁇ 3' exonucleases as well.
  • oligonucleotides as potential therapeutic agents are the oligonucleotides' affinity for DNA (for use in triple helix formation) and RNA. The relative ability of an oligonucleotide to bind to complementary nucleic acids is compared by determining the melting
  • the melting temperature (T m ) denotes the temperature at which 50% of the double helices have dissociated into single stranded molecules. The higher the T m , the greater the strength of the binding of the
  • the oligonucleotides must also usually participate in the formation of a heteroduplex that is recognized by RNase H.
  • RNase H the oligonucleotides
  • hybridization with DNA and RNA does not occur, possibly due to restricted rotation about the trigonal carbamate linkage, thus eliminating their utility.
  • phosphoroamidates nor methyl phosphonates are able to contribute to the formation of RNase H substrates.
  • RNase H cleavage is the predominant route of post-transcriptional control, limiting the usefulness of these compounds as well.
  • the polyamide-containing oligonucleotide on the other hand, has a
  • oligonucleotide substantially increased affinity for nucleic acid, relative to unmodified oligonucleotides.
  • oligonucleotides that contain such oligonucleotides with a high level of selectivity. Any oligonucleotide containing phosphate residues at which one of the peripheral phosphate oxygens are modified, is chiral at the phosphorous. Oligonucleotides, therefore, that contain such
  • modified phosphates are actually made up of a mixture of diastereomers. Given that the number of
  • diastereomers is equal to 2 n , where n is the number of chiral linkages in the oligonucleotide, such a mixture can be very complex.
  • n is the number of chiral linkages in the oligonucleotide
  • oligonucleotide containing 15 modified residues the number of diastereomers in the mixture will be equal to 2 14 , or 16,384.
  • modifications that yield chiral centers can severely affect the oligonucleotides' specificity and the ability to select a single oligomeric entity for administration.
  • phosohorothioate a chiral modification, for example.
  • Oligonucleotides containing such a modification are only partially nuclease resistant, with the level of nuclease resistance being dependent upon both the specific diastereomer being assayed and the specific nuclease being used. Because individual diastereomers do not react in a similar manner to each nuclease, it would be extremely difficult to produce oligonucleotides that are optimally nuclease resistant.
  • phophoramidates and methyl phosphonates also yield chiral centers. Tn addition to the problems involved with chirality, lack of specificity may be exhibited in other ways. Again using phosphorothioates as an example, these compounds, due their hydrophobicity, exhibit a whole range of non-specific effects, including a general inhibition of transcription, translation, DNA replication, and an inhibition of kinase activity. Such effects can be dangerous, even lethal. As stated above, the specificity of the polyamide modified oligonucleotides is also
  • oligonucleotides Before oligonucleotides can be used to inhibit gene expression, the molecules must enter the cell. Unmodified oligonucleotides are highly charged, having roughly one full negative charge per nucleotide residue, which generally results in a reduced rate of transport across membranes, which can limit the oligonucleotides' access to its ultimate site of action. Nonetheless, unmodified oligonucleotides do enter cells at a low, but finite rate (Heikkila et al., 1987, Nature 328:445-449; Loke, S.L. et al..
  • the silyl compounds that have been used as oligonucleotide modifications are both acid and base labile, making their synthesis difficult. Altering the structure of the silyl-containing group in such a way that would make the linkage more stable (e.g., by adding a t-butyl group) is not possible because of an unacceptably high steric hindrance that results due to the direct C-Si bond. No silyl group, to date, has been reported that is useful in oligonucleotide synthesis. Another example of such a modification is the formacetal group. This modification is achiral, neutral and yields
  • oligonucleotides that hybridize to nucleic acid. Its synthesis, however, is prohibitively difficult.
  • Formacetal nucleoside monomer synthesis is very complex, and no effective use of formacetal monomers in the synthesis of oligonucleotides has been
  • formacetal linkages like the silyl-containing ones described above, exhibit very limited flexibility due to steric hindrance, further narrowing the possible composition of oligonucleotides
  • the present invention presents a new class of oligonucleotide analogs that contain one or more stable internucleotide siloxy linkages.
  • Such oligonucleotide analogs may include deoxyribonucleotides or ribonucleotides and may be single or double stranded.
  • oligonucleotides that possess each of the features required for the oligonucleotides to be used as therapeutic drugs, modulators of specific gene
  • nucleic acid hybridization profiles that are useful for determining whether nucleases may be present. These features include ease of and flexibility in synthesis, achiral centers, nuclease resistance, neutral charge, and nucleic acid hybridization profiles that are useful for determining whether nucleases may be present.
  • siloxy-containing monomers provided herein are generally represented as follows:
  • Z is a protecting group
  • Y is a pentose sugar
  • B is a nucleic acid base
  • R 1 , and R 2 are apolar moieties
  • R 3 is a leaving group.
  • R 1 In order for such monomers to be achiral, R 1 must be the same as R 2 .
  • siloxy internucleotide linkages provided herein are generally represented as follows:
  • R 1 and R 2 are apolar moieties
  • Y is a pentose sugar
  • B 1 and B 2 are nucleic acid bases.
  • R 1 must be the same as R 2 .
  • One or more of the phosphodiester linkages of the oligonucleotides in this class may be substituted by such siloxy linkages.
  • Oligonucleotides useful as therapeutic agents and other modulators of specific gene expression range from about 10 to about 75 nucleotides in length, with about 15 to about 35 nucleotides being preferred.
  • FIG. 1 Scheme for the preparation of 5'-3' d(T- T) siloxy dimer phosphoramidite.
  • FIG 2 Photograph of gel showing siloxy links are nuclease resistant. Lanes 1-2 contain unmodified oligonucleotide 21-mers of the sequence depicted in Section 6.4.1 below; lanes 4-6 contain siloxy
  • oligonucleotide analog 21-mers as were described in Section 6.4.1 below.
  • Lanes 1, 4 non-exonuclease-treated controls; lanes 2, 5: partial exonuclease digestions; lanes 3, 6: complete exonuclease
  • a new class of oligonucleotide analogs that contain one or more stable internucleotide siloxy linkages is presented here. These linkages are neutral, provide achiral centers around the silicon atom, and yield oligonucleotides that possess each of the features required for the oligonucleotides to be used as therapeutic drugs, as other modulators of specific gene expression, and as stable hybridization probes for diagnostic applications. In addition to achirality and a more neutral charge, these properties include ease of and flexibility in synthesis, nuclease resistance, and nucleic acid hybridization profiles that are essentially equivalent to those of unmodified oligonucleotides.
  • siloxy oligonucleotide analogs The composition of siloxy oligonucleotide analogs and methods for the synthesis of such analogs is presented below. In addition, the uses for such siloxy oligonucleotide analogs is discussed. Examples are presented of syntheses of siloxy monomers, dimers, and oligonucleotides, and, in addition, it is
  • siloxy oligonucleotide analogs are nuclease resistant and exhibit normal nucleic acid hybridization profiles.
  • internucleotide linkage which is the central feature of this invention, would be unfeasible.
  • siloxy linkages chemically stable.
  • siloxy linkages and the resulting siloxy containing oligonucleotides that contain such linkages are the first class of oligonucleotides described that possess each of the features necessary to make the therapeutic and diagnostic use of oligonucleotides optimally
  • the linkages are uncharged, and therefore contribute to bringing oligonucleotide charge closer to neutral, thus increasing the
  • oligonucleotides ' cell permeation capabilities.
  • siloxy linkages are nuclease resistant, which is a requirement due to the nuclease activity that is prevalent in serum and tissues which quickly degrades unmodified oligonucleotides, severely limiting their effectiveness.
  • the silicon in siloxy linkages is verv similar, or isoteric, to the phosphate in phosphodiester linkages, contributing to excellent nucleic acid hybridization properties for siloxy containing oligonucleotides, which are another prerequisite for success when using oligonucleotides to modulate gene expression as well as when using them as hybridization probes in diagnostic applications.
  • siloxy linkages allow for a large degree of selectivity and flexibility in their composition and in the composition of oligonucleotides that contain such linkages. Because achiral centers around the silicon can be created, specific, pure oligonucleotide compositions can be produced.
  • the siloxy linkages are chemically stable, are easy to synthesize, and, unlike the direct C-Si bond present in silyl compounds
  • hydrophobicity of a given oligonucleotide such that its rate of intracellular uptake is enhanced.
  • siloxy containing monomers can be represented in (I), solely for purposes of illustration and description and not by way of limitation:
  • Z is a protecting group
  • Y is a pentose sugar
  • B is a nucleic acid base
  • R 1 and R 2 are apolar moieties
  • R 3 is a leaving group.
  • the protecting group, Z can include, but is not limited to trityl
  • a pentose sugar may consist of ribose, deoxyribose, altered sugar configurations (e.g., arabinosides or alpha-ribosides), or sugars with halogen
  • the nucleic acid base, B can include, but is not limited to, the naturally occurring bases (e.g., adenine, cytosine, guanine, thymine, or
  • R 1 and R 2 are apolar moieties that can include, but are not limited to straight-chain or branched alkyl groups
  • R 3 the leaving group, can include, but is not limited to a halogen atom, hydroxyl group, amine moiety, or acetate moiety. In order for such monomers to be achiral, the R 1 moiety must be the same as the R 2 moiety.
  • siloxy internucleotide linkages provided herein can be represented as in (II), solely for purposes of illustration and description and not by way of limitation: where R 1 and R 2 are apolar moieties as are described in (I), Y is a pentose sugar as described in (I), and B 1 and B 2 are nucleic acid bases, as is described for B in (I).
  • R 1 and R 2 are apolar moieties as are described in (I)
  • Y is a pentose sugar as described in (I)
  • B 1 and B 2 are nucleic acid bases, as is described for B in (I).
  • One or more of the phosphodiester linkages in an oligonucleotide are substituted by such siloxy
  • Oligonucleotides useful as therapeutic agents and as other modulators of specific gene expression range from about 10 to about 75 nucleotides in length, with about 15 to about 35 nucleotides being preferred.
  • Nucleic acid molecules useful as hybridization probes for diagnostic applications range from about 15 to several thousand nucleotides in length. Molecules up to about 200 nucleotides may be synthesized using standard methods, while molecules longer than this may be obtained by ligating synthesized molecules together and/or by ligating synthesized and naturally occurring molecules together.
  • (III) represents an oligonucleotide consisting of 2 nucleotides connected by an achiral siloxy
  • internucleotide linkage in which Y, the pentose sugar, is deoxyribose, B 1 and B 2 , the nucleic acid bases, are both thymine, and R 1 and R 2 , the apolar moieties added to the siloxy group, are both t-butyl alkyl groups. Syntheses of representative siloxy monomers, dimers, and oligonucleotides are described in the examples in Section 6.
  • oligonucleotides of this invention may be modified to best suit the particular purpose they are to be used for.
  • the siloxy oligonucleotides can be produced in s number of ways.
  • the oligonucleotide must be composed of deoxyribonucleotides.
  • the oligonucleotide must be composed of deoxyribonucleotides.
  • oligonucleotide' s internucleotide linkages can be siloxy linkages.
  • the oligonucleotide should contain a consecutive stretch of at least about four
  • nucleotides and preferably at least about seven nucleotides, connected by unmodified phosphodiester bonds. Most preferably, all the remaining
  • internucleotide linkages flanking the stretch that is to participate in RNase H substrate formation should be siloxy linkages. If antisense oligonucleotides are to be utilized to sterically, rather than
  • the oligonucleotides may contain siloxy linkages at every internucleotide linkage. Oligonucleotides to be used for antisense purposes may range may about 10 to about 75
  • nucleotides with about 15 to about 35 being
  • Oligonucleotides to be used in triplex helix formation should be single stranded and composed of deoxynucleotides .
  • the oligonucleotides may contain siloxy linkages at each of the internucleotide
  • oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Siloxy oligonucleotide sequences may be pyrimidine-based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
  • oligonucleotides provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • oligonucleotides may be chosen that are purine-rich, for example, contain a stretch of G residues. These oligonucleotides will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • Switchback oligonucleotides are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • siloxy oligonucleotides to be used as
  • aptamers can be completely or partially modified.
  • the oligonucleotides may be composed of ribonucleotides or deoxyribonucleotides, and may be either single or double stranded.
  • oligonucleotides to be used as ribozymes must be composed of ribonucleotides, and the siloxy
  • composition of the ribozyme oligonucleotides must include one or more sequences complementary to a target mRNA, and must include the catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat # 5,093,246 (Been, M.D. et al., 1992) which is incorporated by reference herein in its entirety. 5.2 SYNTHESIS OF SILOXY
  • oligonucleotides are synthesized from the 3' to the 5' end of the chain. These methods are discussed in “Oligonucleotide Synthesis: A Practical Approach” (Gait, M.J., ed., 1984, IRL Press, Oxford), which is incorporated in its entirety herein by reference.
  • the first residue is coupled to a solid support, such as polystyrene, silica gel, controlled pore glass beads,
  • nucleoside monomer is added at a time to the 5'-OH group of the growing chain.
  • a block of two or more residues may be added in a single reaction step.
  • oligoribonucleotides are being synthesized
  • Nucleosides may be, for example, reacted with silyl halides in the presence of pyridine and dichloromethane; reacted with silylamines in the presence of pyridine, dichloromethane, and
  • the silicon of the silyl compounds may have alkyl groups, straight chained or branched, of 1 to about 12 carbon atoms, and/or aromatic groups, attached via siloxy linkages (-O-Si-). Representative syntheses of siloxy monomers are presented in the examples in Section 6.2.
  • Siloxy dimers may be synthesized using the siloxy monomers described above, reacted with nucleosides containing 3'-OH protecting groups.
  • the halide, amino, acetate, or hydroxyl groups, (e.g., R 3 , in (I) above) of the monomers react with the unprotected 5'-OH group of the non-siloxy nucleoside to yield a dimer.
  • the 3' protecting groups are then removed using standard techniques known in the art.
  • Siloxy-containing oligonucleotides may be
  • a siloxy monomer may be added, using the same techniques described above for dimer synthesis, to the unprotected 5'-OH end of a growing oligonucleotide chain.
  • a siloxy dimer may be incorporated into a longer chain.
  • the 3' protecting group of the dimer is removed by standard means, at which time a coupling group, such as a phosphite-triester (e.g., phosphoramidite) or a phospho-triester is added.
  • the 5' protecting group of the growing chain is removed and the chain is extended by reacting it with the dimer, in the presence of a catalyst and/or coupling agent.
  • Both siloxy monomers and dimers may be utilized in solution and solid phase oligonucleotide syntheses, and may be used in manual as well as automated, large scale oligonucleotide syntheses.
  • Double stranded oligonucleotides may be produced by synthesizing complementary single stranded oligonucleotides, using the techniques described above, and then allowing these oligonucleotides to anneal.
  • Oligonucleotides to be used as hybridization probes for diagnostic applications may be labeled with radioactive, fluorescent, enzymatic, or chromogenic moieties using standard procedures well known in the art (Ausubel et al. eds., 1989, "Current Protocols in Molecular Biology", Vol. 1, John Wiley Pub., New York; Sambrook et al. eds., 1989, "Molecular Cloning", Cold Spring Harbor Laboratory Press, Cold Spring Harbor). Representative examples of siloxy oligonucleotide syntheses are presented in Section 6.4, while
  • siloxy oligonucleotide analogs of this invention may be utilized for several purposes.
  • siloxy oligonucleotides of the invention in the antisense, aptamer, triplex, or ribozyme configurations described above, may be used as
  • deleterious processes by modulating gene expression include, but are not limited to, viral and bacterial infection and/or replication, and any inherited or acquired genetically induced disorders, including, but not limited to, those genetic lesions that cause malignant, or cancerous, growth to develop.
  • Oligonucleotides designed to enhance specific gene expression in addition to those that are designed to inhibit specific gene expression, may both be useful as therapeutic agents. Oligonucleotide enhancement of gene expression may be brought about, for example, by causing the repression of a negative regulatory transcription factor which, in turn, leads to enhancement of the target gene's expression.
  • siloxy oligonucleotides of the invention may be formulated and administered through a variety of means, including systemic, and localized, or topical, administration. Techniques for formulation and administration may be found in
  • the oligonucleotides of the invention are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or
  • oligonucleotides may be formulated in solid or
  • Systemic administration may also be accomplished by transmucosal, transdermal, or oral means.
  • transmucosal or transdermal For transmucosal or transdermal
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • Transmucosal administration may be through nasal sprays or suppositories.
  • oligonucleotides may be formulated into capsules, tablets, and tonics.
  • topical administration the oligonucleotides of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • the siloxy oligonucleotides of the invention may first be encapsulated into liposomes, then administered as described above.
  • Liposomes are spherical lipid bilayers with aqueous interiors. All molecules that are present in an aqueous solution at the time of liposome formation (in this case,
  • oligonucleotides are incorporated into this aqueous interior.
  • the liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm.
  • siloxy oligonucleotide analogs in antisense, aptamer, triplex or ribozyme configurations, as described above, may be used in any case where it is necessary to modulate gene expression. These cases may include industrial, agricultural, or research applications and may involve cell culture systems in addition to intact, multicellular organisms. As discussed above for therapeutic applications, such siloxy
  • oligonucleotide modulation of gene expression may involve either activation or repression of specific gene activity.
  • siloxy oligonucleotide analogs into organisms and cells for such purposes may be accomplished by several means.
  • mammalian administration each of the techniques described above for therapeutic oligonucleotide purposes may be used.
  • other standard techniques for mammalian administration may be used for mammalian administration.
  • nucleic acids introduction of nucleic acids into cells, including, but not limited to, electroporation, microinjection, and calcium phosphate precipitation techniques may be utilized.
  • the siloxy oligonucleotides of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they bind. Such diagnostic tests may be conducted by hybridization through base pair complementarity or triple helix formation which can then be detected by conventional means.
  • the oligonucleotides may be labeled using radioactive, fluorescent, or chromogenic labels, all of which may be detected using well known procedures.
  • the presence of a triple helix may be detected using antibodies which specifically recognize these forms.
  • the diagnostic use of siloxy oligonucleotides is especially advantageous when applied to procedures in which the specimens to be analyzed may contain
  • nucleases significant levels of nucleases, as, for example, is the case with in situ hybridizations.
  • Nucleosides were obtained from Sigma Chemical Co. (St. Louis, MO) and Peninsula Laboratories Inc.
  • Silylatmg agents were from Aldricn Chemical Co. (Milwaukee, WI). Dichlorometnane and pyridine were anhydrous and were purcnased from
  • PHOSPHORAMIDITE (COMPOUND 4, FIG. 1) 560 milligrams (mg) (1 millimole (mmol), 1.0 equiv) of 5' dimethoxy trityl (DMT) thymidine (T) was weighed in a flask, dried by coevaporation with pyridine, then dissolved in a mixture of 25 ml dry CH 2 Cl 2 and 3 ml pyridine. To this mixture was added 0.24 ml (1 mmol, 1.0 equiv) of Cl 2 Si (OtBu) 2 (compound 1 , FIG. 1). Tbe reaction mixture was then stirred under an inert atmosphere for 3 hours.
  • silica gel thin layer chromatography (TLC) (EtOAc) indicated that all the starting material had reacted and two new spots were seen, corresponding to the desired monomer (compound 2, FIG. 1) and a small amount of 3 '-3' T-T dimer.
  • TLC thin layer chromatography
  • EtOAc silica gel thin layer chromatography
  • the two dimer species were separated by careful flash chromatography on silica gel using a stepwise gradient of CH 2 Cl 2 plus 1.0% triethylamine (TEA) to 1:1 CH 2 Cl 2 plus 1.0% TEA/ 2:1 Et 2 O:CH 2 Cl 2 .
  • the OAc protecting group was removed from the 5'-3' T-T dimer, as described below in Section 6.5.
  • 125 mg of the siloxy dimer phosphoramidite was weighed in a vial. This was dissolved in enough anhydrous acetonitrile (CH 3 CN) to give a 0.1 molar (M) solution, filtered, then placed in the 5th base position of an ABI (Applied Biosystems, Inc.) DNA synthesizer. The normal 0.1 ⁇ mol synthesis cycle was used except that the coupling time was extended to 5 minutes for the dimer. The trityl yield showed that the coupling efficiency of the dimer addition was about 90%. The oligonucleotide was then deblocked using concentrated ammonium hydroxide (NH 4 OH) at 55oC for 7 hours. The product was purified from
  • siloxy oligonucleotide 21-mer described in Section 6.4.1, above, and unmodified oligonucleotides of the same sequence, were used in this study to demonstrate that siloxy internucleotide linkages are nuclease resistant.
  • the gels were stained with Stains-All (Sigma).
  • Unmodified and siloxy-containing 21-mers were reacted separately with calf spleen phosphodiesterase, a 5'-3' exonuclease.
  • the exonuclease digestion reaction conditions were as follows: oligonucleotides were digested in 10 ⁇ l containing 100 mM sodium succinate pH 6.1, 100 ⁇ M EDTA, and 0.2 ⁇ g/ ⁇ l enzyme (Boehringer Mannheim). Partial digests were obtained by digesting the oligonucleotides for 8 minutes at 37°C. More complete digests were obtained by
  • polyacrylamide/7M urea gel run for 3 hours at 400 volts. Gels were then stained with Stains-All.
  • Figure 2 shows the results of one such set of oligonucleotide digests.
  • Lanes 1-3 contain the unmodified oligonucleotide 21-mer, lanes 4-6 are the corresponding lanes containing the siloxy
  • Lanes 1 and 4 are non- exonuclease treated controls showing the intact 21-mers.
  • Lanes 2 and 5 represent partial exonuclease digests which produce a ladder of bands.
  • Lanes 3 and 4 are non- exonuclease treated controls showing the intact 21-mers.
  • Lanes 2 and 5 represent partial exonuclease digests which produce a ladder of bands.
  • Lanes 3 and 4 are non- exonuclease treated controls showing the intact 21-mers.
  • Lanes 2 and 5 represent partial exonuclease digests which produce a ladder of bands.
  • siloxy oligonucleotide 21-mer described in Section 6.4.1, and unmodified oligonucleotides (DNA and RNA) of the same and complementary sequence were used in this study to show that siloxy oligonucleotide analogs and unmodified oligonucleotides exhibit similar nucleic acid hybridization properties.
  • oligonucleotide hybridized to either complementary DNA or RNA sequences, was determined. Melting curves were obtained under the following conditions: A total oligonucleotide concentration of approximately 1 ⁇ M in 150 mM Nacl and 10 mM sodium phosphate buffer, pH 7.3.
  • the siloxy linkage of the 21-mer is at position 10, the middle of the oligonucleotide, which was the position that would have been most likely to affect the oligonucleotide's hybridization properties.

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Abstract

Est décrite une classe d'analogues d'oligodésoxyribonucléotides et d'oligoribonucléotides renfermant une ou plusieurs liaisons siloxy internucléotides stables. Ces liaisons, dans lesquelles le groupe phosphodiester est remplacé par un groupe siloxy, sont neutres, constituent des centres achiraux, et sont totalement résistantes aux nucléases. Ces oligonucléotides à modification siloxy sont faciles à synthétiser, assez souples pour permettre la production d'une famille diverse de composés, et présentent des propriétés d'hybridation de l'acide nucléique sensiblement identiques à celles d'oligonucléotides non modifiés. Cette classe d'oligonucléotides s'emploie idéalement dans le cadre d'une administration thérapeutique et d'applications diagnostiques.
PCT/US1993/008980 1992-09-23 1993-09-22 Analogues d'oligonucleotides a modification siloxy WO1994006811A1 (fr)

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WO1999002673A3 (fr) * 1997-07-10 1999-04-01 Genesense Technologies Inc Sequences oligonucleotidiques antisens servant d'inhibiteurs de micro-organismes
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US6274313B1 (en) 1996-03-21 2001-08-14 Pioneer-Hybrid International, Inc. Oligonucleotides with cationic phosphoramidate internucleoside linkages and methods of use
US6331617B1 (en) 1996-03-21 2001-12-18 University Of Iowa Research Foundation Positively charged oligonucleotides as regulators of gene expression
US6458559B1 (en) 1998-04-22 2002-10-01 Cornell Research Foundation, Inc. Multivalent RNA aptamers and their expression in multicellular organisms
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US7300922B2 (en) 2001-05-25 2007-11-27 Duke University Modulators of pharmacological agents
US7304041B2 (en) 2004-04-22 2007-12-04 Regado Biosciences, Inc. Modulators of coagulation factors
US7312325B2 (en) 2000-09-26 2007-12-25 Duke University RNA aptamers and methods for identifying the same
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Cited By (29)

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Publication number Priority date Publication date Assignee Title
US6646114B2 (en) 1993-07-29 2003-11-11 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6001982A (en) * 1993-07-29 1999-12-14 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6211350B1 (en) 1993-07-29 2001-04-03 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6486312B2 (en) 1993-07-29 2002-11-26 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6870039B2 (en) 1993-07-29 2005-03-22 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
EP0766688A4 (fr) * 1994-05-26 1998-12-30 Isis Pharmaceuticals Inc Synthese d'oligonucleotides
US5998596A (en) * 1995-04-04 1999-12-07 The United States Of America As Represented By The Department Of Health And Human Services Inhibition of protein kinase activity by aptameric action of oligonucleotides
JP2008138005A (ja) * 1995-06-09 2008-06-19 Regents Of The Univ Of Colorado 新規な保護基及びオリゴヌクレオチド合成のための改良方法における該新規な保護基の使用
US6274313B1 (en) 1996-03-21 2001-08-14 Pioneer-Hybrid International, Inc. Oligonucleotides with cationic phosphoramidate internucleoside linkages and methods of use
US6331617B1 (en) 1996-03-21 2001-12-18 University Of Iowa Research Foundation Positively charged oligonucleotides as regulators of gene expression
WO1999002673A3 (fr) * 1997-07-10 1999-04-01 Genesense Technologies Inc Sequences oligonucleotidiques antisens servant d'inhibiteurs de micro-organismes
EP1584681A3 (fr) * 1997-07-10 2005-11-09 GeneSense Technologies Inc. Sequences oligonucleotidiques antisens servant d'inhibiteurs de micro-organismes
US6458559B1 (en) 1998-04-22 2002-10-01 Cornell Research Foundation, Inc. Multivalent RNA aptamers and their expression in multicellular organisms
US7312325B2 (en) 2000-09-26 2007-12-25 Duke University RNA aptamers and methods for identifying the same
US7858591B2 (en) 2000-09-26 2010-12-28 Duke University RNA aptamers and methods for identifying the same
US8143233B2 (en) 2000-09-26 2012-03-27 Duke University RNA aptamers and methods for identifying the same
US7812001B2 (en) 2000-09-26 2010-10-12 Duke University RNA aptamers and methods for identifying the same
US7776837B2 (en) 2000-09-26 2010-08-17 Duke University RNA aptamers and methods for identifying the same
US7776836B2 (en) 2000-09-26 2010-08-17 Duke University RNA aptamers and methods for identifying the same
US7741307B2 (en) 2000-09-26 2010-06-22 Duke University RNA aptamers and methods for identifying the same
US7300922B2 (en) 2001-05-25 2007-11-27 Duke University Modulators of pharmacological agents
US8283330B2 (en) 2001-05-25 2012-10-09 Duke University Modulators of pharmacological agents
US8586524B2 (en) 2001-05-25 2013-11-19 Duke University Modulators of pharmacological agents
US7435542B2 (en) 2002-06-24 2008-10-14 Cornell Research Foundation, Inc. Exhaustive selection of RNA aptamers against complex targets
US7723315B2 (en) 2004-04-22 2010-05-25 Regado Biosciences, Inc. Modulators of coagulation factors
US7531524B2 (en) 2004-04-22 2009-05-12 Regado Biosciences, Inc. Modulators of coagulation factors with enhanced stability
US7304041B2 (en) 2004-04-22 2007-12-04 Regado Biosciences, Inc. Modulators of coagulation factors
US8389489B2 (en) 2004-04-22 2013-03-05 Regado Biosciences, Inc. Modulators of coagulation factors
US8859518B2 (en) 2004-04-22 2014-10-14 Regado Biosciences, Inc. Modulators of coagulation factors

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