Methods and compositions for triple helix formation

• US 6,403,302 B1
• Filed: 12/16/1993
• Issued: 06/11/2002
• Est. Priority Date: 09/17/1992
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Dervan, Peter B., Beal, Peter A.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/168,920
• Time in Patent Office: 3,099 Days
• Field of Search: 435/6, 435/91.1, 435/810, 436/501, 436/811, 536/22.1, 536/23.1, 536/25.3, 935/78, 935/88
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1132 against retroviridae, e.g. HIV
• CPC Class Codes: C12N 2310/15 Nucleic acids forming more than 2 strands, e.g. TFOs
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/3511 intercalating or cleaving agent
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays

Abstract

Improved oligonucleotides and processes for their use for specific recognition of a target sequence in double-stranded nucleic acid through the formation of an alternate strand triple-helix. The triple-helix forming oligonucleotides bind in a parallel and antiparallel orientation, respectively, to target sequences on alternate strands of the double helical nucleic acid. The oligonucleotides are useful as diagnostic or therapeutic agents and can incorporate 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 comprising first and second substantially complementary strands and an oligonucleotide bound to a target sequence within said double-helical nucleic acid, wherein said first strand comprises a first purine-rich sequence and a pyrimidine-rich sequence and said target sequence comprises said first purine-rich target sequence and a second purine-rich target sequence on said second strand based paired with said pyrimidine-rich sequence, and wherein said oligonucleotide has 5′

and 3′

terminal nucleotides and comprises a first binding domain comprising a pyrimidine-rich portion bound to said first purine-rich target sequence in a parallel orientation and a second binding domain comprising a purine-rich portion bound to said second purine-rich target sequence in an antiparallel orientation and a linking domain between said first and said second binding domains having a length which permits each of the said binding domains to bind to purine-rich target sequences.

2. The triple-helix of claim 1 wherein said pyrimidine-rich sequence is adjacent and 5′

to said first purine-rich target sequence and said linking domain comprises two nucleotides.

3. The triple-helix of claim 1 wherein said first binding domain comprises a nucleotide containing a 4-phenylimidazole base when the nucleotide at tie complementary position in said first target sequence comprises T or C.

4. The triple-helix of claim 3 wherein said nucleotide containing a 4-phenylimidazole base comprises a nucleotide containing a 4-phenylimidazole base wherein 3-benzamido is substituted at the C3 position.

5. The triple-helix of claim 3 wherein said first binding domain further comprises a T nucleotide when the nucleotide at the complementary position in said first target sequence is A and a C nucleotide or a nucleotide containing a pyrazolopyrimidine base when the nucleotide at the complementary position in said first target sequences.

6. The triple-helix of claim 1 wherein said first binding domain comprises a nucleotide containing a pyrazolopyrimidine base when said nucleotide at the complementary position in said first target sequence comprises G.

7. The triple-helix of claim 6 wherein said nucleotide containing a pyrazolopyrimidine base comprises a nucleotide containing a pyrazolopyrimidine base linked to ribose at the N1 position.

8. The triple-helix of claim 1 wherein said second target sequence comprises one or more of the pyrimidine nucleotide C and said second binding domain comprises a 2′

-deoxynebularine nucleotide when the nucleotide at the complementary position in said second target sequence is C.

9. The triple-helix of claim 8 wherein said second binding domain further comprises a G nucleotide when the nucleotide at the complementary position in said second target sequence is G and an A or a T nucleotide when the nucleotide at the complementary position in said second target sequence is an A.

10. The triple-helix of claim 1 wherein said oligonucleotide bound to said target sequence within said double-helical nucleic acid comprises at least one modified nucleotide.

11. The triple-helix of claim 10 wherein said modified nucleotide is a detectable moiety or a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.

12. An oligonucleotide capable of forming a triple-helix with a double-helical nucleic acid comprising first and second strands wherein said first strand comprises a first purine-rich sequence and a pyrimidine-rich sequence and a target sequence comprises said first purine-rich sequence and a second purine-rich sequence on said second strand based paired with said pyrimidine-rich sequence, said oligonucleotide comprising a first binding domain comprising a pyrimidine-rich portion capable of binding to said first purine-rich target sequence in a parallel orientation and a second binding domain comprising a purine-rich portion capable of binding to said second purine-rich target sequence in an antiparallel orientation and a linking domain between said first and said second binding domains having a length which permits each of the said binding domains to bind to purine-rich target sequences and w herein said first binding domain comprises a nucleotide containing a 4-phenylimidazole base when the nucleotide at the complementary position in said first target sequence is C or T.

13. The oligonucleotide of claim 12 wherein said nucleotide containing a 4-phenylimidazole base comprises a nucleotide containing a 4-phenylimidazole base wherein 3-benzamido is substituted at the C3 position.

17. The oligonucleotide of claims 12, 13, 14, 15 or 16 wherein said oligonucleotide comprises at least one modified nucleotide containing a detectable moiety or a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.

14. An oligonucleotide capable of forming a triple-helix with a double-helical nucleic acid comprising first and second strands wherein said first strand comprises a first purine-rich sequence and a pyrimidine-rich sequence and a target sequence comprising said first purine-rich sequence and a second purine-rich sequence on said second strand based paired with said pyrimidine-rich sequence, said oligonucleotide comprising a first binding domain comprising a pyrimidine-rich portion capable of binding to said first purine-rich target sequence in a parallel orientation and a second binding domain comprising a purine-rich portion capable of binding to said second purine-rich target sequence in an antiparallel orientation and a linking domain between said first and said second binding domains having a length which permits each of the said binding domains to bind to purine-rich target sequences and wherein said first binding domain comprises a nucleotide containing a pyrazolopyrimidine base when the nucleotide at the complementary position of said first target sequence comprises G.

15. The oligonucleotide of claim 14 wherein said nucleotide comprises a nucleotide containing a pyrazolopyrimidine base linked to ribose at the N1 position.

16. An oligonucleotide capable of forming a triple-helix with a double-helical nucleic acid comprising first and second strands wherein said first strand comprises a first purine-rich sequence and a pyrimidine-rich sequence and a target sequence comprising said first purine-rich sequence and a second purine-rich sequence on said second strand based paired with said pyrimidine-rich sequence, said oligonucleotide comprising a first binding domain comprising a pyrimidine-rich portion capable of binding to said first purine-rich target sequence in a parallel orientation and a second binding domain comprising a purine-rich portion capable of binding to said second purine-rich target sequence in an antiparallel orientation and a linking domain between said first and said second binding domains having a length which permits each of the said binding domains to bind to purine-rich target sequences and wherein said second purine-rich target sequence comprises one or more of the pyrimidine nucleotide C and said second binding domain comprises a 2′

-deoxynebularine nucleotide when the nucleotide at the complementary position in said second target sequence is C.

18. A method for forming a triple-helix comprising of binding an oligonucleotide to a target sequence within a double-helical nucleic acid wherein said double-helical nucleic acid comprises first and second strands and said first strand comprises a first purine-rich sequence and a pyrimidine-rich sequence and said target sequence comprises said first purine-rich sequence and a second purine-rich sequence on said second strand based paired with said pyrimidine-rich sequence said method comprising oligonucleotide containing said double-helical nucleic acid and comprising a first binding domain comprising a pyrimidine-rich portion capable of binding to said first purine-rich sequence in a parallel orientation and a second binding domain comprising a purine-rich portion bound to said second purine-rich sequence in an antiparallel orientation and a linking domain between said first and said second binding domains having a length which permits each of the said binding domains to bind to purine-rich target sequences.

19. The method of claim 18 wherein said pyrimidine-rich sequence is adjacent and 5 ′

to said first purine-rich target sequence and said linking domain comprises two nucleotides.

20. The method of claim 18 wherein said first binding domain comprises a nucleotide containing a 4-phenylimidazole base when the nucleotide at the complementary position in said first target sequence comprises T or C.

21. The method of claim 20 wherein said nucleotide containing a 4-phenylimidazole base comprises a nucleotide containing a 4-phenylimidazole base wherein 3-benzamido is substituted at the C3 position.

22. The method of claim 20 wherein said first binding domain further comprises a T nucleotide when the nucleotide at the complementary position in said target sequence is A and a C nucleotide or a nucleotide containing a pyrazolopyrimidine base when the nucleotide at the complementary position in said first target sequence is G.

23. The method of claim 18 wherein said second binding domain comprises a nucleotide containing a pyrazolopyrimidine base when said nucleotide at the complementary position in said second target sequence comprises G.

24. The method of claim 23 wherein said nucleotide containing a pyrazolopyrimidine base comprises a nucleotide containing a pyrazolopyrimidine base linked to ribose at the N1 position.

25. The method of claim 18 wherein said second target sequence comprises one or more of the pyrimidine nucleotide C and said second binding domain comprises a 2′

-deoxynebularine nucleotide when the nucleotide at the complementary position in said second target sequence is C.

26. The method of claim 25 wherein said second binding domain further comprises a G nucleotide when the nucleotide at the complementary position in said second target sequence is G and an A or a T nucleotide when the nucleotide at the complementary position in said second target sequence is an A.

27. The method of claim 18 wherein said oligonucleotide bound to said target sequence within said double-helical nucleic acid comprises at least one modified nucleotide.

28. The method of claim 27 wherein said modified nucleotide contains a detectable moiety or a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.

29. An oligonucleotide capable of forming a triple helix with a double helical nucleic acid comprising first and second strands and a target sequence within said double-helical nucleic acid, said oligonucleotide comprising a binding domain wherein said binding domain comprises a nucleotide containing a 4-phenylimidazole base when the nucleotide at the complementary position in said target sequence is C or T.

30. The oligonucleotide of claim 29 wherein said nucleotide containing a 4-phenylimidazole base comprises a nucleotide containing a 4-phenylimidazole base wherein 3-benzamindo is substituted at the C3 position.