Targeted mutagenesis in living cells using modified oligonucleotides

• US 6,136,601 A
• Filed: 03/26/1997
• Issued: 10/24/2000
• Est. Priority Date: 08/21/1991
• Status: Expired due to Fees

• Current Assignee: Epoch Pharmaceuticals, Inc.
• Original Assignee: Epoch Pharmaceuticals, Inc.
• Inventors: Gall, Alexander A., Meyer, Rich B. Jr., Kutyavin, Igor V., Gamper, Howard B.
• Primary Examiner(s): LEGUYADER, JOHN L
• Application Number:
  US08/827,117
• Time in Patent Office: 1,308 Days
• Field of Search: 435/6, 435/315, 514/44, 536/24.5, 536/24.3, 536/24.31, 536/24.33
• US Class Current: 435/375
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine

Abstract

A method for introducing a site-specific mutation into a target polynucleotide sequence is presented. The method involves the use of an oligonucleotide capable of binding to the target sequence, either by triplex formation (mediated by Hoogsteen, reverse Hoogsteen or equivalent base pairing) or by Watson/Crick base pairing (in the presence of a recombinase enzyme). The oligonucleotide of the invention is modified by the covalent attachment of one or more electrophilic groups. When a modified oligonucleotide is bound to its target sequence, the electrophilic group is able to interact with a nearby nucleotide in the target sequence, causing a modification to the nucleotide that results in a change in nucleotide sequence. Compositions used in the practice of the method are also disclosed.

Claims

1. A modified oligonucleotide for site-directed mutagenesis of a double-stranded nucleic acid molecule comprising one or more mutagens incorporated into a single-stranded oligonucleotide, said oligonucleotide having a sequence that forms a triple-stranded nucleic acid molecule with a target sequence in the double-stranded nucleic acid molecule.

2. The oligonucleotide according to claim 1 wherein the target sequence is in a chromosomal gene of a plant or animal, a viral genome, or the genome of a pathogen.

3. The oligonucleotide according to claim 1 wherein the mutagen is an electrophilic group.

4. The oligonucleotide according to claim 1 wherein the mutagen is an alkylating agent.

5. The oligonucleotide according to claim 1 wherein two or more mutagens are attached to the oligonucleotide.

6. The oligonucleotide according to claim 5 wherein two or more alkylating agents are attached to the oligonucleotide.

7. A method for site-directed mutagenesis of a nucleic acid molecule comprising the steps of:

a) contacting a modified oligonucleotide to a target sequence of a double-stranded nucleic acid molecule, wherein the modified oligonucleotide comprises one or more mutagens incorporated into a single-stranded nucleic acid that forms a triple-stranded nucleic acid molecule with the target region; and

b) thereby mutating the double-stranded nucleic acid molecule.

8. The method according to claim 7 wherein the target sequence is in a chromosomal gene of a plant or animal, a viral genome, or the genome of a pathogen.

9. The method according to claim 7 wherein the mutagen is an electrophilic group.

10. The method according to claim 7 wherein the mutagen is an alkylating agent.

11. The method according to claim 7 wherein two or more mutagens are attached to the oligonucleotide.

12. The method according to claim 7 wherein two or more alkylating agents are attached to the oligonucleotide.

13. The method according to claim 7 wherein the mutagenesis alters the activity of the double-stranded nucleic acid molecule.

22. The method according to claim 7 wherein the oligonucleotide comprises at least about 26 nucleotides and wherein the oligonucleotide forms a triple-stranded complex with the target according to the rules of Watson/Crick base pairing,

the method further comprising contacting the oligonucleotide with a recombinase.

14. A method of producing a mutagenic oligonucleotide comprising the steps of:

a) synthesizing an oligonucleotide which forms a triple-stranded nucleic acid molecule with a target sequence of a double-stranded nucleic acid molecule; and

b) covalently attaching one or more mutagens to the oligonucleotide.

15. The method according to claim 14 wherein the target sequence is in a chromosomal gene of a plant or animal, a viral genome, or the genome of a pathogen.

16. The method according to claim 14 wherein the mutagen is an electrophilic group.

17. The method according to claim 14 wherein the mutagen is an alkylating agent.

18. The method according to claim 14 wherein two or more mutagens are attached to the oligonucleotide.

19. The method according to claim 14 wherein two or more alkylating agents are attached to the oligonucleotide.

20. The method according to claim 14 wherein the mutagen is incorporated into the oligonucleotide during synthesis of the oligonucleotide.

21. The method according to claim 14 wherein the mutagen is incorporated into the oligonucleotide subsequent to synthesis of the oligonucleotide.