WO1997048715A1

WO1997048715A1 - Modulation of tetraplex formation by chemical modifications of a g4-containing oligonucleotide

Info

Publication number: WO1997048715A1
Application number: PCT/US1997/009842
Authority: WO
Inventors: Jia Liu Wolfe; John Goodchild
Original assignee: Hybridon, Inc.
Priority date: 1996-06-19
Filing date: 1997-06-09
Publication date: 1997-12-24
Also published as: AU3379597A

Abstract

This invention provides oligonucleotides having a region of four or more G nucleotides at or near their 5' end and in which from one to all of the 5'-most nucleotides bears a 2' substituent, preferably methoxy, the 3'-terminal nucleotide of the oligonucleotide bears a hydrophobic moiety, preferably cholesterol, or both the 3'-terminal nucleotide bears a hydrophobic moiety and the 5'-most nucleotides are 2'-substituted. Both attachment of a hydrophobic moiety to the 3'-terminal nucleotide of the oligonucleotide and 2'-substitution at the 5'-end results in increased tetraplex stability. The presence of these Hoogsteen base-paired G-quartet structures, however, does not hamper their ability to recognize and form Watson-Crick base-paired duplexes with complementary oligonucleotides. Accordingly, the invention also provides methods of using the disclosed oligonucleotides for nucleic acid expression inhibition.

Description

Modulation of Tetraplex Formation by Chemical Modifications of a G₄-Containing Oligonucleotide

BACKGROUND OF THE INVENTION

Field of the Invention This invention relates to compounds and methods useful in the study of tetraplex forming oligonucleotides as, inter alia, anti-viral and antisense agents. Summary of the Related Art

Antisense oligodeoxynucleotides can inhibit cellular and viral gene expression in a sequence specific manner. Their specificity, stemming from Watson-Crick base pairing with targeted RNA, provides opportunities for the development of therapeutics for human diseases including cancer and viral infections. For a review, see Field and Goodchild, J. Exp. Opin. Invest. Drugs 4, 799 (1995). For example, it has been previously reported that a 20 residue phosphorothioate oligonucleotide 5'-d(TGGGGCTTACCTTGCGAACA)-3' (PS (SEQ ID NO 2) in Table 1 ) is a potent antisense inhibitor of human cytomegalovirus (HCMV). Smith and Pari, J. Virol. 69, 1925 (1995); Pari et al., Antimicrob. Agents Chemother. 39, 1157 (1995).

PS contains four contiguous guanosine residues (G₄) near its 5'-end. Such runs of consecutive guanine bases in RNA or DNA can self-assemble into four-stranded tetraplexes via guanine-guanine Hoogsteen base pairs. Williamson et al., Cell 59, 871 (1989); Sen and Gilbert, Nature 344, 410 (1990). These tetraplexes contain stacks of guanine quartet (G-quartet) planes with the phosphate backbones running in either parallel or antiparallel orientation; both have been observed by X-ray and NMR analysis. Aboul-ela et al., Nature 360, 280 (1992); Laughlan et al., Science 265, 520 (1994); Kang, Nature 356, 126 (1992); Smith, Biochemistry 32, 8682 (1993).

In addition to antisense inhibition (Higgins, Proc. Natl. Acad. Sci. USA 90, 9901 (1993); Pari et al., Antimicrob. Agents Chemother. 39, 1157 (1995)), G₄-containing oligonucleotides can show other biological activity. A G-quartet structure made of phosphorothioate oligonucleotides inhibited cell fusion of HIV via interactions with a virus envelope protein (Wyatt et al., Proc. Natl. Acad. Sci. USA 91, 1356 (1994)), and a non-antisense effect of G₄ was responsible for the antiproliferative activity of c-myb and c-myc "antisense" oligonucleotides. Burgess, Proc. Natl. Acad. Sci. USA 92, 4051 (1995). Oligonucleotides capable of G-quartet formation might possess special biological properties in vivo, as implicated by discoveries of proteins that promote the formation of tetraplex structures (Fang and Cech, Cell 74, 875 (1993)) and elicit specific cleavages in their vicinity (Liu and Gilbert, Cell 77, 1083 (1994)). The antisense and/or antiviral efficacy of a G₄-containing oligonucleotide could therefore be influenced by its tendency to form tetraplexes.

In view of the potentially important role that oligonucleotide tetraplex structures may play in a variety of biological processes, compounds and methods to aid in the elucidation of tetraplex function are desirable.

SUMMARY OF THE INVENTION

The present invention provides novel compounds and methods useful as scientific tools for the study of oligonucleotide tetraplex structures. The compounds of the invention form more stable oligonucleotide tetraplexes than previously known compounds, making them superior for the study of the function of oligonucleotide tetraplexes. Concomitantly, the instant invention also provides methods for studying tetraplex function in antisense inhibition of nucleic acid expression and other biological process in which tetraplex structures are thought to play a role.

Oligonucleotides according to the invention also form duplexes with complementary RNA and are therefore useful as antisense agents. Accordingly, the present invention also comprises methods of using these oligonucleotides to inhibit nucleic acid expression.

In particular, the compounds of the invention comprise oligonucleotides having four or more contiguous G nucleotides at or near their 5' end. In addition, from one to all of the G nucleotides of the aforementioned four or more contiguous G nucleotides (along with any nucleotides 5' to the contiguous G nucleotide sequence ) is 2'-0-substituted (preferably with methyl), or the 3' end of the oligonucleotide bears a hydrophobic (preferably cholesterol) moiety, or the oligonucleotide has both of these structural features.

The foregoing merely summarizes certain aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any manner. All patents and other publications are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 display the effect of chemical modifications on the relative amounts of tetra- stranded vs. single-stranded oligonucleotides.

Fig. 2 displays formation of mixed-stranded tetraplexes.

Fig. 3 displays hybridization of various oligonucleotides with Sense RNA (SEQ ID NO 1 ).

Fig. 4 displays two tetraplex structures formed by oligonucleotides according to the invention. DETAILED DESCRIPTION OF THE INVENTION

2'-0-methylated oligonucleotides have been shown to hybridize to complementary RNA with increased affinity, possibly resulting from stabilization of its A-form conformation. Cummins et al., Nucleic Acids Res. 23, 2019 (1995). A cholesterol substituent in oligonucleotides has been shown to stabilize duplexes and triplexes by inter-strand hydrophobic interactions. Letsinger, et al., J. Am. Chem. Soc. 115, 7535 (1993); Gryaznov and Lloyd, Nucleic Acids Res. 21, 5909 (1993). We have found that one or both of the foregoing structural features in G₄-containing oligonucleotides enhance the stability of tetraplexes formed by such oligonucleotides. The oligonucleotides of the invention are generally from 8 to 50 nucleotides in length, and preferably 20 to 35 nucleotides in length. They may comprise any of the purine or pyrimidine bases, including, but not limited to, any of their equivalents or derivatives known to those skilled in the art. Similarly, the oligonucleotide backbone may comprise any of the known internucleotide linkages or their equivalents or derivatives, including, but not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, alkyl- or aryl- phosphonates, phosphoramidates, carbamates, etc. Similarly, the sugar moiety may be modified, for example at the 2' position with alkyl (e.g., methyl) or other groups known by those skilled in the art to be useful. As used herein, the term "oligonucleotide" is intended to encompass molecules having one or more of any of the foregoing structural features. The oligonucleotides of the invention have at least two distinctive structural features.

First, they contain a sequence of contiguous G nucleotides at or near the 5' end, which sequence is sufficient to enable the oligonucleotide to form a tetraplex structure in conjunction with other oligonucleotides having a contiguous G nucleotide sequence at or near its 5' end. In general, this sequence of contiguous G nucleotides will be four or more G nucleotides in length. As used herein, the phrase "at or near the 5' end" means that the G nucleotide sequence is from 0 to 4 nucleotides from the 5' end. In other words, the contiguous G nucleotide sequence is "at" the 5' end (i.e., 0 nucleotides from the 5' end) when the 5' terminal nucleotide of the oligonucleotide is also the 5'-most G of the contiguous G sequence. When the G nucleotide sequence is "near" the 5' end, there are from 1 to 4 nucleotides 5' to the G nucleotide sequence (i.e., the contiguous G nucleotide sequence is from 1 to 4 nucleotides from the 5' end of the oligonucleotide). Preferably, the sequence of G nucleotides is four G nucleotides long and is 0 or 1 nucleotide from the 5' end. In one embodiment of the present invention, the second structural feature of the oligonucleotides of the invention consists of 2' substituents on from one to all of the 5'-most nucleotides. Any chemical substituent that stabilizes the C3'-endo conformation when bound at the 2' position can be used in the present invention. A number of these are known to those skilled in the art and include, -N₃, -F, -Cl, and -OR where R is methyl, ethyl, propyl, allyl, and methoxyethoxy. When more than one of the 5'-most nucleotides bears a 2' substituent, each of the 2' substituents may be the same as or different from the other 2' substituents. In a preferred embodiment of the invention from one to all of the 5'-most nucleotides are modified with a methyl group at the 2'-0 position, i.e., the 2'-substituent is a methoxy moiety. As used herein, the phrase "the 5 '-most nucleotides" means all the nucleotides in the sequence from the 5'-most nucleotide through (in the 3'-direction) the 3'-most G of the contiguous G nucleotide sequence. As demonstrated herein, the more 2'-0-substituted nucleotides at the 5'-end, the more stable the tetraplex formed by the oligonucleotide. Accordingly, by varying the number of 2'-O-substituted nucleotides at or near the 5'-end, the skilled artisan can finely tune the stability of the tetraplex under consideration to the desired degree.

In another embodiment of the present invention, the second structural feature comprises a hydrophobic moiety linked to the oligonucleotide at its 3' end. Such hydrophobic moiety can be, but is not limited to cholic acid, retinoic acid, long hydrocarbon chains (e.g., C_π or C,₂), Vitamin E, phospholipids, glycerol based compounds such as l ,2-di-0-hexadecyl-3-glyceryl, other steroidal compounds such as cholanic acid, and long chain fatty acids. A number of these hydrophobic moieties are known in the art and can be found, for example, in Kabanov et al., FEB 259, 327 (1990), Saison-Behmoaras et al., EMBO J. 10, 1 1 1 1 (1991 ), Jvlackellar et al., Nucleic Acids Res. 20, 341 1 (1992), Shea et al., Nucleic Acids Res. 18, 3777 (1990), and references cited in each of the foregoing. Preferably, the hydrophobic moiety is a cholesterol group.

In another embodiment of the present invention, both of the foregoing second structural features are present. That is, in this embodiment, one or more of the 5' most nucleotides is 2'- substituted and the 3' end of the oligonucleotide bears a hydrophobic moiety. In a preferred embodiment of this aspect of the invention, the hydrophobic group is a cholesterol moiety, and the G-containing sequence consists of four 2'-0-methylated G nucleotides, which G-containing sequence is one nucleotide away from the 5' end.

Synthesis of the compounds according to the invention can be accomplished by any of the art known methods. E.g., Shea et al., supra. Oligonucleotides of each of the foregoing embodiments have been found to form tetraplexes having superior stability as compared to tetraplexes formed from oligonucleotides without either of the second structural features disclosed herein. Oligonucleotides according to the invention having a 3'-hydrophobic moiety (with or without also having one or more of the 5'- most nucleotides 2'-substituted) generally form more stable tetraplexes than those having only the 2'-substituted second structural feature. The degree of tetraplex stability may be adjusted, therefore, by varying the number of 2'-substituted nucleotides and including or omitting a 3'- hydrophobic moiety.

Oligonucleotides of the invention will form homo-tetraplexes (i.e., tetraplexes comprised of four identical oligonucleotides), or hetero-tetraplexes (i.e.. tetraplexes comprised of two or more different oligonucleotides).

From the foregoing the skilled artisan will appreciate that the oligonucleotides of the present invention are valuable scientific research tools for use in the study of oligonucleotide tetraplex function in a variety of biological settings. By enhancing the stability of oligonucleotide tetraplexes, the oligonucleotides of the invention can be used to probe the effects of tetraplexes and increase the efficacy of antisense and/or antiviral applications of tetraplexes. Furthermore, the enhanced stability of this structural motif might result in favorable pharmacokinetic properties such as cellular uptake, distribution, and metabolism, thereby further emphasizing the importance of studying tetraplexes and the resultant need for useful tools to study them.

Oligonucleotides according to the invention also are capable of forming duplexes with complementary RNA. Tetraplexes of oligonucleotides having the additional 2'-substituted second structural feature readily dissociate to form duplexes with complementary RNA. Tetraplexes of oligonucleotides having 3'-hydrophobic moieties (with or without the 2'- substituted structural feature) dissociate somewhat slower, initially having about half of the tetraplexes remaining intact in the presence of complementary RNA. Prolonged incubation increases duplex formation, indicating that although the kinetics are less favorable, duplex formation is still thermodynamically favored. This feature is useful for prolonged or "timed- release" delivery of oligonucleotides. In view of their ability to form duplexes with complementary RNA, those skilled in the art will appreciate that oligonucleotides according to the invention are also useful for inhibiting nucleic acid expression, both in vitro and in vivo. In vitro, the present oligonucleotides are useful tools for modulating gene expression to determine the role of a gene of interest in certain biological processes. See, e.g., Holt et al., Mol. Cell Biol. 8, 963 (1988) ("To study the role of a nuclear proto-oncogene in the regulation of cell growth and differentiation, we inhibited HL-60 c-myc expression with a complementary antisense oligomer.") In vivo, the present oligonucleotides are useful for inhibiting expression of nucleic acids from viruses and other pathogens. See, e.g., Simons et al. (Nature 359, 67 (1992)) (Reporting that a phosphorothioate "antisense c-myb 18-mer locally delivered to a rat with an injured left common carotid artery suppressed c-myb mRNA concentrations 2 weeks after injury and blocked the accumulation of intimal smooth muscle cells"); Ratajczak et al. (Proc. Natl. Acad. Sci. U.S.A. 89, 11823 (1992)) (reporting that "24-mer [phosphorothioate] oligonucleotides targeted to the human c- myb mRNA were infused, through a mini-osmotic pump, into scid mice bearing the human K562 chromic myeloid leukemia cell line. Mean survival times of the mice treated with the antisense oligonucleotide were six- to eightfold longer than those of mice untreated or treated with the sense controls. . . . "); and Kitajima et al. (Science 258, 1792 (1992)) (reporting that "after injecting IP 3 '-[phosphorothioate] modified [phosphodiester] chimeric oligonucleotides that were complementary to the initiation codon region of the NF-κB mRNA (p65), they observed complete tumor involution in 13 out of 13 antisense-treated [transgenic] mice [having that gene] . Untreated or sense-treated mice died by 12 weeks, whereas the treated animals had no recurrence for at least 5 months. ")

In view of the foregoing utilities, therefore, the present invention also comprises methods for using oligonucleotides of the present invention to study the activity and mechanism of oligonucleotide tetraplexes in various biological environments. Such methods comprise introducing one or more oligonucleotides according to the invention into the biological system, either in vitro or in vivo. Additional methods according to the invention comprise inhibiting nucleic acid expression in vitro or in vivo by contacting a nucleic acid with one or more of the oligonucleotides according to the invention.

The following Examples are presented for illustrative purposes only and are not intended, nor should they be construed, to limit the invention in any manner. Those skilled in the art will appreciate that variations on the following can be made without exceeding the spirit or scope of the present invention.

EXAMPLES The oligonucleotides listed in Table 1 were used to demonstrate the tetraplex stability- enhancing nature of the present oligonucleotides. All oligonucleotides were synthesized using standard techniques. Table l^a

^Λ The synthetic oligonucleotides in this table were purified either by HPLC or denaturing PAGE, then precipitated from solution in 0.3 M and 0.1 M NaCl using ethanol and qunatified by UV aborbance at 260 nm. u

Sense RNA has phosphodiester backbones, and the rest contain phosphorothioate backbones unless otherwise indicated. ^c Underlined bases represent unmethylated RNA residues, bold faced bases represent T-O- methyl RNA residues, I represents inosine, and * represents a phosphoramidate linkage P- NH(CH₂)₆NH-CO-O-cholesteryl. Lestsinger et al., J. Am. Chem. Soc. 115, 7535 (1993).

The experiments presented herein demonstrate how 2'-0-methyl and 3'- and/or 5'- cholesteryl modifications on PS influence the formation of G-quartets and affect their ability to form duplexes with complementary RNA.

Non-denaturing PAGE analysis was applied throughout. Each sample analyzed by non- denaturing PAGE contained 0.1 mM oligonucleotide, 10 mM Tris pH 7, 1 mM EDTA, 50 mM NaCl, and was incubated at room temperature for 1 hour before analysis. 4 mL of 20% glycerol were added to each (20 mL) sample before loading onto a 20% (19: 1 acrylamide:bisacrylamide) gel containing 0.5xTBE (45 mM Tris-Borate pH 8, 1 mM EDTA) and 50 mM NaCl. The gel was run in 0.5xTBE/50 mM NaCl buffer at constant voltage of 75 V so that the temperature of the gel remained below 30 °C. After orange G dye in a separate lane reached bottom, the gel was placed on a fluorescent TLC plate and photographed under UV illumination (254 nm). Analyzed by denaturing polyacrylamide gel electrophoresis (PAGE), all the oligonucleotides ran as a single band with the expected mobility. When analyzed by non- denaturing PAGE, however, 4x4 OMe (SEQ ID NO 3) showed formation of a much lower mobility species (lane 8 of Figure 1 ) whose regeneration following heat denaturation was favored more in the presence of KC1 than NaCl of same concentration, typical for tetraplex structures. Raghuraman and Cech, Nucleic Acids Res. 18, 4543 ( 1990).

To unambiguously demonstrate its tetraplex identity, 4x4 OMe (SEQ ID NO 3) was mixed with a shorter, partially 2'-O-methylated lOmer (SEQ ID NO 13), then heat denatured and annealed in the presence of K\ Non-denaturing PAGE analysis showed expected mixed- stranded tetraplexes with five bands of varying intensity (lane 4 of Figure 2). In the mixed strand experiments whose results are displayed in Fig. 2, aqueous solutions (10 mL) containing either 2 nmoles of one oligonucleotide or a mixture of two oligonucleotides (2 nmoles each) were heated at 90 °C for 3 minutes and placed on ice immediately thereafter. Samples were brought to 20 mL containing 10 mM Tris pH 7, 1 mM EDTA, and 200 mM KC1, reheated at 90 °C for 3 minutes and cooled to 30 °C over 2-3 hours before analysis. Non-denaturing PAGE was conducted as described previously. Similar results were observed for 4x0 OMe(SEQ ID NO 7)/10mer (SEQ

ID NO 13) and 4RxO (SEQ ID NO 14)/10mer (SEQ ID NO 13), but 3'-Chol (SEQ ID NO l iyiOmer (SEQ ID NO 13) showed only two distinct bands corresponding to individual tetraplexes of 3'-Chol (SEQ ID NO 1 1) and lOmer (SEQ ID NO 13) (lane 5 of Figure 2). The degree of tetraplex formation depended on the extent of 2'-(9-methylation at the 5'- end: whereas PS (SEQ ID NO 2) and 0x4 OMe (SEQ ID NO 6) did not afford any detectable tetraplexes, the intensity of tetraplex bands increased in the order of 4x4 OMe (SEQ ID NO 3) > 2x4 OMe (SEQ ID NO 4) > 1x4 OMe (SEQ ID NO 5) (lanes 4-8 of Figure 1 ). As expected for Hoogsteen base paired G-quartet structures, replacing a guanosine by inosine dramatically reduced tetraplex formation (compare lane 7, 8 with 9, 10 in Figure 1). It has been demonstrated that an RNA tetraplex (UGGGGU (SEQ ID NO 15))₄ is much more stable than its DNA counterpart (Cheong and Moore, Biochemistry 31, 8406 (1992)), and that RNA forms more stable duplexes with 2'-O-methylated RNA than with DNA. Cummins et al, supra. The results presented herein indicate that 2'-O-methylation of guanine also promotes the formation of G- quartet structures, with either 2'-0-methylated or unmodified guanines (as in 4Rx0 (SEQ ID NO 14)/10mer (SEQ ID NO 13)). Taken together, C3'-endo sugur pucker might have contributed to stablize tetraplex structures.

3'-Chol (SEQ ID NO 1 1 ) ran predominantly as a tetraplex on non-denaturing PAGE, whereas 5'-Chol (SEQ ID NO 10) ran exclusively single stranded (lanes 1 , 3 of Figure 1 ). In 3'- Chol (SEQ ID NO 1 1), the inter-strand hydrophobic interaction of cholesteryl groups could help assemble the tetraplex (Model B, Fig. 4). In the case of 5'-Chol (SEQ ID NO 10), steric effects might impede G-quartet formation due to the proximity of cholesteryl groups to the G₄ motif.

The tetraplex of 3'-Chol (SEQ ID NO 1 1) migrated slightly faster than that of 2'-O- methylated oligonucleotides, although the mobility of the single strand oligomers are similar (compare lane 1 with others in Figure 1 ). An oligonucleotide possessing both modifications, 4xOMe 3'-Chol (SEQ ID NO 12), showed similar mobility to 3'-Chol (SEQ ID NO 1 1 ) (lanes 7, 9 of Figure 3). These findings are consistent with the parallel-stranded tetraplex models depicted as A and B in Fig. 4. A represents the tetraplex structure formed by 4x4 OMe (SEQ ID NO 3), with negatively charged phosphate backbones separated as far as possible. B illustrates that the hydrophobic interactions amongst cholesteryl groups might overcome the charge-charge repulsion and stabilize the tetraplex structure. The observed mobility differences support this proposal: the more compact complex B should migrate faster than A. Circular dichroism spectra of these oligonucleotides are similar, with single maxima at -265-270 nm, consistent with characteristic spectrum reported for parallel-stranded tetraplexes (Chen, J. Biol. Chem. 270, 23090 (1995)) that are generally more stable than corresponding antiparallel structures. Aboul- ela, supra. To probe the effect of tetraplex formation on their ability to hybridize with complementary RNA, these oligonucleotides were incubated with sense RNA (SEQ ID NO 1) in buffered aqueous solutions with NaCl or KC1. Each sample contained 0.1 mM oligoncleotide(s) (antisense:sense=l : l where applicable), 10 mM Tris pH 7, 1 mM EDTA, 100 mM KC1, and was incubated at 37 °C for 1 hour before analysis. PAGE analysis was conducted as described previously. PAGE analysis revealed that tetraplexes of 1x4 OMe (SEQ ID NO 5), 2x4 OMe (SEQ ID NO 4), 4x4 OMe (SEQ ID NO 3) readily dissociated to form duplexes (examples shown in lanes 5, 6 of Figure 3), whereas about half of 3'-Chol (SEQ ID NO 1 1) and 4xOMe 3'- Chol (SEQ ID NO 12) remained as tetraplexes (lanes 7-10). Prolonged incubation increased duplex formation, indicating a slow exchange amongst monomers, duplexes, and tetraplexes. The increased tetraplex stability of 3 -Chol (SEQ ID NO 1 1) and 4xOMe 3'-Chol (SEQ ID NO 12) probably resulted from decreased accessibility of 3'-ends for duplex formation.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Wolfe Ph.D., Jia Liu Goodchild Ph.D., John

(ii) TITLE OF INVENTION: Modulation of Tetraplex Formation by Chemical Modifications of a G4-Containing Oligonucleotide

(iii) NUMBER OF SEQUENCES: 14

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Banner & Allegretti, Ltd.

(B) STREET: 10 South Wacker Drive, Suite 3000

(C) CITY: Chicago

(D) STATE: IL

(E) COUNTRY: USA (F) ZIP: 60606

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Greenfield Ph.D. , Michael S. (B) REGISTRATION NUMBER: 37,142

(C) REFERENCE/DOCKET NUMBER: 96,583

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (312)715-1000 (B) TELEFAX: (312)715-1234

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(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

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(ii) MOLECULE TYPE: other nucleic acid

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCUGCACCCC GAAUGGAACG CUUGUCUGCC 30

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(C) STRANDEDNESS. single

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(iv) ANTI-SENSE. YES (xi) SEQUENCE DESCRIPTION SEQ ID NO:3 :

UGGGGCTTAC CTTGCGAACA 20

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : UGGGGCTTAC CTTGCGAACA 20

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

TGGGGCTTAC CTTGCGAACA 20 (2) INFORMATION FOR SEQ ID NO:7:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 :

UGGGGCTTAC CTTGCGAACA 20

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : UGGGICTTAC CTTGCGAACA 20

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(xi) SEQUENCE DESCRIPTION SEQ ID NO 11 TGGGGCTTAC CTTGCGAACA 20

(2) INFORMATION FOR SEQ ID NO: 12

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12 : UGGGGCTTAC CTTGCGAACA 20

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

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(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

UGGGGCTTAC 10 (2) INFORMATION FOR SEQ ID NO: 14:

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(iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

UGGGGCTTAC CTTGCGAACA 20

Claims

We claim:

1. An oligonucleotide of from 8 to 50 nucleotides comprising a sequence of 4 or more contiguous G nucleotides at or near the oligonucleotide's 5' terminal end, wherein the oligonucleotide has one or both of the following two structure features: (a) a C3'-endo conformation stabilizing 2'-substituent on from 1 to all of the

5'-most nucleotides, and (b) a 3 '-terminal hydrophobic moiety.

2. The oligonucleotide according to claim 1 wherein all of the C3'-endo conformation stabilizing 2'-substituents are methoxy.

3. The oligonucleotide according to claim 2 wherein the oligonucleotide has structural feature (a) but not (b).

4. The oligonucleotide according to claim 2 wherein the oligonucleotide has both structural feature (a) and (b).

5. The oligonucleotide according to claim 1 wherein 3'-terminal hydrophobic moiety is cholesterol.

6. The oligonucleotide according to claim 5 wherein the oligonucleotide has structural feature (b) but not (a).

7. The oligonucleotide according to claim 5 wherein the oligonucleotide has both structural feature (a) and (b).

8. The oligonucleotide according to claim 4 wherein the 3'-terminal hydrophobic moiety is cholesterol.

9. The oligonucleotide according to claim 1 wherein the sequence of contiguous G nucleotides is 0 or 1 nucleotide from the 5' end.

10. The oligonucleotide according to claim 3 wherein the sequence of contiguous G nucleotides is 0 or 1 nucleotide from the 5' end.

1 1. The oligonucleotide according to claim 6 wherein the sequence of contiguous G nucleotides is 0 or 1 nucleotide from the 5' end.

12. The oligonucleotide according to claim 8 wherein the sequence of contiguous G nucleotides is 0 or 1 nucleotide from the 5' end.

13. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 1.

14. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 2.

15. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 3.

16. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 4.

17. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 5.

18. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 6.

19. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 7.

20. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 8.

21. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 9.

22. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 10.

23. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 1 1.

24. A method of inhibiting nucleic acid expression comprising contacting the nucleic acid with an oligonucleotide according to claim 12.