| US4547569A | 1985-10-15 | |||
| EP0330221A2 | 1989-08-30 |
BIOCONJUGATE CHEMISTRY, Vol. 1(3), issued May/June 1990 (Easton, PA), GOODCHILD, "Conjugates of Oligonucleotides and Modified Oligonucleotides: A Review of their Synthesis and Properties", pp. 166-186.
BIOCHEMIE, Vol. 67, issued 1985 (France), MILLER et al., "Control of Ribonucleic Acid Function by Oligonucleoside Methylphosphonates", pp. 769-776.
NUCLEIC ACIDS RESEARCH, Vol. 16(9), issued 1988 (Oxford, GB), COOK et al., "Synthesis and Hybridization of a Series of Biotinylated Oligonucleotides", pp. 4077-4095.
BIOCHEMISTRY, Vol. 12(2), issued 1973 (Easton, PA), HARVEY et al., "Use of Phosphate-Blocking Groups in Ligase Joining of Oligodeoxyribonucleotides", pp. 208-214.
| 1. | An oligonucleotide, wherein at least one nucleotide unit of the oligonucleotide includes a phosphonate moiety having the following structural formula:O = O -, wherein X is:(R) -CO -, -(R) - 2-Xl O XI-SOw, , or -(R)X -PO, , wherein R is a hydrocarbon, and n is 0 or 1. |
| 2. | The oligonucleotide of Claim 1 wherein R is an alkyl group. |
| 3. | The oligonucleotide of Claim 2 wherein R is an alkyl group having from 1 to 15 carbon atoms. |
| 4. | The oligonucleotide of Claim 3 wherein R is methylene. |
| 5. | The oligonucleotide of Claim 4 wherein X is (CH2)n-COO~. |
| 6. | The oligonucleotide of Claim 1 wherein said oligonucleotide is a deoxyribonucleotide. |
| 7. | The oligonucleotide of Claim 1 wherein said oligonucleotide is a ribonucleotide. |
| 8. | A composition for binding to an RNA, a DNA, a protein, or a peptide, comprising:(a) an oligonucleotide, wherein at least one nucleotide unit of the oligonucleotide includes a phosphonate moiety having the following structural formula:O O = P - 0 -, wherein X is: X -(R) Xi,-COO", -(R)XX-SO«3" or -(R)XI-PO*,32", wherein R is a hydrocarbon, and n is 0 or 1; and(b) an acceptable pharmaceutical carrier, wherein said oligonucleotide is present in an effective binding amount to an RNA, a DNA, a protein, or a peptide. |
| 9. | The composition of Claim 8 wherein R is an alkyl group. |
| 10. | The composition of Claim 9 wherein R is an alkyl group having from 1 to 15 carbon atoms. |
| 11. | The composition of Claim 10 wherein R is methylene. |
| 12. | The composition of Claim 11 wherein X is (CH2)n-COθ". |
| 13. | The composition of Claim 8 wherein the oligonucleotide is a deoxyribonucleotide. |
| 14. | The composition of Claim 8 wherein the oligonucleotide is a ribonucleotide. |
| 15. | In a process wherein an oligonucleotide is administered for binding to an RNA, a DNA, a protein, or a peptide, the improvement comprising: administering to a host an effective binding amount of an oligonucleotide, wherein at least one nucleotide unit of the oligonucleotide includes a phosphonate moiety having the following structural formula:✓ AO = P - 0 -, wherein X is: - 2-(R) X-X-COO , -(R)X-SO, , or -( )XI-PO, , wherein R is a hydrocarbon, and n is 0 or 1. |
| 16. | The process of Claim 15 wherein R is an alkyl group. |
| 17. | The process of Claim 16 wherein R is an alkyl group having from to 1 to about 15 carbon atoms. |
| 18. | The process of Claim 17 wherein R is methylene. |
| 19. | The process of Claim 18 wherein X is (CH2) -C00~. |
| 20. | The process of Claim 15 wherein the oligonucleotide is a deoxyribonucleotide. |
| 21. | The process of Claim 15 wherein the oligonucleotide is a ribonucleotide. |
| 22. | An. oligonucleotide, wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having the following structural formula: I / o 0 = P - 0 -V wherein X is : - (R)n-S02- ( L)p-W, orwherein R is a hydrocarbon, n is 0 or 1, L is a linker group, p is 0 or 1, and W is a detectable marker. |
| 23. | The oligonucleotide of Claim 22 wherein R is an alkyl group. |
| 24. | The oligonucleotide of Claim 23 wherein R is an alkyl group having from 1 to 15 carbon atoms. |
| 25. | The oligonucleotide of Claim 24 wherein R is methylene. |
| 26. | The oligonucleotide of Claim 22 wherein L is an -NH- group. |
| 27. | The oligonucleotide of Claim 22 wherein W is selected from the group consisting of colorimetric markers, fluorescent markers, enzyme markers, luminescent markers, radioactive markers, and ligand recognition reporter groups. |
| 28. | The oligonucleotide of Claim 22 wherein the oligonucleotide is a deoxyibonucleotide. |
| 29. | The oligonucleotide of Claim 22 wherein the oligonucleotide is a ribonucleotide. |
This invention relates to oligonucleotides which bind to R A (such as mRNA), DNA, proteins, or peptides, including, for example, oligonucleotides which inhibit mRNA function. More particularly, this invention relates to oligonucleotides in which one or more of the nucleotides include a modified anionic moiety.
Watson-Crick base pairing enables an oligonucleotide to act as an antisense complement to a target sequence of an mRNA in order to block processing or effect translation arrest and regulate selectively gene expression. (Cohen,
,Oligodeoxynucleotides, CRC Press, Boca Raton, Florida (1989)); Uhlmann, et al., Chem. Rev. , Vol. 90, pgs. 543-584 (1990)). Oliogonucleotides have also been utilized to interfere with gene expression directly at the DNA level by formation of triple-helical (triplex) structures in part through Hoogsteen bonding interactions (Moffat, Science, Vol. 252, pgs 1374-1375 (1991)). Furthermore, oligonucleotides have been shown to bind • specifically to proteins (Oliphant, et al., Molec. Cell. Biol. Vol 9, pgs. 2944-2949 (1989)) and could thus be used to block undesirable protein function.
Natural oligonucleotides, which include phosphodiester moieties ("all PO" moieties), and which are negatively charged, however, are relatively ineffective as therapeutic agents due to their poor penetrability into the cell, and their susceptibility
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to degradation by nucleases in vivo. Therefore, relatively high concentrations of natural oligonucleotides are required in order to achieve a therapeutic effect.
To overcome the above shortcomings, various strategies have been devised. One approach is to modify the phosphodiester backbone in order to prevent degradation. U.S. Patent No. 4,469,863, issued to Miller, et al. , discloses the manufacture of nonionic nucleic acid alkyl and aryl phosphonates, and in particular nonionic nucleic acid methyl phosphonates. U.S. Patent No. 4,757,055, also issued to Miller, et al., discloses a method for selectively controlling unwanted expression of foreign nucleic acid in an animal or in mammalian cells by binding the nucleic acid with a nonionic oligonucleotide alkyl or aryl phosphonate analogue.
Oligonucleotides have also been synthesized in which one non-bridging oxygen in each phosphodiester moiety is replaced by sulfur. Such analogues sometimes are referred to as phosphorothioate (PS) analogues, or "all PS" analogues, (Stein, et al., Nucl. Acids Res., Vol. 16, pgs. 3209-3221 (1988)). Oligonucleotide phosphorodithioates in which both non-bridging oxygen atoms attached to phosphorus are replaced by sulfur, have also been prepared. (Brill, et al. J. Amer. Chem. Soc, Vol. Ill, pg. 2321 (1989)). Other backbone modified oligonucleotides previously prepared include phosphoramidates in which non-bridging oxygen atoms have been replaced by nitrogen. (Froehler, et al., Nucleic Acids Res. , Vol. 16, pgs. 4831-4839 (1988)). These compounds, however, are generally less stable due to the presence of labile phosphorus-nitrogen bonds.
Backbone modifications have also been made in which the phosphorus atoms are replaced by other atoms such as carbon or silicon. Examples of such oligonucleotides include oligonucleotide carbamates (Stirchak, et al, J. Org. Chem., Vol. 52, pgs. 4202-4206 (1987)), and silyl esters (Cormier, et al.,
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Nucle c Acids Res., Vol. 16, pg. 4583 (1988)). A review of modified oligonucleotides previously synthesized is given in Uhlmann, et al., Chemical Reviews, Vol. 90, pgs. 543-584 (1990).
Phosphonoacetic acid derivatives of nucleosides have been prepared and evaluated as potential antiviral and/or antineoplastic agents in U.S. Patent No. 4,056,673, issued to Heimer, et al. This patent, however, does not disclose phsophonoacetic acid oligonucleotides.
In accordance with an aspect of the present invention, there is provided an oligonucleotide wherein at least one of the nucleotide units of the oligonucleotide includes a phosphonate moiety having the following structural formula: 0
0 = P - 0 -
Y wherein X is:
-(R)xi-COO -, -(R)n-SO, , or -(R)xi-P0 2- «,-3 R is a hydrocarbon, preferably an alkyl group, and more preferably an alkyl group having from 1 to 15 carbon atoms, still more preferably from 1 to 3 carbon atoms, and most preferably R is methylene. n is 0 or 1. In one embodiment n is 1, and in another embodiment, n is 0.
The oligonucleotides of the present invention thus include a modified anionic moiety, and are in an anionic state when at physiological pH.
In one embodiment, X is (R) -C00~, wherein R is methylene, and n is 0 or 1. Thus, in such an embodiment, at least one of the nucleotide units of the oligonucleotide has a phosphonate
When the oligonucleotide includes at least one nucleotide unit having an anionic phosphonate moiety having one of the structures hereinabove described, such an oligonucleotide may be
employed in the formation of an oligonucleotide having a detectable marker. The detectable marker may be attached to the oligonucleotide at the carboxyl (COO-) group, the SO,~ group, or the PO- 2- group, by means known to those skilled in the art; e.g., through the use of a linker group or by direct attachment. Thus, for example, when the marker or label includes an amino group, the marker or label may be attached by a condensation reaction.
Thus, in accordance with another aspect of the present invention, there is provided an oligonucleotide wherein at least one nucleotide unit of the oligonucleotide includes a phosphonate moiety having the following structural formula: O
O O -, wherein X is:
(R) n -CO-(L) p -W,-(R) n -S0 2 -(L) p -W, or
O -(R) n - -(L) p -w,
0 wherein R is a hydrocarbon as hereinabove desribed, n is 0 or 1, L is a linker group, p is 0 or 1, and W is a detectable marker. Suitable linker groups which may be employed include, but are not limited to, an -NH- group, an NH (CH 2 ) 5 -C group, and an NH-NH-CO-(CH 2 ) 5 -NH group.
Detectable markers which may be employed include, but are not limited to, colorimetric markers,, fluorescent markers, enzyme markers, luminescent markers, radioactive markers, or ligand recognition reporter groups. Specific examples of detectable markers which may be employed include, but are not limited to, biotin and derivatives thereof (such as, for example, e-amino-caproyl biotin, and biotin amidocaproyl hydrazide), fluorescein (including derivatives such as fluorescein amine), rhodamine, alkaline phosphatase, horseradish peroxidase, and 2,4-dinitrophenyl markers. Such oligonucleotides which include a
detectable marker may be used as DNA or RNA probes. The probes may be used as diagnostics as known in the art.
The term "oligonucleotide" as used herein means that the oligonucleotide may be a ribonucleotide or a deoxyribonucleotide; i.e., the oligonucleotide may include ribose or deoxyribose sugars. Alternatively, the oligonucleotide may include other 5-carbon or 6-carbon sugars, such as, for example, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof.
The oligonucleotide also include any natural or unnatural, substituted or unsubstituted, purine or pyrimidine base. Such purine and pyrimidine bases include, but are not limited to, natural purines and pyrimidines such as adenine, thymine, uracil, guanine, cytosine, or other purines and pyrimidines, such as isocytosine, 6-methyluracil, 4,6- di-hydroxypyrimidine, hypoxanthine, xanthine, 2, 6-diaminopurine, azacytosine, 5-methyl cytosine, and the like.
In general, the oligonucleotide includes at least two, preferably at least 5, and most preferably from 5 to about 30 nucleotide units.
The substituted phosphonate moieties hereinabove described are attached to at least one nucleotide unit of the oligonucleotide. In one embodiment, a substituted phosphonate moiety is attached to one or more oligonucleotide units at the 3\' end and/or the 5 1 end of the oligonucleotide. In one embodiment, a substituted phosphonate moiety is attached to alternating nucleotide units of the oligonucleotide. In another embodiment, a substituted phosphonate moiety is attached to each nucleotide unit of the oligonucleotide.
The oligonucleotides may have certain modifications to the 3\' or 5\' termini to improve the pharmacological properties of the oligonucleotides, such as polyethylene glycol, polylysine, acridine, long chain aliphatic groups, and cholesterol.
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The oligonucleotides of the present invention may be employed to bind to RNA sequences by Watson-Crick hybridization, and thereby block RNA processing or translation. For example, the oligonucleotides of the present invention may be employed as "antisense" complements to target sequences of mRNA in order to effect translation arrest and regulate selectively gene expression.
The oligonucleotides of the present invention may be employed to bind double-stranded DNA to form triplexes, or triple helices. Such triplexes inhibit the replication or transcription of DNA, thereby disrupting gene transcription. Such triplexes may also protect DNA binding sites from the action of enzymes such as DNA methylases.
The RNA or DNA of interest, to which the oligonucleotide binds, may be present in a prokaryotic or eukaryotic cell, a virus, a normal cell, or a neoplastic cell. The sequences may be bacterial sequences, plasmid sequences, viral sequences, chromosomal sequences, mitochondrial sequences, or plastid sequences. The sequences may include open reading frames for coding proteins, mRNA, ribosomal RNA, snRNA, hnRNA, introns, or untranslated 5\'- and 3\'-sequences flanking open reading frames. The target sequence may therefore be involved in inhibiting production of a particular protein, enhancing the expression of a particular gene by inhibiting the expression of a repressor, or the sequences may be involved in reducing the proliferation of viruses or neoplastic cells.
The oligonucleotides may be used in vitro or in vivo for modifying the phenotype of cells, or for limiting the proliferation of pathogens such as viruses, bacteria, protists, Mvcoplasma species, Chlamvdia or the like, or for inducing morbidity in neoplastic cells or specific classes of normal cells. Thus, the oligonucleotides may be administered to a host subject to or in a diseased state, to inhibit the transcription and/or expression of the native genes of a target cell.
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Therefore, the oligonucleotides may be used for protection from a variety of pathogens in a host, such as, for example, enterotoxigenic bacteria, Pneumococci, Neisseria organisms, Giardia organisms, Entamoebas, neoplastic cells, such as carcinoma cells, sarcoma cells, and lymphoma cells; specific B-cells; specific T-cells, such as helper cells, suppressor cells, cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, etc.
The oligonucleotides may be selected so as to be capable of interfering with transcription product maturation or production of proteins by any of the mechanisms involved with the binding of the subject composition to its target sequence. These mechansims may include interference with processing, inhibition of transport across the nuclear membrane, cleavage by endonucleases, or the like.
The oligonucleotides may be complementary to such sequences as sequences expressing growth factors, lymphokines, immunoglobulins, T-cell receptor sites, MHC antigens, DNA or RNA polymerases, antibiotic resistance, multiple drug resistance (mdr), genes involved with metabolic processes, in the formation of amino acids, nucleic acids, or the like, DHFR, etc. as well as introns or flanking sequences associated 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 Diseases: Antivirals, Human AIDS, Herpes, CMV Antivirals, Animal Chicken Infectious Bronchitis Pig Transmissible Gastroenteritis Virus
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Antibacterial, Human Drug Resistance Plasmids, E.coli
Antiparasitic Agents Malaria
Sleeping Sickness
(Trypanσsomes)
Cancer
Direct Anti-Tumor Oncogenes and their products
Agents
Adjunctive Therapy Drug-resistant Tumors, genes and their products
Autoimmune Diseases T-cell receptors Rheumatoid Arthritis Type I Diabetes Systemic Lupus Multiple sclerosis
Organ Transplants Kidney-OTK3 cells cause GVHD
The oligonucleotides of the present invention may be employed for binding to target molecules, such as, for example, proteins including, but not limited to, ligands, receptors, and or enzymes, whereby such oligonucleotides inhibit or stimulate the activity of the target molecules.
The above techniques in which the oligonucleotides may be employed are also applicable to the inhibition of viral replication, as well as to the interference with the expression of genes which may contribute to cancer development.
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The oligonucleotides of the present invention are administered in an effective binding amount to an RNA, a DNA, a protein, or a peptide. Preferably, the oligonucleotides are administered to a host, such as a human or non-human animal host, so as to obtain a concentration of oligonucleotide in the blood of from about 0.1 to about 100 μmole/1. It is also contemplated, however, that the oligonucleotides may be administered in vitro or ex vivo as well as in vivo.
The oligonucleotides may be administered in conjunction with an acceptable pharmaceutical carrier as a pharmaceutical composition. Such pharmaceutical compositions may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Such oligonucleotides may be administered by intramuscular, intraperitoneal, intravenous or subdermal injection in a suitable solution. Preferably, the preparations, particularly those which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees and capsules, and preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration parenterally or orally, and compositions which can be administered bucally or sublingually, including inclusion compounds, contain 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.
The pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art. For example, the pharmaceutical preparations may be made by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes. The process to be used will depend ultimately on the physical properties of the active ingredient used.
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Suitable excipients are, in particular, fillers such as sugar, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch or paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are flow- egulating agents and lubricants, such as, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores may be provided with suitable coatings which, if desired, may be resistant 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, solutions 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 combinations of active compound doses.
Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the oligonucleotide in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or
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lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols. In addition, it is also posible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition, suspensions of the active compounds as appropriate oil injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. Optionally, the suspension may also contain stabilizers.
Additionally, the compounds of the present invention may also be administered encapsulated in liposomes, wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active ingredient, depending upon its solubility, may be present both in the aqueous layer, in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively,
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comprises phospholipids such as lecithin and sphingomycelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. The diameters of the liposomes generally range from about 15 nm to about 5 microns.
The invention will now be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.
Example 1
Synthesis of 5\'-dimethoxytrityl-2\'-deoxynucleoside -3\'ethoxycarbonyl phosphonates.
Ethyl phosphonoacetate or ethyl phosphonoformate is treated with triisopropyl benzenesulfonyl chloride in dry pyridine, and 5\'-dimethoxytrityl (DMTr) 2\'deoxynucleoside having the following structure:
Affr -6 -y * A
otf
(wherein B- is thymine, N^-benzoylcytosine, N 2 -isobutyrylguanine, or Ng-benzoyladenine) is added. The mixture is stirred at room temperature overnight and treated with cold water. The solution is then evaporated to dryness and partitioned between ethyl acetate and water, and the ethyl acetate layer is washed with water and then dried overnight over sodium sulfate. The solution is filtered, evaporated to dryness, and the residue is purified by silica column chromatography, using methylene chloride/methanol/triethylamine as an eluant. The fractions containing a 5\'-dimethoxytrityl 2\'-deoxynucleoside
-3 \' -ethoxycarbonyl- phosphonate, which has the following structural formula:
, wherein B. is as hereinabove described and n is 0 or 1, are combined, and evaporated to dryness.
Example 2 Synthesis of a dinucleoside-carboxy-phosphonate A DNA synthesis column, 1 umol size, containing
5\'-dimethoxytrityl thymidine attached to controlled pore glass
(CPG), and having the following structural formula:
VλtTr-0-γoA
(B. is as hereinabove described), obtained from Applied Biosystems Inc., Foster City, California, is installed on an Applied Biosystems DNA synthesizer (Model #394) and synthesis of a modified dinucleoside phosphonate having the following structural formula:
Pi 7i-o -γ 0 y
tPc-
iε carried out by treatment of the supported nucleoside with a solution of a 5\'-dimethoxytrityl- 2\'deoxynucleoside -3\'-ethoxycarbonyl- phosphonate and l-(2- mesitylenesulfonyl) 3-nitro -1,2,4- triazole (MSNT) in acetonitrile. The support is treated with a solution of dichloroacetic acid (DCA) in methylene chloride to remove the dimethoxytrityl protecting group,, and then the nucleoside is cleaved from the support by treatment with ammonia using a standard deprotection cycle. Further prolonged treatment with ammonia or alternatively with dilute aqueous sodium hydroxide or trimethylamine and water hydrolyzes the ethyl ester. After neutralization with Amberlite CG 50 ion exchange resin (H+ form) the solution is evaporated to dryness to give the nucleoside dimer having the following structure:
wherein B 2 is thymine, cytosine, adenine, or guanine.
Example 3 Synthesis of a 3\' modified pentadecanucleotide
The oligonucleotide synthesis column containing a dinucleoside phosphonate having the following structure:
(B-,, is as hereinabove described, n is 0 or 1), prepared as described in Example 2, is treated with dichloroacetic acid in methylene chloride to remove the dimethoxytrityl protecting group to give a dinucleoside phosphonate having the following
This material is then subjected to thirteen cycles of oligonucleotide synthesis on a DNA synthesizer (Applied Biosystems Model No. 394) using cyanoethyl phosphoramidites and reagents as supplied by the manufacturer, Applied Biosystems. At the conclusion of the synthesis, the modified oligonucleotide is cleaved from the support using concentrated ammonia, and the
a monia solution is heated at 55°C for 12 hours to remove the base protecting groups. The solution is then evaporated to dryness and treated with dilute aqueous sodium hydroxide or trimethylamine and water to hydrolyze completely the ethyl ester. After neutralization with Amberlite CG50 ion exchange resin (H+ form), the solution was evaporated to dryness to give a 3\' modified oligonucleotide having the following structure:
wherein B, is thymine, cytosine, guanine, or adenine.
Example 4 Attachment of a Reporter Molecule (Biotin) to an Oligonucleotide containing a Modified Anionic Group The pentadecanucleotide possessing a modified anionic moiety as described in Example 3 (0.5 umol) is dissolved in water containing imidazole hydrochloride (100 mM, pH 4, 1 ml) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.8 mg, 25 umol) and treated with a solution of biotinamidocaproyl hydrazide (25 umol, 9.29 mg, Sigma Chemical Co.) in dimethylsul oxide (1 ml) . The solution is stored at room
temperature overnight and passed through a Sephadex G 25 column (1.5 x 50 cm). Fractions of 1 ml are collected, assayed by UV spectroscopy and the tubes containing the oligonucleotide are combined, concentrated, and purified by high pressure liquid chromatography using a reversed phase C18 column (9.4 x 250 mm) with a linear gradient of 0.1M triethylammonium acetate pH 7: acetonitrile as eluant. The later eluting peak of biotinylated oligonucleotide is collected, evaporated to dryness, and lyophilized overnight to give the biotinylated oligonucleotide having the following structure:
Advantages of the present invention include improved resistance of the modified anionic oligonucleotide to nucleases, as compared with natural "all PO" oligonucleotides. Also, the modified anionic oligonucleotides of the present invention are taken up by the cell and are less readily degraded because of their modified backbones.
In addition, the modified anionic moieties can be used as a linkage site for the attachment of conjugate groups, such as polyethylene glycol, polylysine, acridine, long chain aliphatic groups, or cholesterol.
It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.
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