Triple helices and processes for making same

• US 5,874,555 A
• Filed: 11/22/1993
• Issued: 02/23/1999
• Est. Priority Date: 10/30/1987
• Status: Expired due to Term

• 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:
  US08/156,957
• Time in Patent Office: 1,919 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: 536/23.1
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays

Abstract

Oligonucleotides and processes for their use for specific recognition of a target sequence in double-stranded nucleic acid through the formation of a triple helix. The oligonucleotides can bind in a parallel or antiparallel orientation to one of the strands of the target sequence depending upon the sequence composition of the target sequence and oligonucleotide used. The oligonucleotides are used as diagnostic or therapeutic agents through incorporation of an appropriate moiety in one or more nucleotides in the triple helix forming oligonucleotide.

Claims

1. A triple helix comprising a double-helical nucleic acid having more than 500 nucleotide base pairs and comprising first and second strands and a heteropolymeric oligonucleotide bound to a target sequence within said double-helical 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 a G respectively, on said first strand.

2. The triple helix of claim 1 wherein said oligonucleotide comprises a pyrimidine-rich oligonucleotide and said target sequence comprises a purine-rich sequence on one of the strands of said double-helical nucleic acid.

3. The triple helix of claim 2 wherein said pyrimidine-rich oligonucleotide binds to said target sequence in a parallel orientation to said purine-rich target sequence.

4. The triple helix of claim 1 wherein said oligonucleotide is bound in said parallel orientation to said first strand.

5. The triple helix of claim 1 wherein said oligonucleotide comprises a purine-rich oligonucleotide and said target sequence comprises a purine-rich sequence on one of the strands of said double-helical nucleic acid.

6. The triple helix of claim 5 wherein said purine-rich oligonucleotide binds to said target sequence in an antiparallel orientation to said purine-rich target sequence.

7. The triple helix of claim 6 wherein said purine-rich oligonucleotide contains a G when the nucleotide at the complementary position in said purine-rich target sequence is G and an A when the nucleotide at the complementary position in said purine-rich target sequence is A.

8. The triple helix of claim 7 wherein said purine-rich target sequence contains up to about 40% of the pyrimidine nucleotide C and said purine-rich oligonucleotide contains the pyrimidine nucleotide T when the nucleotide at the nucleotide at the complementary position in said purine-rich target sequence is C.

9. The triple helix of claim 8 wherein said purine-rich target sequence contains up to about 25% C.

10. The triple helix of claim 6 wherein said purine-rich target sequence contains one or more or the pyrimidine nucleotide C and said purine-rich oligonucleotide contains a G when the nucleotide at the complementary position in said purine-rich target sequence is G, an A or a T when the nucleotide at the complementary position in said purine-rich target sequence is an A and a nebularine nucleotide when the nucleotide at the complementary position in said purine-rich target sequence is C.

11. The triple helix of claim 1 wherein said oligonucleotide bound to said target sequence within said double-helical nucleic acid further comprises at least one nucleotide to which a moiety is attached.

12. The triple helix of claim 11 wherein said moiety is a detectable moiety.

13. The triple helix of claim 11 wherein said moiety is a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.

14. The triple helix of claim 1 wherein said double-helical nucleic acid is an isolated nucleic acid.

15. The triple helix of claim 1 wherein said double-helical nucleic acid is substantially pure.

16. The triple helix of claim 1 wherein said target sequence comprises at least one purine nucleotide and at least one pyrimidine nucleotide.

17. The triple helix of claim 1 wherein said oligonucleotide is at least eleven nucleotides in length.

18. The triple helix of claim 1 wherein said oligonucleotide is at least fifteen nucleotides in length.

19. The triple helix of claim 1 wherein said oligonucleotide at least eighteen nucleotides in length.

20. A process for forming a triple helix wherein a heteropolymeric oligonucleotide is bound to a target sequence within a double-helical nucleic acid having more than 500 nucleotide base pairs and comprising first and second strands, comprising contacting said double-helical nucleic acid with a heteropolymeric oligonucleotide capable of binding to a target sequence contained within said double-helical 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 a G respectively, on said first strand.

21. The method claim 20 wherein said oligonucleotide comprises a pyrimidine-rich oligonucleotide and said target sequence comprises a purine-rich sequence on one of the strands of said double-helical nucleic acid.

22. The method of claim 21 wherein said pyrimidine-rich oligonucleotide binds to said target sequence in a parallel orientation to said purine-rich target sequence.

23. The method of claim 20 wherein said oligonucleotide is bound in said parallel orientation to said first strand.

24. The method of claim 20 wherein said oligonucleotide comprises a purine-rich oligonucleotide and said target sequence comprises a purine-rich sequence on one of the strands of said double-helical nucleic acid.

25. The method of claim 24 wherein said purine-rich oligonucleotide binds to said target sequence in an antiparallel orientation to said purine-rich target sequence.

26. The method of claim 25 wherein said purine-rich oligonucleotide contains a G when the nucleotide at the complementary position in said purine-rich target sequence is G and an A when the nucleotide at the complementary position in said purine-rich target sequence is an A.

27. The method of claim 26 wherein said purine-rich target sequence contains up to about 40% of the pyrimidine nucleotide C and said purine-rich oligonucleotide contains the pyrimidine nucleotide T when the nucleotide at the nucleotide at the complementary position in said purine-rich target sequence is C.

28. The method of claim 27 wherein said purine-rich target sequence contains up to about 25% C.

29. The method of claim 25 wherein said purine-rich target sequence contains one or more or the pyrimidine nucleotide C and said purine-rich oligonucleotide contains a G when the nucleotide at the complementary position in said purine-rich target sequence is G, an A or a T when the nucleotide at the complementary position in said purine-rich target sequence is an A and a nebularine nucleotide when the nucleotide at the complementary position in said purine-rich target sequence is C.

30. The method of claim 20 wherein said oligonucleotide bound to said target sequence within said double-helical nucleic acid further comprises at least one nucleotide to which a moiety is attached.

31. The method of claim 30 wherein said moiety is a detectable moiety.

32. The method of claim 30 wherein said moiety is a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.

33. The process of claim 20 wherein said double-helical nucleic acid is an isolated nucleic acid.

34. The process of claim 20 wherein said double-helical nucleic acid is substantially pure.

35. A process for forming a triple-helix comprising contacting a triple-helical nucleic acid having more than 500 nucleotide base pairs with an oligonucleotide comprising a nebularine-containing nucleotide which is capable of binding to a target sequence contained within said double-helical nucleic acid.