CA2153057A1 - Novel oligonucleotides modified with non-nucleotide bridging groups - Google Patents

Abstract

An oligonucleotide having a structural formula selected from the group consisting of (a) and (b). S1, S2, S3, S4, and S5 are oligonucleotide strands, and each of X1 and X2 is a bridging moiety which may be a nucleotide strand or a non-nucleotide bridging moiety.
Each of X1 and X2 may be the same or different, with the proviso that when one of X1 and X2 is a nucleotide strand, the other of X1 and X2 is a non-nucleotide bridging moiety. Each of X1 and X2 independently is a bridging moiety having first and second termini that each binds independently with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety. Such oligonucleotides are designed to have increased resistance to exonucleases and endonucleases, greater thermal stabilities, improved cellular uptake, and improved binding to target proteins and nucleic acids.

Description

~094/15620 2 1 $ 3 ~ 5 7 PCT~S94/00585 NOVEL OLIGONUCLEOTIDES MODIFIED WITH NON-NUCLEOTIDE
BRIDGING GROUPS

This invention relates to oligonucleotide duplexes.
More particularly, this invention relateJ to &ingle or double-stranded oligonucleotides in which the 5' and 3' end~
of opposing oligonucleotide strand~ are linked by novel ~ridging groups.
The synthe~is of single or double-stranded oligonucleotides with natural nucleotide bridging groups has been deJcribed in the literature. Erie, et al., Biochemistry, Vol. 28, 268-273 (1989) describe the synthe~is of a 26-residue double-stranded circular oligonucleotide using five thymidylate re~idues for each bridging group.
Formation of the closed circular form wa~ carried out using T4 DNA liga~e. A~hley, et al., BiochemistrY~ Vol. 30, pgs.
2927-2933 (1991), di~close the con~truction of cyclic oligonucleotide~, or dumbbells, which employ four thymidyllc acid r~Jidue~ a~ the bridging group~. Cyclization of the linear ollgonucleotide precursor wa~ achieved using a carbodiimidc as the coupling agent. Amaratunga, et al., Biopolymer~, Vol. 32, pgq. 865-879 (1992), diJclo~e the preparation of S~tJ of double-~tranded oligonucleotides having 16 b~se pair~ in which the number of thymidylate bridging group6 wa~ varied between 2 and 14. These oligonucleotideJ were also circularized u~ing an enzymatic W094/15620 ~53~ -2- PCT~S94tO058~

method. Single-Stranded circular oligonucleotides have also been d~cribed in Kool, J. Am. Chem. Soc., Vol. 113, pg~.
6265-6266 (1991), by Prakash, et al., J. Chem. Soc. Chem.
Commun., pgs. 1161-1163 (1991), and by Prakash, et al., J.
Am. Chem. Soc., Vol. 114, pgs. 3523-3527 (1992).
Chu, et al., Nucl. Acids Res., Vol. 19, pg. 6958, disclo~e the binding of hairpin and dumbbell DNA sequences to transcription factors. These DNA sequence~ employed natural nucleotide~ for the bridging group~.
Durand, et al., Nucl. Acids Re~., Vol. 18, pgs.
6353-6359 (1991) dlsclo~e self-complementary oligonucleotides containing a hairpin loop, or bridging group. The loop consists of a hexaethylene glycol chain, and the oligonucleotide could form a hairpin structure a~
effectively a~ the analogous oligonucleotide possessing thymidylate re~idues for the bridging group~.
Glick, et al., J. Am Chem. Soc., Vol. 114, pgs, 5447-544B (1992), di~close the formation of a double-stranded oligonucleotide which is cros~linked through linker arms attached to the ba~es rather than the sugar phosphate backbone.
In accordance with an a~pect of the pr~ent invention, there is provided an oligonucleotide having a structural formula ~elected from the group consi~ting of:

51 ' I - S,~_ Xl X2 and X1 X~2 L

Sl, S2, 53, S4, and S5 are oligonucleotlde strands.
Each of X1 and X2 is a nucleotide ~trand, such as a nucleotide bridging loop, or a non-nucleotide bridging W094/15620 ~ S 3 0~ 7 PCT~S94/00~85 moiety. Each of X1 and X2 may be the same or different, and when one of X1 and X2 is a nucleotide strand, the other of X1 and X2 i8 a non-nucleotide bridging moiety. Thus, each of X1 and X2 independently is a bridging moiety ha~lns flrst and second termini that each bind~ independently with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety Examples of terminal moieties that bind with phosphate moieties; e.g., terminal phosphates of nucleic acid sequences, include, but are not limited to, -OH groups, -NH2 groups, and -SH groups.
Examples of terminal moieties that bind with a hydroxyl moiety, particularly the terminal hydroxyL moiety of a nucleic acid ~equence (e.g., the ribose of a 3' terminal nucleotide), including, but are not limited to, _po32 group~, -SO3 groups, and -COO groups.
When Xl and/or X2 is a non-nucleotide bridging moiety, the non-nucleotide bridging moiety may have the following structural formula:
Tl - ~ - T2, whereas each of T1 and T2 independently binds with a nucleotide pho~phate moiety or a hydroxyl moiety. R i~ ~elected from the group con~isting of (a) ~aturated and un~aturated hydrocarbons; (b) polyalkylene glycols; (c) polypeptides; (d) thiohydrocarbons; (e) polyalkylamine~; tf) polyalkylene thioglycols; (g) polyamide~; (h) dicub~titute~ monocyclic or polycyclic aromatic hydrocarbons; (i) intercalatinq agents; (j) mono~accharidea; and ~k) oligo~accharides; or mixtures thereof.
In one embodiment, one of Tl and T2 binds with a nucleotide pho~phate moiety, and the other of T1 and T2 binds with a nucleotide hydroxyl moiety.

W094/15620 2 1 S 3 ~ S 7 PCT~S94/00585 In one embodiment, the oligonucleotide has the structural formula:
- - Sl-Xl X2 S

In one embodiment, at least a portion of S1 is complementary to S2. In another embodiment, all of S1 and 52 are complementary to each other ~uch that Sl and 52 bind to form a double-stranded region.
In yet another embodiment, 51 is not complementary to 52~ and the oligonucleotide molecule exists a~ an unpaired oligonucleotide.
In another embodiment, the oligonucleotide has the structural formula:

Xl X2 s4 S5 In one embodiment, at least a portion of 53 is complementary to portions of S4 and/or S5. In another embodiment, all of 54 and S5 are complementary to 53 such that S4 ~nd S~ bind to 53 to form double-~tranded regions.
In yet another embodiment, S3 is not complementary to S4 and S5, and formation of double-stranded regions is not possible, ~o that the oligonucleotide molecule exists as an unpaired oligonucleotide.
The term "ollgonucleotide" as used herein means that the oligonucleotide may be a ribonucleotide, deoxyribonucleotide, or a mixed W094/lS620 21 S3D~ 7 PCT~S94/00585 ribonucleotide/deoxyribonucleotide; i.e., the oligonucleotide may include ribo~e or deoxyribose sugars or both. Alternatively, the oligonucleotide may include other 5-carbon or 6-carbon sugars, such as, for example arabinose, xylose, glucose, galactose, or deoxy deri~atives thereof or any mixture of sugars.
In one embodiment, each of Sl, and S2, or S3, and 54 and S5 combined may include from about 5 to about lOO
nucleotide units, and preferably from about lO to about lO0 nucleotide units.
The phosphorus containing moieties of the oligonucleotide may be, for example, a phosphate, phosphonate, alkylphosphonate, aminoalkyl phosphonate, thiophosphonate, phosphoramidate, phosphordiamidate phosphorothioate, phosphorothionate, phosphorothiolate, phosphoramidothiolate, and pho~phorimidate. It i~ to be understood, however, the scope of the present invention is not to be limited to any specific phosphorus moiety or moieties. The pho~phorus moiety be modified with cationic, anionic, or zwitterionic moieties. The oligonucleotides may also contain backbone linkages which do not contain phosphorus, such as carbonates, carboxymethyl e~ter~, acetamidates, carbamates, acetals, and the like.
The oligonucleotides may include any natural or unnatural, Jub~tltuted or unsubstituted, purine or pyrimidine ba~e. Such purine and pyrimidine bases include, but are not limited to, natural purines and pyrimidines, such a~ adenine, cytosine, thymine, guanine, uracil, or other purines and pyrimidines, such as isocytosine, 6-methyluracil, 4, 6-di-hydroxypyrimidine, hypoxanthine, xanthine, 2, 6-diaminopurine, 5-azacyto~ine, 5-methyl cytosine and the like.
In one embodiment, each of X1 and X2 i~ a non-nucleotide bridging moiety having the formula T1-R-T2, W094/15620 PCT~S94/00585 2153~57 -6-as hereinabove de8cribed. In one embodiment, R i8 a ~aturated or un~aturated hydrocarbon, and preferably R is a polyalkylene moiety wherein the polyalkylene group has from 5 to lOO carbon atoms, preferably from 5 to 20 carbon atoms.
Most preferably, the polyalkylene is a polymethylene moiety The bridging moiety including the polyalkylene group may be attached to the sugar phosphate backbone of the oligonucleotide.
In another embodiment, R i8 a polyalkylene glycol. In particular, the polyalXylene glycol has the structural formula (R~O)n, wherein R is an alkylene group having from 2 to 6 carbon atoms, preferably from 2 to 3 carbon atom~, and n is from 1 to 50, preferably from 3 to 6. In one embodiment, the polyalkylene glycol i~ polyethylene glycol, and preferably the polyethylene glycol is he~aethylene glycol.
Bridging moieties including polyalkylene glycols may be attached to the oligonucleotide by converting the polyalkylene glycol into a material which may be employed in a DNA ~ynthesizer. For example, the polyalkylene glycol may be converted to its mono-dimethoxytrityl ether, which is then reacted with chloro-N, N-dii~opropylamino-cyanoetho~y-pho~phine to produce a bridging group phosphoramiditc. For oligonucleotide synthe~is, a pho~phorylating agent is attached to a solld ~upport of a DNA ~ynthe~izer, and a series of DNA ba~e~ i~ delivered in order, dc ~ ing upon the sequence required for binding to the target DNA, RNA, protein or peptide. The bridging group phosphoramidite i8 then added, followed by the addition of a further sequence of DNA bases. Optionally, another bridging group is then attached, and a further sequence of DNA bases is added to complete the oligonucleotide ~equence.
At the conclu~ion of the synthe~is, the oligonucleotlde is cleaved from the ~olid support wlth ammonia to give a WO94/15620 ~57 PCT~S94/00585 crude trityl-containins oligonucleotide pos~e~sing a 3'-pho~phate group. Purification i8 carried out u~ing rever~ed pha~e HPLC and the later eluting, trityl-containing oligonucleotide i8 collected. The oligonucleotide is detritylated ucing acetic acid, extracted with ethyl acetate to remove trityl alcohol, and lyophilized to give an oligonucleotide which can hybridize to it~elf to form an open chain 3'-phocphorylated oligonucleotide. Reaction of thi~ open chain oligonucleotide with a carbodiimide coupling agent in an aqueous buffer produces a clo~ed circular oligonucleotide. In one procedure, small portion~ of the carbodiimide coupling agent are added at infreguent interval~ a~ de~cribed in Example 2. In another procedure, a large exces~ of coupling agent is added at the beginning of the procedure as described in Exumple 4.
Unpaired open chain oligonucleotide~ can be circularized using a coupling agent such a~ cyanogen bromide in the pre~ence of a complementary oligonucleotide ~plint as described by Praka~h, et al., J. Chem. Soc. Chem. Commun., pgs. 1161-1163 (1991) or using a water ~oluble carbodiimide in the pre~ence of a complementary oligonucleotlde ~plint as described by Dolinnaya, et al, Nucl. ACidc Re~., Vol. 16, pgc 3721-373B (1988).
In another embodiment, R i~ a polypeptide.
Polypeptid-- whlch can be included in the bridging groups include, but are not limited to, hydrophobic poly~peptides such a~ (Ala)n, basic polypeptides Cuch a~ (Lysn), and ac~dic polypeptldes ~uch as (Glu)n, wherein n ic from 3 to 50, preferably from 4 to 10. In an alternative embodiment, the polypeptideo may contain mixture~ of amino acids.
Such bridging groups including polypeptides may be attached to the oligonucleotide by procedures such as that given in Example 7 hereinbelow.

WO94/15620 PCT~S94/00585 215~7 In yet another embodiment, R is a poLyalkylamine.
Polyalkylamine~ which may be included in the bridging groups include, but are not limited to tho9e having the following structural formula:
R3NHl(cH2)m NHRl]p ~ [(CH2)n MHR2lq wherein each of Rl, R2, and R3 is hydrogen or an alkyl group having from 2 to lO carbon atoms, and wherein m and n are from 2 to lO, preferably from 2 to 4, and p and q each are from 2 to 20, preferably from 3 to 6. Rl, R2 and R3 may be the same or different, m and n can be the ~ame or different, and p and q can be the ~ame or different. Examples of such polyamines include polyethylene imine, which has the formula H2N(CH2 CH2 NH)rH, wherein m and n each are 2, p+q=r, and Rl, R2, and R3 are H; and spermidine, which has the

2 ( H2)4 NH-(CH2)3-NH2, wherein m i~ 4, n is 3 p is l, and q i 8 l, and each of Rl, R2, and R3 i~ hydrogen Ex~mples of polyalkylene thioglycols which may be included in the bridging groups include, but are not limited to,

3,6 dithio-l,8-octanediol,HOCH2CH2SCH2CH25CH2CH2OH, 2-mercaptoethyl sulfide, (HSCH2CH2)2S, 3,3'-thiodipropanol, S(CH2CH2CH2OH)2, 2-mercaptoethyl ether, (HSCH2CH2)2O, and 2,2'-dithiodlethanol S(CH2CH2OH)2.

E~ample~ of thiohydrocarbons such as polyalkylene disulfides which may be employed ln the bridging groups include, but are not limited to, 2-hydroxyethyl di~ulfide (HOCH2CH2)252 An example of incorporation of one of these moietie~ as a bridging group i~ described in Example 9 hereinbelow.
In another embodiment, R i~ a polyamide.
Polyamides which may be included in the bridging group~
include tho~e having the following structural formula:

~094/15620 1S30S~ PCT~594/0~585 H~NH(CH2) -CO1 OH
wherein m i8 from l to 6, and n is from 3 to 50.
Bridging groups containing such polyamides may be attached to the oligonucleotide by procedures such as those given in Example 7 hereinbelow.
In yet another embodiment, R is a disubstituted monocyclic aromatic. Disubstituted monocyclic aromatics which may be included in the bridging groups include those having the following structural formula:
(CH2)m(-x ~ Y)n-(CH2)m, wherein each of X and Y
is -CONH, or X is -NHCO and Y is COMH, m is from l to lO and n is from l to 5. In another embodiment,-the disubstituted monocyclic aromatic may have the following structural formula:
( O X)n, wherein X i5 oxygen, sulfur, or -OCH2, and n is from l to lO.
In another embodiment, R is a disubstituted polycyclic aromatic hydrocarbon. Disubstituted polycyclic aromatic hydrocarbon~ which may be employed include, but are not limited to, di~ubstituted naphthalenes, anthracenes, phenanthrenes, fluorenes, and pyrene~. Bridging moieties containing di~ubstituted aromatics may be attached to the oligonucleotide by procedures such as given in Example lO
hereinbelow.
In yet another embodiment, R i~ an intercalating agent.
Intercalating agent~ which can be included in the bridging moieties include, but are not limited to, acridine~, phenanthrid~nes, anthracyclinones, phenazines, phenothiazine~, ~nd qu~nollnes. Bridging moieties including such agents may be attached to the oligonucleotide by procedure~ such as those given in Example ll hereinbelow.
In another embodiment, R is a monosaccharide, or in yet another embodiment, R is a oligosaccharide. Monosaccharides and oligosaccharide~ which may be included in the bridging W094/15620 PCT~S94/00585 ~1S3~7 -lo-group~ include, but are not limited to, glucose, mannoce, and galactose; disaccharides such as cellobiose and gentobio~e; trisaccharides such a~ cellotriose; and larger oligosaccharides such as cellotetraose, cellopentaose, and pentamannose.
Bridging moieties containing such monosaccharides and oligosaccharide~ may be attached to the oligonucleotide by procedures such as those given in Example 12 hereinbelow.
The oligonucleotides of the present invention may be employed to bind to RNA ~equences by Watson-Crick hybridization, and thereby block RNA proce~sing or translation. For example, the oligonucleotide~ of the present invention may be employed as "antisense" complements to target ~e~uences of mRNA in order to effect translation arrest and selectively regulate protein production.
The oligonucleotides of the pre~ent invention may be employed to bind RNA or DNA to form triplexe~, or triple helice~. Single stranded oligonucleotide~ have been described to bind double-stranded DNA and thereby interfere with transcription in Maher, et al., 8iochemistrY, Vol. 29, pgs. B820-8826 (1990) and in Orson et al., Nucleic Acids ReE ., Vol. 19, pgs. 3435-3441 (1991).
Similarly, ~uch triplex formation could be expected to interfere with replication. SingLe-stranded oligonucleotide~ could also be envisaged to bind to double-ctranded (e.g., viral) RNA to form triplexes which block transcription or reverse transcription. Circular paired oligonucleotides may be employed to form "rever~e triplexes" in which the paired oligonucleotides form triplexe~ with a ~ingle-stranded RNA or DNA target, thereby blocking transcription, replication or reverse transcriptior of said RNA or DNA target.
Triple helix formation by oligonucleotides in a ~equence-~pecific manner is normally re~tricted to O 94/1~620 ~S PCT/llS94/00585 ?

polypurine tract9 of duplex DNA. In order to increase the number of targets for triple helix formation, Froehler et al., in BiochemistrY, Vol. 31, pg~. 1603-1609 (1992) and Horne, et al., in J. Am. Chem. Soc., Vol. 112, pgs.
2435-2437 (1990) utilized oligonucleotides containing a 3',3' internucleotide junction or linker group to allow for bindlng to opposite strands of DNA. Unpaired circular oliqonucleotides of the pre~ent invention can be employed to form "~witchover" complexe~ with double-stranded DNA or RNA
as shown in the following ~tructure:

nucleic acid target oligonucleotide In such complexes, pyrimidines on one portion of the bridged cyclic oligonucleotide interact via Hoogsteen interaction~ with purine~ on one strand of the nucleic acid target, while pyrimidines on another portion of the oligonucleotide interact with purines on the other strand of the target. Structure~ of this type do not need an interveninq linker group and have the added advantage that stacking interactions are maintained. The bridging residues and the cyclic nature of the oliqonucleotide serves to minimize degradation by nuclea~es. Unpaired cyclic oligonucleotide~ forminq switchover comple~e~ as described herein can be envi~aged to occur with target double-stranded WO94/15620 215 3 ~ 5 ~ PCT~S94/00~85 DNA or RNA, thereby blocking transcription, replication, or rever~e transcription.
The paired or unpaired circular oligonucleotides of the pre~ent invention may be employed to bind specifically to target protein~, or to selected regions of target proteins 80 a~ to block function or to restore functions that had been lost by a protein a~ a result of mutation. For example, the oligonucleotide~ of the pre~ent invention may be used to block the interaction between a receptor and its ligand(s) or to interfere with the binding of an enzyme to its sub~trate or cofactor or to interfere otherwise with the catalytic action of an enzyme. Convercely, the oligonucleotides of the pre~ent invention may be employed to restore lost function to a mutated protein, for example, by eliciting conformational alteration of such a protein through formation of a complex with that protein.
In another embodiment, the oligonucleo'ide~ may bind to tran~criptional activators or suppre~sors. Such factors might, for example, enhance transcription of cellular DNA, in order to regulate cellular gene expression. As a further example, the oligonucleotide~ may inhibit the action of the protein encoded by the mvb oncogene, which act~ as a tran~criptional activator (Gabriel~en, et al., Science, Vol 253, pgs. 1140-1143 (1991)). When this protcin is inappropriately expressed, it can activate genes leading to the formatlon of a cancer. 8inding of the myb protein to the oligonucleotide~ of the pre~ent invention would block the gene activation and block the growth of the cancerous cell~.
Alternatively, the oligonucleotides may bind to viral transcription factors. For example, the oligonucleotides may inhlbit human immunodeficlency (HIV) tran~criptional activatorr or enhancer~ or bovine or human papilloma virus tran~criptional activators or enhancer~. Alternatively, the WO94/15620 1 S3 ~ ~ 7 PCT~S94/00585 -l3-oligonucleotide~ may activate gene expression by binding to and preventing activity of, transcriptional repressors.
Bielinska, et al., Science, Vol. 250, pgs. 997-lOOO
(November 16, l99O), disclose double-stranded phosphorothioate oligonucleotides which bind to transcription factors or enhancers of viru~es such as HIV
Such oligonucleotides may also be added to Jurkat leukeml a T- cells in order to inhlbit interleukin-2 secretion.
Androphy, et al., Nature, Vol. 325, pgs. 70-73 (January 1, 1987), disclose a 23 ba~e pair oligonucleotide which prevents binding of the E2 protein of bovine papilloma virus (BPV) to the upstream regulatory region of the BPV genome, which immediately precedes the early genes of the 8PV
genome. European Patent Application No. 302,758 discloses double-stranded oligonucleotide~ which bind to transcription enhancers of bovine papilloma virus or human papilloma virus, thereby repressing the transcription of the DNA of the virus and lnhibiting the growth of the viru~. The above oligonucleotides disclosed in the above-mentioned publications may be modified to include the bridging moieties of the pre~ent invention and still be employed for binding to transcription factors or en~an~er~.
The RNA, DNA, protein or peptide target of interest, tc which the oligonucleotide binds, may be present in or on a prokaryotic or eukaryotic cell, a virus, a normal cell, or a neoplastic cell, in a bodi!y fluid or in stool. The target nucleic acid~ or proteins may be of plasmid, viral, chromo~omal, mitochondrial or plastid origin. The target sequence~ may include DNA or RNA open reading frames encoding proteins, mRNA, ribosomal RNA, JnRNA, hnRNA, introns, or untranslated 5'- and 3'-sequences flanking DNA
or RNA open readlng frames. The modified oligonucleotide may therefore be involved in inhibiting production or function of a particular gene by inhibiting the express~on W094/15620 PCT~S94/00~85 ~I53~7 -14-of a repressor, enhancing or promoting the function of a particular mutated or modified protein by eliciting a conformational change in that protein, or the modified oligonucleotide may be involved in reducing the proliferation of viruses, microorganisms, or neoplastic cells. The oligonucleotides may al~o target a DNA origin of replication or à rever~e tranqcription initiation site.
The oligonucleotide~ may be u~ed in vitro or in vivo for modifying the phenotype of cells, or for limitinq the proliferation of pathoqens such as viruses, bacteria, protists, Mycoplasma species, Chlamydia or the like, or for killing or interfering with the growth of neoplastic cells or specific classes of normal cells. Thus, the oligonucleotideR may be administered to a host subject in a diseased or susceptible state to inhibit the transcription and/or expre~sion of the native genes of a target cell, or to inhibit function of a protein in that cell. Therefore, the oligonucleotides may be used for protection from, or treatment of, a variety of pathogens in a host, such as, for example, enterotoxigenic bacteria, Pneumococci, Neisseria organism~, Giardia organisms, or Entamoebas, etc. Such oligonucleotides may also inhibit function, maturation, or proliferation of neoplastic cells, such as carcinoma cells, sarcoma cells, and lymphoma cells; specific B-cell~;
~pecific T-cell~, such as helper cells, suppre~sor cells, cytoto~ic T-lymphocytes (CTL), natural killer (NK) cells, etc.
The oligonucleotide~ may be selected ~o as to be capable of interfering with RNA processing (transcription product maturation) or production of proteins by any of the mechanisms involved with the binding of the subject composition to its target ~equence. These mechanisms may include interference with processing, inhibition of W094/15620 215 3 ~ ~ 7 PCT~S94/0058~

transport across the nuclear membrane, cleavage by endonucleases, or the like.
The unpaired, circular oligonucleotides may contain sequence~ complementary to those present in growth factors, lymphokines, immunoglobulins, T-cell receptor sites, MHC
antigens, DNA or RNA polymerases, antibiotic resistance, multiple drug resistance (mdr), genes involved with metabolic proces~es, in the formation of amino acids, nucleic acids, or the like, DHFR, etc. as well as introns or flankinq sequence~ a~sociated with the open reading frames.
The following table is illustrative of some additional applications of the subject compositions.

Area of Application Specific APplication Targets Infectious Disea~es:
Antivirals, Hum-n HIV, HSV, CMV, HPV, VZV
infections Antivirals, Animal Chicken Infectious Bronchitis Pig Transmissible Gastroenteritis Virus infections Antibacterial, Human Drug Resistance Plasmids Antipara~itic Agent~ Malaria Sleeping Sicknes~
(Trypanosomes) Cancer Direct Anti-Tumor Oncogenes and their products Agent~ Tumor Suppre~sor genes and their products Adjunctive T~erapy Drug Resistance genes and their products W094/15620 PCT~S94/00585 215305~ -16-Auto Immune Diseases T-cell receptors or Rheumatoid Arthritis autoantibodie~ Type I Diabetes Systemic Lupus Multiple 5C lerosi 8 Organ Transplants OKT3 cells causing GVHD

The oligonucleotides of the present invention may be employed for binding to target molecules, such a8, for example, proteins including, but not limited to, li~ands, receptors, and or enzymes, whereby such oligonucleotide~
inhibit the activity of the target molecules, or restore activity lost through mutation or modification of the tar~et molecules.
The oliyonucleotides of the present invention are adminlstered in an effective binding amount to an RNA, a DNA, a protein, or a peptide. Preferably, the oligonucleotides are administered to a host, such a~ a human or non-human animal host, 80 as to obtain a concentration of oligonucleotide in the blood of from about 0.1 to about 100 ~mole/l. It i~ also contemplated that the oligonucleotides may be admini~tered in vitro or ex vivo a~ well as in vivo The oligonucleotide~ may be admini~tcred ln coniunction with an acceptable pharmaceutical carrier a~ a pharmaceutical composition. Such pharmaceutical composition~ may contain suitable excipients and auxiliaries which facilitate processin~ of the active compound~ into preparations which can be u~ed pharmaceutically. Such oligonucleotide~ may be administered by intramuscular, intraperitoneal, intraveneou~, or ~ubdermal injection in a quitable ~olution. Preferably, the preparation~, particularly those which can be administered orally and ~0 94/15620 21 S3 o ~ 7 PCT/US94/00585 --17-- ~

which can be used for the preferred type of administration, such as tablet~, drageeQ and capsules, and preparations which can be administered rectally, such as suppositorie~, as well as ~uitable 601utions for administration parenterally or orally, and compositions which can be administered ~ ~c ally or sublingually, including inclusion compounds, ccnr~-_n from about 0.1 to 99 percent by weight of active ingredients, together with the excipient. It is also contemplated that the oligonucleotides may be administered topically in a suitable carrier, emul~ion, or cream, or by aerosol.
The pharmaceutical preparation~ of the present invention are manufactured in a manner which is itself well known in the art. For example, the pharmaceutical preparation~ may be made by mean~ of conventional mixing, granulating, dragee-making, di~olving or lyophilizing processes. The process to be used will depend ultimately on the physical properties of the active ingredient u~ed.
Suitable excipients are, in particular, fillers such as sugar, for example, lactose or sucro~e, mannitol or sorbitol, cellulo~e preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch or paste, using, for e~cample, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydro~cypropylmethylcellulose, sodium carboxypropylmethyl-cel~ulo~e, ~odium carboxymethylcellulo~e, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, ~uch a~ the above-mentioned starche~ a~ well as carboxymethyl-~tarch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are flow-regulatlng agents and lubricants, such as, for example, silica, talc, ~tearic acid or salts thereof, such as magnesium stearate or calcium W094/15620 PCT~S94/00585 2~3~5~ -18-stearate, and/or polyethylene glycol- Dragee coreq may be provided with suitable coatinsfi which, if desired, may be re~istant to gastric juices. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solution~ of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Dyestuffs and pigments may be added to the tablets of dragee coatings, for example, for identification or in order to characterize different combination~ of active compound do~e~.
Other pharmaceutical preparation~ which can be used orally include push-fit cap~ule~ made of gelatin, as well as soft, ~ealed cap~ules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contaln the oligonucleotides in the form of granule~ which may be mixed with filler~ such as lactose, binders such as starche~, and/or lubricants such ac talc or magne~ium stearate and, optionally, ~tabilizer~. In soft capsules, the active compoundq are preferably dissolved or suspended in suitable liguids, such as fatty oils, liquld paraffin, or liquid polyethylene glycolQ. In addition, stabilizer~ may be added.
Po~s$ble pharmaceutical preparations which can be used rectally include, for example, suppo~itories, which consis~
of a combination of the active compounds with a suppository base. Suitable ~uppo~itory ~ase~ are, for example, natural or synthetic tr~glycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols. In addition, i t is also possible to u~e gelatin rectal capsules which consist of a combination of the active compounds with a ba~e. Possible ba~e materials include, for example, liqu~d ~094/15620 ~S 7 PCT~S94/00585 triglycerides, polyethylene glycols, or paraffin hydrocarbon~.
Sultable formulations for parenteral administration include aqueous ~olutions of the active compounds in water-soluble or water-di~persible form. In addition, suspension~ of the active compounds as appropriate oil injection suspensions may be admini~tered. Suitable lipophilic solvent~ or vehicle2 include fatty oil~, for ex~mple, sename oil, or synthetic fatty acid esters, for example, ethyl oleate or triglyceride~. Aqueous injection suspensions may contain sub~tance~ which increase the viscosity of the ~uspen~ion including, for example, ~odium carboxymethyl celluloJe, ~orbitol and/or dextran Optionally, the su~pension may also contain stabilizers.
Additionally, the compound~ of the preacnt invention may also be admini~tered encapsulated in llpo~omes, wherein the active inqredient is contained either disper~ed or variourly preoent in corpuscle~ conci~ting of aqueou~
concentric layera adherent to lipidic layer~. The active ingredient, depending upon its solubility, may be presen~
both in the aqueous layer, in the lipidic layer, or in what is generally termed a liposomic ~u~pencion. The hydrophobic layer, generally but not exclu~ively, compri~es phospholipid~ ~uch a~ lecithin and ~phingomycelin, steroids such a~ chole~terol, surfactants such a~ dicetylphosphate, ~tearylamlne, or phosphatidic acid, and/or other materials of a hydrophobic nature. The diameter~ of the liposomes generally range from about 15 nm to about 5 microns.
A variety of functional groups, such a~ -OH,-NH2, -COOH, or -SH, can be attached to the bridging moieties through linker arms and u~ed to attach con~ugate molecules which might confer favorable properties to the adduct.
Examples of favorable properties include increased uptake into the cell, increased lipophilicity or improved binding WO94/15620 PCT~S94/00585 ~1~3~ 20-to cell surface receptorE. Examples of 8uch conjugate groupc include, but are not limited to, biotin, folic acid, chole~terol, epidermal growth factor, and acridine.
The oligonucleotide~ may be used as a diagno~tic probe Hapten~, such as, but not limited to, 2, 4-dinitrophenyl group~; vitamins such as biotin and iminobiotin;
streptavidin; fluore~cent moieties such as fluorescein and FITC; or enzymes such a~ alkaline pho~phata~e, acid phosphatace, or hor~eradiJh peroxidase, may be attached to the oligonucleotides. Other labels include, but are not limited to, detectable marker~ such a~ radioactive nuclides;
and chemical markers including, but not limited to, biotinated moletie~, antigens, sugar~, fluors, and pho~phor~, apoenzymes and co-factors, ligands, allosteric effectors, ferritin, dyes, and micro~phere~. The~e label~
can be attached to any portion of the oligonucleotide which is not e~ential for binding to its target. Preferably, the marker i~ attac~ed to the bridging group~. In general, the bridging group has no biological function, and therefore, attachment of the label to the bridging group does not interfere with the therapeutic or diagnostic applications of the oligonucleotides.
The invention will now be de~cribed with re~pect to the following e~umple~, the scope of which doe~ not limit the invention. In particular, the sequences of the paired oligonucleotidee in the examples hereinafter deccribed compr~ee a DNA binding ~equence of the tumor ~uppre~sor protein, p53. Thi~ protein, which i~ mutated in a number of human cancer~, wae identified a~ a ~equence-~pecific DNA-binding-protein by Kern, et al., Science, Vol. 252, pgs 1708-1711 (1991). Thc ~ubject of p53 mutations in human cancers ha~ al~o bee reviewed in Hollstein, et al., Sclence Vol. 253, pg~. 49-53 (199O).

W094/15620 -21-` 5 ExamPle 1 SYnthesis of an open chain oliqonucleotide with two hexaethYlene qlYcol bridqing qroups.
An oligonucleotide with the following structure:
5'-DMTr-AGCATGCCXG~CATGCTCAGACATGCCXGGCA~ , whereln A
i8 adenine, C is cytosine, G i8 guanine, T is thymine, X
i-Q hexaethylene glycol phosphodiester, Y is phosphate, and DMTr is dimethoxytrityl, was synthesized using a DNA
synthesizer.
Synthesis was carried out on a l umole scale using conventional cyanoethyl pho~phoramidities and other reagents as a~ follows: The 3'-phosphate was intraduced using (2-cyanoethoxy)-2-(2'-0-4,4'-dimethoxytritylo~yethyl-sulfonyl) ethoxy-N, N-diisopropylamino-phosphine (Horn and Urdea, Tetrahedron Letters, Vol. 27 pgs. 4705-4708 (19~6)) as the phosphoramidite, the reagent being coupled directly to controlled-pore gla88 ~olid support to which a deoxycytidine re~idue was attached (i.e., a C-column). The hexaethylene glycol bridging groups were introduced u-~ing

4,4'-dimethoxytrityloxy-hexaethyleneoxy-2-cyanoethoxy-N,N'-diisopropylaminopho~phine (Durand et al, Nucleic Acids Re~earch, Vol. 18, pgs. 63S3-6359 (l990)). After cleavage from the solid zupport, the agueous ammonia solution wa~
heated at 55C to remove protecting groups and ammonia was removed by pa-~ing a stream of nitrogen over the solution.
The Jolution wa~ then lyophilized and dissolved in 0.02 M
triethylummonium bicar~onate, pH 7.6. The crude trityl-on oligonucleotide wa~ purified by reversed phase HPLC (C4 Radial ~ak cartridge, 25 X lO0 mm, 15u, 300A) using a linear gradient of O.l M triethylammonium acetate (TEAA)/acetonitrile, with the concentration of acetonitrile being varied from 2 to 20% over 55 minutes. The peak eluting between 43 and 50 minutes, corresonding to the tritylated oligonucleotide, was collected and lyophilized to W094t15620 PCT~S94/00585 2153~ 22-remove buffer and detritylated by treatment with O.lM acetlc acid ~olution for 10 minutes at room temperature. The product was directly extracted with ethyl acetate (3x) followed by ether (6x) and lyophilized to dryne~s. The residue wa~ converted into the sodium salt by dissolution in water (1 mL) and pa~age through a column of ion exchange resin (Dowex AGSOW-X8, 7 x lSO mm). The eluate was evaporated to dryness to give an open chain oligonucleotide having the following structural formula:
~G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C

C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G

5' HO po32 3~
wherein X i~ O(C~2 CH20)6 -PO 3-The above sequence corresponds to a portion of the DNA~equence which i~ known to bind to the p53 protein encoded by the p53 tumor suppre~sor gene.

Example 2 Formation of a clo~ed circular oliqonucleotide with two hexaethYlene qlycol bridqing groups.
The oligonucleotide isolated from Example 1 (10 OD260 units) was dlaaolved in sodium 4-morpholine-ethanesulfonate buffer (MES, 0.05 M,pH 6.0, 22 uL) containing 20 mM
magneaium chlorlde and treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) (EDC, 6.4 mg). The mixture wa~ briefly vortexed, ~tored at 4C and analyzed by RPLC u~ing a Dionex PA-100 column, 4 x 250 mm.
(Buffer A:25 mM tri~-chloride, pH 8, containing 0.5X
acetonitrile; buffer B: 25mM tris-chloride, lM ammonium chloride, pH 8, containing 0.5~ acetonltrile. Flow rate 1.5 mL/min, gradient: 15X B to 70/0 B over 5 min., then 70-80~
5-20 min.) Since analysis after 15 hour~ indicated that a ~094/15620 PCT~S94/00585 ~;g substantial amount of ~tarting material remained, the oligonucleotide wa8 precipitated by addition of absolute ethanol (85 uL), redissolved in MES buffer (22 uL) and treated with additional EDC (~3.7 mg). After storage for 2 days at 4C, the ethanol precipitation procedure was repeated and the oligonucleotide was treated for a third time with EDC (5.3 mg) in MES (22uL) for 24 hours at 4C.
HPLC analy~is at this point indicated that no ~tarting material remained. The oligonucleotide was precipitated by addition of ethanol (B5 uL), (washed with 300 uL absolute ethanol), and dried to give a clo~ed, circular, oligonucleotide having the following structural formula:
~ G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C ~
X X
~C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G~
wherein X is O(CH2CH20)6 3 Analy~i~ by polyacrylamide gel electrophoresis (15%
acrylamide, 7M urea, tris borate/EDTA buffer), followed by W detection, revealed a ~ingle, fa~ter running band as compared to the unligated material.

Example 3 Synthesis of an Open Chain Double-Stranded Oliqonucleotide with Two Dodecanediol ~ridqinq Group~
a) Synthe~i~ of 4,4'-dimethoxytrityldodecanediol-2-cyanoethoxy-N,N-di-i~opropylamino-phosphine.
Dodecanedlol (1.012 g, 5 mmol) wa~ dried by coc~poration wlth di~tilled pyridine (2 x 10 mL), dissolved in distilled pyridine (20 mL) under nitrogen, and while being stirred wa~ treated with dimethoxytrityl chloride (1.694 g, 5 mm~1). The reaction was monitored by TLC uslng methanol/methylene chloride (1:9) as the solvent system and after 3.5 hour~ at room temperature the mi~ture wa~
partitioned between methylene chloride (100 mL) and 5%

W094/15620 PCT~S94/00585 ~1S3~7 aqueou~ sodium bicarbonate (80 mL)- The organic layer was wa~hed with 5% sodium bicarbonate (2 x 80 mL) followed by ~aturated sodium chloride (80 mL) and concentrated to a gum The sample wa~ purified by column chromatography on silica gel (80 g, 230-400 mesh) u~ing a linear gradient of methanol in methylene chloride/triethylamine (99.8:0.2). The concentration of methanol was rai~ed in a stepwise manner from 0.5-7~. The appropriate fractions were combined and evaporated to yield 1.16 g (2.30 mmol, 46%) of mono-(4,4'-dimethoxytrityl)-dodecanediol as a yellowish gum.
A sample of mono-(4,4'-dimethoxytrityl)-dodecanediol (1.16 g, 2.30 mmol) was dis~olved in dimethylethylamine (1.24 mL, 5x) and methylene chloride (15 mL) under nitrogen and, while being stirred wa~ treated with 2-cyanoethyl N, N-dii~opropylamino-chloropho~phine (1 g, 4.225 mmol, 1.8 x).
After 2.5 hr~. at room temperature, the reaction was checked by TLC u~ing ethyl aeetate/triethylamine (95:5) as the solvent system, and ~ince the reaction was incomplete, additional 2-cyanoethyl-N, N-dii~opropylamino-chloropho~phine (lg, 4.225 mmol, 1.8 x) was added. After an additional 0.5 hr., TLC ~howed the reaction to be ~ub~tantially complete. The mixture wa~ partitioned between ethyl acetate (80 mL) and 5% sodium bicarbonate (100 mL) and the organic layer was washed with 5% ~odium bicarbonate (2 x 100 mL) followed by ~aturated ~odium chloride (100 mL) and concentrated to gum. The sample was purified by column chromatoqraphy on ~ilica gel (50 g, 230-400 me~h) using ethyl acetate/triethylamine (99.8:0.2). The appropriate fraction~ were combined and evaporated to yield 1.454 g (2.06 mmol, 89.6X) of dimethoxytrityldodecanediol-2-cyanoethoxy-N,N-di-isopropylamino-pho~phine as a yellowish gum.

V094/15620 ~ S PCT~S94/00585 ~ 3~S~

b) Oligonucleotide synthesis An oligodeoxynucleotide with the following ~tructure ~s synthesized using a DNA ~ynthesizer:
5'-DMTr-AGCATGCCTXAGGCATGCTCAGACAlG~lXAGGCAl~l~l~Y, where A = adenine, C = cyto~ine, G = guanine, T = thymine, X =
dodecanediol-phosphodiester brid~ing group, Y = phosphate and DMTr = dimethoxytrityl.
Synthesis is carried out on a 1 umole scale using conventional cyanoethyl phosphoramidites and other reagents as follows: The 3' phosphate is introduced as described in Example 1, the reagent being coupled directly to controlled-pore glass solid support to which a deoxycytidine residue was attached (i.e. a C-column). The dodecanediol-pho~phodiester bridging groups are introduced using 4,4'-dimethoxytrityloxy-dodecanediol-2-cyanoethoxy-N,N'-diisopropylamino-pho~phine. After cleavage from the solid support, the aqueous ammonia solution is heated at 55C to remove protecting groups and ammonia is removed by passing a stream of nitrogen over the solution. The solution is then lyophilized and di~solved in 0.02 M triethylammonium bicarbonate, pH 7.6. The crude, trityl-on oligonucleotide is purified by rever~ed phase HPLC (C4 Radial Pak cartridge, 25 x 100 mm, 15u, 300A) u~ing a linear gradient of 0.1 M
triethylammonium acetate (TEM )/acetonitrile, with the concentration of acetonitrile being varied from 2 to 20 %
over 55 minute~. The peak corresponding to the tritylated oligonucleotide is collected and lyophilized to remove buffer and detritylated by treatment with O.lM acetic acid solution for 10 minutes at room temperature. The product is directly extracted with ethyl acetate (3x) followed by ether (6x) and lyophilized to dryness. The re~idue is converted into the sodium salt by dissolution in water (1 mL) and passage through a column of ion exchange re~in (Dowex AGSOW-X8, 7 x 150 mm). The eluate is evaporated to dryness W0 94/15620 21~ 3 ~ ~ ~ PCT/US94/00~85 to give the open chain double-stranded oligonucleotide with two dodecanediol bridging groups. The purity of the product is examined by reinjection into an analytical ion exchange Dionex PA-100 column, 4 x 250 mm. Buffer A: 25 mM
tris-chloride, pH 8 containing 0.5% acetonitrile, buffer B:
25 mM tris-chloride, lM sodium chloride, pH 8 containing 0.5 % acetonitrile. Flow rate 1.5 mL/min. Gradient: 15% B to 70% B over 5 min, then 70-80% B 5-20 min. This material of the following structure is suitable for chemical ligation as described hereinbelow in Example 4.
~A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T ~
X X
~T-C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G-A

5'H0 opo32 3, where X is 0-(CH2)12-0-PO3 Example 4 Synthesis of a Closed, Circular Double-Stranded Oliqonucleotide with Two Dodecanediol Brid~inq Groups The oligonucleotide isolated from Example 3 (10 OD260 units) i~ di~solved in sodium 4-morpholine-ethanesulfonate buffer (MES, 0.05 M, pH 6.0, lmL) containing 20 mM magnesium chloride and treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 100 mg) The mixture i~ briefly vortexed, stored at 4C for 2 days, and the oligonucleotide is precipitated by addition of absolute ethanol (9 mL), washed with 1 mL absolute ethanol, and dried to give the closed circular oligonucleotide.
Analysis by polyacrylamide gel electrophoresis (15%
acrylamide, 7M urea, tris borate/EDTA buffer, W detection), reveal~ a single, faster running band as compared to the unligated material. HPLC analysis employed a Dionex PA-100 column, 4 x 250 mm (Buffer A: 25 mM tri~-chloride, pH 8 W094/15620 ~ PCT~S94/~585 s3~ ~--27- ~ ~

containing 0.5X acetonitrile: Buffer B: 25 mM
tris-chloride, lM ~odium chloride, pH 8 containing 0.5%
acetonitrile. Flow rate: 1.5 mL/min. Gradient: 15% B to 70% B over 5 min. then 70-80x B 5-20 min). This procedure provides material having the following structure:
~A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T \
X X
~T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A~
where X i 8 O- (CH2)12-0-P03 Example 5 Synthe~is of an Open Chain, Double-Stranded Oliqonucleotide with Biotin Attached to one of the Bridqing Groups An oligodeoxynucleotide with the following structure is prepared using a DNA synthesizer:
S'-DMTr-AGCA~ XAGGCATGCTCAGACA~L~AGGCA~ Y, _ where A i~ adenine, C is cytosine, G is guanine, T i 5 thymine, W i~ hexaethylene glycol pho~phodiester, X is triethylene glycol, Y is phosphate, Z is 2(4-biotinamidopentyl)-1,3-propanediol-phosphodiester, and DMTr = 4,4'-dimethoxytrityl.
Syntho~is i8 carried out on a 1 umole ~cale using conventional cyanoethyl phosphoramidites and other reagents as follows: The 3'- phosphate and the hexaethylene glycol group are introduced as described in Example 1 and the triethylene glycol groups are introduced u~ing 4,4'-dimethoxytrityloxy-triethyleneoxy-2-cyanoethoxy-N,N'-diisopropylaminopho~phine, obtalned from Glen Research Corporation, Sterling, Virginia. The biotin is introduced using l-(4,4'-dimethoxytrityl)-2~4-biotinamidopentyl)-1,3-propanediol-3- (2-cyanoethyl~-N, N-diisopropylamino-chlorophosphine, al~o obtalned from Glen Research Corporation.

W094/15620 PCT~S94/00585 21.53 057 28 After cleavage from the solid support, the aqueous ammonia solution is heated at 55C to remove protecting groups and ammonia is removed by passing a ~tream of nitrogen over the ~olution. The solution is then lyophilized and purified by rever~ed-phase HPLC. The product is directly extracted with ethyl acetate (3x) followed by ether (6x) and lyophilized to dryness. The residue is converted into the sodium salt by dissolution in water (1 mL) and passage through a column of ion exchange resin (Dowex AG50W-X8, 7 x 150 mm). The eluate i8 evaporated to dryness to give the open chain double stranded oligonucleotide having the following ~tructure, with biotln in one of the bridging groups.
~ X
Z A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T
W
~X -T-C-C-~-T-A-C-G-A G-T-C-T-G-T-A-C-G-G-A ~
l l 5' H0 po32 where W is O(CH2CH20)6 P03 X i~ O(CH2CH20)3 P03 Z i ~ OCH2CHCH20-P03 CH2CH2CH2CH2NH-Biotin Thi~ material is suitable for chemical ligation as de~cribed in Example 6.
Example 6 Synthe~is of a Closed, Circular Double-Stranded Oliqonucleotide with Biotin Attached to one of the Bridqing Groups The oligonucleotide isolated from Example 5 (10 OD260 units) is di~olved in sodium 4-morpholine-ethane~ulfonate WO94/15620 ~S~O PCT~S94/00585 buffer (MES, 0.05 M, pH 6-0, 22 uL) containing 20 mM
magnesium chloride and treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 6.4 mg) The mixture is briefly vortexed, stored at 4C for 15 hrs.
and the oligonucleotide is precipitated by addition of absolute ethanol (85 uL), redissolved in MES buffer (22 uL) and treated with additional EDC (8.7 mg). After storage for 2 day~ at 4C, the ethanol precipitation procedure is repeated and the oligonucleotide is treated for a third tlme with EDC (5.3 mg) in MES (22uL) for 24 hours at 4C. The oligonucleotide is precipitated by addition of ethanol (85 uL), washed with 300 uL absolute ethanol, and dried to give the closed circular oligonucleotide having the following structure:
~ X~
Z A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T
\ T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A
~X~

where W is o(CH2cH2o)6 PO3 X is OlCH2CH2O)3-PO3 Z is OCH2CHCH2O-PO3 \

CH2CH2CH2CH2NH-Biotin Example 7 SYnthe~is of an oliqonucleotlde with peptide bridqing qroup~.
a) Synthe~iq of a peptide phosphoramidite.
The tripeptide Ala-Ala-Ala (4 mmol) is treated with the N-hydroxysuccinimide ester of 3-hydroxybutyric acid (4 mmol) in dimethylformamide (20 mL) at room temperature for 4 hours. After removal of solvent, the residue is dissolved in pyridine and treated with 4,4'-dimethoxytrityl chloride W094/15620 PCT~S94/00585 2l53~7 _30_ (2 mmol) at room temperature for 18 hours. The product is evaporated to dryness, partitioned between ethyl acetate and aqueous sodium bicarbonate and the organic layer is washed with sodium bicarbonate (1 x) followed by water (2 x) and dried over magne 8 ium sulfate. The solution is filtered, evaporated to drynes~, and purified by silica column chromatography uqing methylene chloride/methanol/
triethylamine as the solvent to give the dimethoxytritylated trlpeptide of the following structure:

DMTrO-CH(CH3)CH2-CO-NHCH(CH3)CO-NHCH(CH3)CO-NHCH(CH3)COO~

This material ~2 mmol) is dissolved in dimethylformamide and treated with 6-aminohexanol (2 mmol) at room temperature for 1~ hours using dicyclohexylcarbodiimide (5 mmol) as the coupling agent. The urea is removed by filtration and the product is evaporated to dryness, partitioned between ethyl acetate and aqueous sodium bicar~onate, and the organic layer is washed w1th sodium bicarbonate (1 x) followed by water (2 x) and dried over magnesium sulfate. The solution is filtered, evaporated to dryness, and purified by silica column chromatography using methylene chloride/methanol/triethylamine as the solvent to give the dimethoxytr1tyl-ted tripeptide aminohexanol derivative of the following ~tructure:

DMTrO-C~(CH3)CH2-CO-NHCH(CH3)CO-NHCH(CH3)CO-NHCH(CH3)CO-NH(CR2)6 OH

The aminohexanol derivative (1 mmol) is dissolved in methylene chloride containinq diisopropylethylamine (2 mmol) and treated w1th 2-cyanoethyl-N,N-diisopropylamino-chlorophosphine (2 mmol) at room temperature for 20 min.
The ~olution is poured into ethyl acetate, extracted with 5~0 WO94/15620 ~ S PCT~S94/00585 -31- 5~

aqueou~ sodium bicarbonate (2 x) followed by saturated aqueous sodium chloride (2 x) and dried over magnesium chloride overnight. The solid is removed by filtration and the solution is evaporated to dryness and purified by sillca column chromatography using methylene chloride/methanol/triethylamine as the solvent to give the phosphoramidite with the following structure:

DMTrO-CH(CH3)CH2CO-NHCH(CH3)CO-NHCH(CH3)CO-NHCH(CH3)CO-( H2)6 O-p(ocH2cH2cN)N(ipr)2 This material is u~ed to attach the bridging groups to the oligonucleotide in the DNA synthesizer.
b) Oliqonucleotide synthesis The procedures outlined in Examples 1 and 2 are followed to synthesize oligonucleotides with peptide bridging groups, except that the peptide pho~phoramidite of the present example i~ ~ubstituted for the bridging group phosphoramidite described in Example 1.
This procedure can also be used to attach polyamides as bridging groups for oligonucleotides.

Example 8 Synthesis of an oliqonucleotide with polYamine bridqinq qroups The polyamine spermidine (4 mmol) is treated with ~ -butyrolactone to give the disubstituted derivative of the following structure:

HO(CH2)3CO-NH(CH2)3NH(CH2)4NH-CO(CH2)3-OH

This material is dissolved in pyridine (lO mL) and treated with trifluoroacetic anhydride (2 mL) overnight at room temperature. The solution is treated with water at 0C

WO94/15620 2 15 3 0 5 ~ - 32- PCT~S94/00585 for 4 hours and evaporated to dryness to give the N-trifluoroacetyl derivative which is treated with 4,4-dimethoxytrityl chloride (4 mmol) for 19 hours at room temperature and evaporated to dryness. The residue l5 partitioned between ethyl acetate and aqueous sodium bicarbonate and the organic layer is washed with water (2 x) and dried over magnesium sulfate overnight. The solld i 5 removed by filtration and the solution is evaporated to dryness and pur~fied by silica column chromatography.
FractionQ containing the monotrityl derivative are combined, evaporated to dryness and 1 mmol of this material is di~solved in methylene chloride containing diisopropylethylamine (2 mmol) and treated with 2-cyanoethyl-N,N-diisopropylamino-chlorophosphine (2 mmol) at room temperature for 20 min. The ~olution is poured into ethyl acetate, extracted with 5~ aqueou~ sodium bicarbonate (2 x) followed by saturated aqueou~ ~odium chloride (2 x) and dried over magnesium chloride overnight. The aolid is removed by filtration and the solution i8 evaporated to dryne~s and purified by silica column chromatography u~ing methylene chloride/methanol as the ~olvent to give the phosphoramidite with the following structure:

DMTr-O(CH2)3CO-NH(CH2)3~H(CH2)4NH CO(CH2)3 2 2 ~OCF3 N (iPr)2 This material i~ used in the DNA synthesizer to introduce bridging groups into the oligonucleotide. The procedures outlines in Examples 1 and 2 are followed to synthesize oligonucleotides with polyamine bridging groups, except that the bridginq group phosphoramidite of the present example is substituted for the bridgin~ group phosphoramidite deqcribed in Example 1.

W094/15620 2~ PCT~S94/00585 s30~

Example 9 IncorPoration of PolYalkylene thioqlYcol bridqinq qroups 3,6-Dithio-1,8-octanediol is treated with 4,4-dimethoxytrityl chloride in pyridine and then converted into a phosphoramidite derivative of the following structure by reaction with 2-cyanoethyl-N,N-diisopropylamino-chlorophosphine using the procedure described in Example 3:

H2cH2scH2cH2scH2cH2O-P-N(ipr) This material is employed as the bridging group phosphoramidite in the synthesi~ of an oligonucleotide as described in Example 1.

Example lO
Synthesis of Double-Stranded Oligonucleotides with Two Di~ub~tituted Aromatic ~rid~inq Groups An oligodeoxynucleotide with the following structure 15 ~ynthesized-uJing a DNA synthesizer:

5'-DMTr-AGCA-~L~AGGCATGCTCAGACA~ ~AGGCA~

wherein A = adenine, C = cytosine, G = guanine, T = thymlne X =0-(CH2)6-NHCO-C6H5-CONH-(CH2)6-OP03 bridging group, Y = pho~phate and DMTr = dimethoxytrityl.
Synthesis i8 carried out on a 1 umol scale using conventional cyanoethyl pho3phoramidites and other reagent~
as follows: The 3' phosphate is introduced a~ de~cribed i~
~xample 1, and the aromatic bridging groups are introduced u~ing N-(6-(4,4'-dimethoxytrityloxy)hexyl)-N' -(6(2-cyanoethoxy-N,N'- diisopropylamino-WO94/15620 PCT~S94/00585 2 lS 3 ~ _34_ pho~phinyloxy)hexYl)terePhthalamide as de~cribed by Cashmanet al in the Journal of the American Chemical Society, Vol 114, pqs 8772-8777 (1992) After cleavage from the solid support, the oligonucleotide i9 proce~ed as described ln Example 1 to give an open chain oligonucleotide duplex with two aromatic bridging groups Formation of the closed circular duplex i~ carried out using the procedure outlined in Example 2 Example 11 Incorporation of an intercalating aqent as a bridqing ~roup The intercalating agent acriflavine is treated with

6-bromo-1-hexanol to form a disubstituted derivative which is then treated with one equivalent of 4,4-dimethoxytrityl chloride to give the monotrityl compound having the following structure ~r~O~~Cff~ ,fLk_o~

After purification and i~olation by ~ilica column chromatography, the monotrityl compound i5 treated with trifluoroacetic nhydride in pyridine followed by aqueous workup to ~ive the N-trifluoroacetyl derivative which is then converted into a phosphoramidite of the following structure by tre-tment with 2-cyanoethyl-N,N-diisopropylamino-chlorop ~ s described in Example 3 D~T~-o~ ) ocf~zChCl/

WO94/15620 ~ PCT~S94100585 -35- ~

The phosphoramidite is incorporated into an oligonucleotide by the procedure outlined in Example 1.

ExamPle 12 IncorPoration of a carbohydrate bridqinq moiety 6-O-~-D glucopyranosyl-D-qlucopyranose (B-gentobiose) is treated w~th t-butyl-dimethylsilyl chloride to produce the 6'-silyl compound which is converted into the acetobromo derivative by a conventional method using acetic anhydride followed by hydrogen bromide in acetic acid. The l-bromo derivative i~ then treated with 1,6- hexanediol to give the glycoside which i9 reacted with 4,4- dimethoxytrityl chloride to give a compound having the following structure:

~4to~ 7~0~,--~C~f2)C_o~

The 8ilyl group i~ ~c...ovc~ u~ing fluoride ion and the 6-hydroxy compound is treated with 2-cyanoethoxy-N,N-dii opropylamino-chlorophosphine to give a phosphoramidite having the following ~tructure:
C~f~z C~ O~

tir~ ~ ~ ~ ~ t~ )6- ~ ~

~C ~o ~C

W094/15620 2 1 5 3 5~ PCT~S94/00585 This material is employed in the DNA synthesizer to introduce bridging groups as described in previous examples.
Example 13 Thermal Denaturation of Bridqed, Double-Stranded OliqonucLeotides The thermal denaturation temperatures (Tm's) of some of the oligonucleotide~ of the present invention were measured on a Gilford spectrometer at 260 nm in order to determine their relative ~tabilitie fi . Approximately 1 OD260 unit of each oligonucleotide was dis~olved in O.9 mL of 10 mM
disodium pho~phate buffer, pH 7.0, and each sample was heated briefly at 100C, and allowed to cool 910wly to room temperature. Melting profiles were obtained by increasing the temperature of the ~ample~ from 25C to 100C at a rate of 0.8C per minute, followed by measurement of optical absorption at each time interval. The oligonucleotides examined in thie study are as follows:
A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T X

\T-C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G-A

5' HO ~po32 3~

Oligonucleotide 1. X = pentathymidylate (T5) Oligonucleotide 2. X = triethylene glycol phosphodiester ~ -G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T
X X
~T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A

Oligonucleotide 3. X = T5 Oligonucleotide 4. X = triethyiene glycol phosphodiester WO94/15620 . ~ PCT~S94/00585 A-G-G-C-A-T-G-C-T-C-~-G-A-C-A-T-G-C-C-T
Oligonucleotide 5.
T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A
Oligonucleotide 6.

The thermal denaturation temperature~ of OligonucleotideR l through 6 are given in Table I below.

Table I

Oligo- 3'-Terminal Bridging Tm nucleotide Group GrouP (X~ ~C) 1 Pho~phomono- T5 61.5 ester 2 Phosphomono- Triethylene 68 e~ter glycol pho~phodie~ter 3 None T~ 82.5 4 None Triethylene 89.5 glycol pho~phodie~ter 5 and 6 N/A None 62 Thi~ e~periment demonstrates that an open chain, double-~tranded ollgonucleotide with triethylene glycol bridging group~ i~ more stable towardq thermal denaturation than either the ~ame ~equence with pentathymidylate bridgin~
group~, or an unmodified duplex without any bridging groups.
The clo~ed, circular double-stranded oligonucleotide with triethylene glycol bridging groups iq also more stable than the same ~equence with pentathyamidylate bridge~.

W094/15620 1 PCT~S94/00585 30~

Example 14 EnzYmatic Stability of a Double-Stranded Oliqonucleotide with Hexaethylene Glycol Bridqing Groups Enzymes:
Exonuclease - Exonuclea~e III
Endonuclease - Mung Bean Nuclea~e Buffers:
For Exonuclease III: 50 mM Tri~-HCl, pH 7.5; 5mM MgC12;
5 mM DTT; 50 mg/mL BSA.
For Mung Bean Nuclease. 30 mM NaOAc, pH 5.0; 50 mM NaCl;
1 mM ZnC12; S~ glycerol.

The enzymatic degradation of the detritylated oligonucleotide of Example 1, having hexaethylene glycol bridging groups, was compared to a duplex of the same sequence without any bridging groups. A 1 OD260 sample of each oligonucleotide was digested in a mixture of 95 ~L of the reaction buffer and 5 ~L of the enzyme solution containing 1 unit of the enzyme. The mixture~ were incubated at 37C (for Exonuclease III) or room temperature (for Mung Bean Nuclea~e) and the extent of degradation was monitored by ~PLC wtth a Dionex ion-exchange column, uslng a linear gradient of ammonium chloride (O-lM) in tris hydrochloride. The time required for ~O% degradation (tl/2) for each sample was determined and the following results were obtained:

WO94/15620 Sob; PCT~S94/00585 Oliqonucleotide Mung Bean Nuclease Exonucleaqe II' (tl/2) (t~/2) Detritylated 68 hours > 80 hours Oligonucleotide of Example l Unmodified 3 hour~ 2.5 hours duplex This experiment demonstrate~ that the oligonucleotide of Example l, posse~sing hexaethylene gly~ol bridging groups, i8 considerably more re~istant to degradation than an unmodified duplex of the same sequence.

Example 15 Bindinq of an Open Chain Double-stranded Oliqonucleotide with Two Hexaethylene Glycol Bridqinq Groups to pS3 Tumor Supprersor Gene Protein The following oligodeoxynucleotider were prepared for bind to pS3 protein.

l. G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C

~ C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G
Il 5' HO OH 3' where X O(CH2CH2)6 P 3 2. G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C

3. C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G

W094/15620 21~ 3 a 5 ~ PCT~S94/00585 Oligonucleotide 1 was prepared by the procedure outlined in Example 1, except that the 3'-phosphoryla~ion reagent was omitted and the hexaethylene glycol bridging groups were introduced using 4,4'-dimethoxytrityloxy-hexaethyleneoxy-2-cyanoethoxy-N,N'-diisopropylaminophosphine as the bridging group reagent. A
92 base pair natural duplex containing a randomized internal 60 base pair region wa~ used as the control oligonucleotide.
The oligonucleotides were radiolabeled with 32p using a standard protocol as de~cribed in "Molecular Cloning, a Laboratory Manual" by Sambrook, FritRch and Maniatis, page 11.31, Cold Spring Harbor Pre~ (1989). The radiolabeled oligonucleotides were then purified and u~ed in an immunoprecipitation assay to evaluate binding efficiency to p53 tumor suppressor gene protein. The immunoprecipitation assay was performed with 2.0 pmole~ purified p53, 0.25 pmoles radiolabeled oligonucleotide, 100ng poly dl-dC, and 400ng each of anti-p53 antibodie~ pAb421 and pAbl801 (purchased from Oncogene Science~, Inc.), incubated in 100 ~1 of binding buffer containing 100 mM NaCl, 20 mM Tris pH

7.2, 10% glycerol, 1~ NP40, and 5 mM EDTA a 4C for 1 hour.
The DNA-pS3-anti-pS3 antibody complexes were precipitated following the addition of 30 ~1 of a 50X slurry of protein A
~epharo~e and mixing at 4C for 30 minutes. After removal of tho aupernatant, the immunoprecipitate was waRhed three times with binding buffer. ~ound oligonucleotide was then quantified by direct Cerenkov counting. Specific binding wa~ evaluated by comparl~on to an immunoprecipitation performed in the ab~ence of pS3. The results are summarlzed below.

WO94/15620 PCT~S94/0058~
-41- ~S30~7 p53 Bindinq of natural and modified oliqonucleotide duplexes Oliqonucleotide Percent Bound (vs unmodified duplex~

2 + 3 lOO

l 43.5 control 3.2 This experiment shows that the open chain, double-~tranded Oligonucleotide l, with he~aethylene glycol bridging groups, i~ capable of binding to p53 protein although somewhat le~ efficiently than an unmodified duplex of the same sequence. This result, taken together with the results of Ex~mple 14 (which demonstrated ~at an open chain double-~tranded oligonucleotide with hexaethylene glycol bridging group~ was considerably more stable towards nucleases than an unmodified duplex), indicates that an oligonucleotide with bridging group~ of this type has considerably greater pharmacological potential.
Advantage~ of the present invention include increased resistance of the circular oligonucleotide~ to enzymes which degrade ol~gonucleotide~ by attack at the 5' and/or 3' termini, ~uch a~, for example, 3' exonuclea~es. In addition, double-~tranded oligonucleotide~ of the present invention are re~iJtant to enzymes which degrade single-~tranded regions of DNA because the non-nucleotide bridging group~ cannot be recognized by such enzymes. The bridging group~ can be constructed from simple, readily available starting materials, and may be lncorporated easlly into an oligonucleotide using a DNA synthe~izer. In addition,.both the open chain and the clo~ed, circular, W094/15620 .~ PCT~S94/00585 2l53~5 l paired oligonucleotides with non-nucleotide bridging groups are capable of forming more stable hydrogen-bonded structures than the corresponding sequences with nucleotlde (pentathymidylate) bridging groups or with natural duplexes without bridging groups and thus provide binding to target protein~ which are capable of binding double-stranded oligonucleotides. ~ecause the paired oligonucleotides also remain hydrogen-bonded at higher temperatures this could be advantageous for diagnostic applications. Unpaired circular oligonucleotides might possess a significant advantage over single-stranded oligonucleotides in binding to double-~tranded target DNA or RNA by forming Hoog~teen nteractions with both strands of the target DNA or RNA.
Also, the bridging moieties of the double-stranded oligonucleotides may be modified to introduce favorable properties into the molecules, such as increaJed lipophilicity, or be modified to introduce materials which assist in the delivery of the oligonucleotide into the cell, such as cationic group~ or molecules which are recognized by cell surface receptor~.
It is to be understood, however, that the qcope of the present invention i~ not to be limited to the specific embodiments described above. The invention may be practiced other than a~ particularly described and still be within the scope of the accompanying claims.

Claims (69)

What is Claimed is:

1. An oligonucleotide having a structural formula selected from the group consisting of:

and wherein S1, S2, S3, S4, and S5 are oligonucleotide strands, and each of X1 and X2 is a bridging moiety which may be a nucleotide strand or a non-nucleotide bridging moiety, and each of X1 and X2 may be the same or different, with the proviso that when one of X1 and X2 is a nucleotide strand, the other of X1 and X2 is a non-nucleotide bridging moiety, and each of X1 and X2 independently is a bridging moiety having first and second termini that each binds independently with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety.

2. The oligonucleotide of Claim 1 wherein said non-nucleotide bridging moiety has the following structural formula:
T1-R-T2, wherein each of T1 and T2 independently binds with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety, and R is selected from the group consisting of: (a) saturated and unsaturated hydrocarbons; (b) polyalkylene glycols; (c) polypeptides; (d) thiohydrocarbons; (e) polyalkylamines; (f) polyalkylene thioglycols; (g) polyamides; (h) disubstituted monocyclic or polycyclic aromatic hydrocarbons; (i) intercalating agents; (j) monosaccharides; and (k) oligosaccharides; or mixtures thereof.

3. The oligonucleotide of Claim 2 wherein R is a polyalkylene having from about 5 to about 100 carbon atoms

4. The oligonucleotide of Claim 3 wherein R is polymethylene.

5. The oligonucleotide of Claim 2 wherein R is a polyalkylene glycol.

6. The oligonucleotide of Claim 5 wherein said polyalkylene glycol is polyethylene glycol.

7. The oligonucleotide of Claim 2 wherein R is a polypeptide.

8. The oligonucleotide of Claim 2 wherein R is a thiohydrocarbon.

9. The oligonucleotide of Claim 2 wherein R is a polyalkylamine.

10. The oligonucleotide of Claim 2 wherein R is a polyalkylene thioglycol.

11. The oligonucleotide of Claim 2 wherein R is a polyamide.

12. The oligonucleotide of Claim 2 wherein R is a disubstituted monocyclic or polycyclic aromatic hydrocarbon.

13. The oligonucleotide of Claim 2 wherein R is an intercalating agent.

14. The oligonucleotide of Claim 2 wherein R is a monosaccharide.

15. The oligonucleotide of Claim 2 wherein R is an oligosaccharide.

16. A composition for binding to a DNA, an RNA, a protein, or a peptide, comprlsing:
(a) an oligonucleotide having a structural formula selected from the group consisting of:

and wherein S1, S2, S3, S4, and S5 are oligonucleotide strands, and each of X1 and X2 is a bridging moiety which may be a nucleotide strand or a non-nucleotide bridging moiety, and each of X1 and X2 may be the same or different, with the proviso that when one of X1 and X2 is a nucleotide strand, the other of X1 and X2 is a non-nucleotide bridging moiety and each of X1 and X2 independently is a bridging moiety having first and second termini that each binds independently with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety; and (b) an acceptable pharmaceutical carrier, wherein said oligonucleotide is present in an effective amount for binding to a DNA, and RNA, a protein, or a peptide .

17. The composition of Claim 16 wherein said non-nucleotide bridging moiety has the following structural formula:
T1-R-T2, wherein each of T1 and T2 independently binds with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety, and R is selected from the group consisting of: (a) saturated and unsaturated hydrocarbons; (b) polyalkylene glycols; (c) polypeptides; (d) thiohydrocarbons; (e) polyalkylamines; (f) polyalkylene thioglycols; (g) polyamides; (h) disubstituted monocyclic or polycyclic aromatic hydrocarbons; (i) intercalating agents; (j) monosaccharides; and (k) oligosaccharides; or mixtures thereof.

18. The composition of Claim 17 wherein R is a polyalkylene having from about 5 to about 100 carbon atoms.

19. The composition of Claim 18 wherein R is polymethylene.

20. The composition of Claim 17 wherein R is a polyalkylene glycol.

21. The composition of Claim 20 wherein said polyalkylene glycol is polyethylene glycol.

22. The composition of Claim 17 wherein R is a polypeptide.

23. The composition of Claim 1 wherein R is a thiohydrocarbon.

24. The composition of Claim 17 wherein R is a polyalkylamine.

25. The composition of Claim 17 wherein R is a polyalkylene thioglycol.

26. The composition of Claim 17 wherein R is a polyamide.

27. The composition of Claim 17 wherein R is a disubstituted monocyclic or polycyclic aromatic hydrocarbon.

28. The composition of Claim 17 wherein R is an intercalating agent.

29. The composition of Claim 17 wherein R is a monosaccharide.

30. The composition of Claim 17 wherein R is an oligosaccharide.

31. In a process wherein an oligonucleotide is administered for binding to a DNA, an RNA, a protein, or a peptide, the improvement comprising:
administering to a host an oligonucleotide having a structural formula selected from the group consisting of:

and wherein S1, S2, S3, S4, and S5 are oligonucleotide strands, and each of X1 and X2 is a bridging moiety which may be a nucleotide strand or a non-nucleotide bridging moiety, and each of X1 and X2 may be the same or different, with the proviso that when one of X1 and X2 is a nucleotide strand, the other of X1 and X2 is a non-nucleotide bridging moiety, and each of X1 and X2 independently is a bridging moiety having first and second termini that each binds independently with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety, said oligonucleotide being administered in an amount effective for binding to a DNA, an RNA, a protein, or a peptide .

32. The process of Claim 31 wherein said non-nucleotide bridging moiety has the following structural formula:
T1-R-T2, wherein each of T1 and T2 independently binds with a nucleotide phosphate moiety or a nucleotide hydroxyl moiety, and R is selected from the group consisting of: (a) saturated and unsaturated hydrocarbons; (b) polyalkylene glycols; (c) polypeptides; (d) thiohydrocarbons; (e) polyalkylamines; (f) polyalkylene thioglycols; (g) polyamides; (h) disubstituted monocyclic or polycyclic aromatic hydrocarbons; (i) intercalating agents; (j) monosaccharides; and (k) oligosaccharides; or mixtures thereof.

33. The process of Claim 32 wherein R is a polyalkylene having from about 5 to about 100 carbon atoms.

34. The process of Claim 33 wherein R is a polymethylene.

35. The process of Claim 32 wherein R is a polyalkylene glycol.

36. The process of Claim 35 wherein said polyalkylene glycol is polyethylene glycol.

37. The process of Claim 32 wherein R is a polypeptide.

38. The process of Claim 32 wherein R is a thiohydrocarbon.

39. The process of Claim 32 wherein R is a polyalkylamine.

40. The process of Claim 32 wherein R is a polyalkylene thioglycol.

41. The process of Claim 32 wherein R is a polyamide.

42. The process of Claim 32 wherein R is a disubstituted monocyclic or polycyclic aromatic hydrocarbon.

43. The process of Claim 32 wherein R is an intercalating agent.

44. The process of Claim 32 wherein R is a monosaccharide.

45. The process of Claim 32 wherein R is an oligosaccharide.

46. The oligonucleotide of Claim 2 wherein at least one of X1 and X2 includes a conjugate group selected from the group consisting of biotin, folic acid, cholesterol, epidermal growth factor, and acridine.

47. The oligonucleotide of Claim 2 wherein at least one of X1 and X2 includes a detectable marker.

48. The composition of Claim 17 wherein at least one of X1 and X2 includes a conjugate group selected from the group consisting of biotin, folic acid, cholesterol, epidermal growth factor, and acridine.

49. The composition of Claim 17 wherein at least one of X1 and X2 includes a detectable marker.

50. The process of Claim 32 wherein at least one of X1 and X2 includes a conjugate group selected from the group consisting of biotin, folic acid, cholesterol, epidermal growth factor, and acridine.

51. The process of Claim 32 wherein at least one of X1 and X2 includes a detectable marker.

52. The oligonucleotide of Claim 1 wherein said oligonucleotide has the following structural formula:

53. The oligonucleotide of Claim 52 wherein S1 and S2 combined include from about 5 to about 100 nucleotide units.

54. The oligonucleotide of Claim 53 wherein S1 and S2 combined include from about 10 to about 100 nucleotide units.

55. The oligonucleotide of Claim 1 wherein said oligonucleotide has the following structural formula:

56. The oligonucleotide of Claim 55 wherein S3, S4, and S5 combined include from about 5 to about 100 nucleotide units.

57. The oligonucleotide of Claim 56 wherein S3, S4, and S5 combined include from about 10 to about 100 nucleotide units.

58. The composition of Claim 16 wherein said oligonucleotide has the following structural formula:

59. The composition of Claim 58 wherein S1 and S2 combined include from about 5 to about 100 nucleotide units.

60. The composition of Claim 59 wherein S1 and S2 combined include from about 10 to about 100 nucleotide units.

61. The composition of Claim 16 wherein said oligonucleotide has the following structural formula:

62. The composition of Claim 61 wherein S3, S4 and S5 combined include from about 5 to about 100 nucleotide units.

63. The composition of Claim 62 wherein S3, S4 and S5 combined include from about 10 to about 100 nucleotide units.

64. The process of Claim 31 wherein said oligonucleotide has the following structural formula:

65. The composition of Claim 64 wherein S1 and S2 combined include from about 5 to about 100 nucleotide units.

66. The composition of Claim 65 wherein S1 and S2 combined include from about 10 to about 100 nucleotide units.

67. The process of Claim 31 wherein said oligonucleotide has the following structural formula:

68. The process Claim 67 wherein S3, S4 and S5 combined include from about 5 to about 100 nucleotide units.

69. The process Claim 68 wherein S3, S4 and S5 combined include from about 10 to about 100 nucleotide units.