Process for identifying nucleic acids and triple helices formed thereby

• US 5,789,155 A
• Filed: 11/12/1993
• Issued: 08/04/1998
• 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): Marschel, Ardin H.
• Application Number:
  US08/152,250
• Time in Patent Office: 1,726 Days
• Field of Search: 435/5, 435/6, 435/810, 436/501, 536/22.1, 536/23.1, 536/24.1, 536/24.31-24.33, 536/25.3, 935/77, 935/78, 935/88
• 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 process for identifying the presence of a subregion within a large double-helical nucleic acid, comprising the steps of:

(a) contacting a double-helical nucleic acid comprising first and second strands with an oligonucleotide under conditions which permit the formation of a triple-helix when said subregion is present in said double-helical nucleic acid, wherein said oligonucleotide contains at least one nucleoside to which is attached at least one moiety capable of being detected when bound to said subregion, wherein when said oligonucleotide is bound in a parallel orientation to said first strand, said oligonucleotide comprises a T or a C⁺ bound to an A or G respectively, on said first strand; and

(b) detecting said moiety as an indication of the presence of said subregion within said double-helical nucleic acid.

2. The process of claim 1 wherein said oligonucleotide comprises a substantially homopyrimidine oligonucleotide and said double-helical sequence comprises a homopyrimidine-homopurine tract.

3. The process of claim 1 wherein said double-helical nucleic acid comprises nucleic acid coding for a virus and said oligonucleotide is capable of binding to a subregion within said virus.

4. The process of claim 1, wherein said double-helical nucleic acid comprises nucleic acid coding for an oncogene and said oligonucleotide is capable of binding to a subregion within said oncogene.

5. The process of claim 1 wherein said moiety comprises a radioactive isotope.

6. The process of claim 1 wherein said moiety comprises a fluorescent molecule.

7. The process of claim 1 wherein said moiety comprises at least one enzyme capable of detectably reacting with a substrate.

8. The process of claim 1 wherein said large double-stranded nucleic acid sequence comprises deoxyribonucleic acid.

9. The process of claim 1 wherein said large double-stranded nucleic acid sequence comprises ribonucleic acid.

10. The process of claim 1 wherein said double-stranded nucleic acid sequence comprises a hybrid duplex with one strand of deoxyribonucleic acid and one strand of ribonucleic acid.

11. The process of claim 1 wherein said large double-helical nucleic acid is from a chromosome, a gene, or other natural source.

12. Nucleic acid comprising a large double-helical nucleic acid comprising first and second strands, and an oligonucleotide bound to a double helical subregion of said nucleic acid to form a triple-helix, wherein when said oligonucleotide is bound in a parallel orientation to said first strand, said oligonucleotide comprises a T or a C⁺ bound to an A or G respectively, on said first strand, said oligonucleotide comprising at least one modified nucleotide containing a moiety capable of being detected when bound to said subregion.

13. The nucleic acid of claim 12 wherein said oligonucleotide comprises a substantially homopyrimidine oligonucleotide and said double-helical subregion in said nucleic acid comprises a substantially homopyrimidine-homopurine tract.

14. The nucleic acid of claim 12 wherein said moiety comprises a radioactive isotope.

15. The nucleic acid of claim 12 wherein said moiety comprises a fluorescent molecule.

16. The nucleic acid of claim 12 wherein said moiety comprises at least one enzyme capable of detectably reacting with a substrate.

17. The nucleic acid of claim 12 wherein said large double-helical nucleic acid comprises a double-stranded deoxyribonucleotide acid.

18. The nucleic acid of claim 12 wherein said large double-helical nucleic acid comprises ribonucleic acid.

19. The nucleic acid of claim 12 wherein said double-helical nucleic acid comprises a hybrid duplex between one strand of deoxyribonucleic acid and one strand of ribonucleic acid.

20. The nucleic acid of claim 12 wherein said large double-helical nucleic acid is from a chromosome, a gene, or other natural source.

21. A triple helix comprising a large double-helical nucleic acid comprising first and second strands and an oligonucleotide bound to a specific sequence within said nucleic acid, wherein when said oligonucleotide is bound in a parallel orientation to said first strand, said oligonucleotide comprises a T or a C⁺ bound to an A or G respectively, on said first strand.

22. The triple helix of claim 21 wherein said oligonucleotide comprises a substantially homopyrimidine oligonucleotide and said specific sequence comprises a substantially homopyrimidine-homopurine tract.

23. The triple helix of claim 21 wherein said large double-helical nucleic acid comprises double stranded deoxyribonucleic acid.

24. The triple helix of claim 21 wherein said large double-helical nucleic acid comprises ribonucleic acid.

25. The triple helix of claim 21 wherein said double-helical nucleic acid comprises a hybrid duplex between one strand of deoxyribonucleic acid and one strand of ribonucleic acid.

26. The triple helix of claim 21 wherein said oligonucleotide has a length between 11 and 15 nucleotides.

27. The triple helix of claim 21 wherein said double-helical nucleic acid is an isolated nucleic acid.

28. The triple helix of claim 21 wherein said oligonucleotide comprises at least one modified nucleotide.

29. The triple helix of claim 21 wherein said large double-helical nucleic acid is from a chromosome, a gene, or other natural source.

30. A process for forming a triple helix comprising contacting a large double-helical nucleic acid comprising first and second strands with an oligonucleotide under conditions which permit the formation of a triple-helix wherein said oligonucleotide is bound to a specific sequence within said double-helical nucleic acid, and wherein when said oligonucleotide is bound in a parallel orientation to said first strand, said oligonucleotide comprises a T or a C⁺ bound to an A or G respectively, on said first strand.

31. The process of claim 30 wherein said oligonucleotide has a length between 11 and 15 nucleotides.

32. The process of claim 30 wherein said double-helical nucleic acid is an isolated nucleic acid.

33. The process of claim 30 wherein said oligonucleotide comprises at least one modified nucleotide.

34. The process of claim 30 wherein said large double-helical nucleic acid is from a chromosome, a gene, or other natural source.