Triple helix recognition of DNA

• US 6,432,638 B2
• Filed: 08/04/1998
• Issued: 08/13/2002
• Est. Priority Date: 10/30/1987
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Moser, Heinz E., Dervan, Peter B.
• Primary Examiner(s): LeGuyader, John L.
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/128,732
• Publication Number:
  US 20010002314A1
• Time in Patent Office: 1,470 Days
• Field of Search: 435/375, 435/6, 435/29, 435/325, 435/455, 514/44, 536/24.5, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.1, 536/25.3
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays

Abstract

Probes and processes for their use for specific recognition and/or cleavage of double-stranded DNA or RNA at sequence specific desired loci through the intermediacy of a triple helix are disclosed. These probes may also be used as diagnostic chemotherapeutic agents through incorporation of a radiolabeled, fluorescing, or otherwise detectable molecule. Preferred assay conditions are also provided for recognition of homopurine-homopyrimidine double-helical tracts within large DNA by triple helix formation under physiological conditions. Hybridization probes for double-stranded recognition with binding site sizes that range >8 base pairs are also provided.

Claims

1. A method for forming a triple helix in a cell cultured in vitro wherein said cell comprises a double-stranded nucleic acid involved in a gene-based disease caused by a virus, genetic defect, or oncogene, said method comprising the steps of:

contacting a subregion in said nucleic acid with an oligonucleotide to form a triple helix such that when said oligonucleotide binds in a parallel orientation to a first strand of said nucleic acid, said oligonucleotide comprises a T or a C+ bound to an A or a G respectively on said first strand, and wherein said oligonucleotide also contains a nucleoside to which is attached a moiety that alters the natural expression of said nucleic acid.

2. The method of claim 1, wherein said double-stranded nucleic acid comprises deoxyribonucleic acid.

3. The method of claim 1, wherein said double-stranded nucleic acid comprises ribonucleic acid.

4. The method of claim 1, wherein said double-stranded nucleic acid comprises a hybrid duplex with one strand of deoxyribonucleic acid and one strand of ribonucleic acid.

5. The method of claim 1, wherein said oligonucleotide comprises a pyrimidine-rich oligonucleotide and said double-stranded nucleic acid comprises a purine-rich subregion on one of the strands.

6. The method of claim 1, wherein said oligonucleotide comprises a purine-rich oligonucleotide and said large double-stranded nucleic acid comprises a purine-rich subregion on one of the strands.

7. The method of claim 1, wherein said large double-stranded nucleic acid is from a chromosome, a gene or other natural source.

8. The method of claim 1, wherein said large double-stranded nucleic acid comprises a nucleic acid encoding a viral protein and said oligonucleotide is capable of binding to a subregion within said nucleic acid encoding said viral protein.

9. The method of claim 1, wherein said large double-stranded nucleic acid comprises a nucleic acid encoding an oncogenic protein and said oligonucleotide is capable of binding to a subregion within said nucleic acid encoding said oncogenic protein.

10. The method of claim 1, wherein said moiety comprises a radioactive isotope.

11. The method of claim 1, wherein said moiety comprises a radioactive fluorescent molecule.

12. The method of claim 1, wherein said moiety comprises at least one enzyme that detectably reacts with a substrate.

13. The method of claim 1, wherein said moiety comprises a cleaving moiety that causes cleavage of said large double-stranded nucleic acid.

14. The method of claim 1, wherein said moiety comprises a chemotherapeutic agent.

15. The method of claim 1, wherein said oligonucleotide has a length between 11 and 15 nucleotides.

16. The method of claim 1, wherein said oligonucleotide has at least one modified nucleotide.

17. The method of claim 1, further comprising detecting said moiety as an indication of the presence of said subregion within said double-stranded nucleic acid.

18. The method of claim 13, further comprising cleaving said double-stranded region by said cleaving moiety.

19. The method of claim 14, further comprising permitting said chemotherapeutic agent to act.

20. The method of claim 14, wherein said chemotherapeutic agent is an artificial or natural gene repressor to specific site of interest in said double-stranded nucleic acid involved in a gene-based disease.