Antisense oligonucleotides with increased RNase sensitivity

• US 7,790,877 B2
• Filed: 05/08/2002
• Issued: 09/07/2010
• Est. Priority Date: 10/02/1997
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Brown, Bob D., Riley, Timothy A., Arnold, Lyle J.
• Primary Examiner(s): Chong; Kimberly
• Application Number:
  US10/142,566
• Publication Number:
  US 20030119016A1
• Time in Patent Office: 3,044 Days
• Field of Search: 435/6, 435/91.1, 435/91.3, 435/325, 435/375, 536/23.1, 536/24.5, 536/24.3, 536/24.31, 536/24.33, 514/44
• US Class Current: 536/24.5
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/313 Phosphorodithioates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3533 Halogen

Abstract

Combinatorial libraries comprise first oligonucleotide analogs and second oligonucleotide analogs which are coupled together to form antisense molecules capable of binding target polynucleotides and activating an RNase, and ribozymes capable of cleaving polynucleotides.

Claims

1. An antisense oligonucleotide having improved RNase activation characteristics comprising an RNase activation region and a plurality of adjacent universal bases, wherein said antisense oligonucleotide recruits and/or activates an RNase when hybridized to a target polynucleotide, wherein each said universal base comprises an unnatural nucleobase analog, and wherein said plurality of adjacent universal bases increase the activity of said RNase to cleave a target molecule compared to an antisense oligonucleotide without said plurality of adjacent universal bases.

2. The antisense oligonucleotide of claim 1, further comprising an oligonucleotide analog moiety.

3. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises a phosphorothioate.

4. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises 2′

-O-hydrocarbyl.

5. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises 2′

-F.

6. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises 2′

-O-methoxyethyl.

7. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises MMI.

8. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises methylphosphonate.

9. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprising morpholine.

10. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises peptide nucleic acid.

11. The antisense oligonucleotide of claim 2, wherein said oligonucleotide analog moiety comprises 3′

-amidate.

12. The antisense oligonucleotide of claim 1, further comprising a flexible linker disposed between said plurality of universal bases and said RNase activation region.

13. The antisense oligonucleotide of claim 12, wherein said flexible linker comprises polyethylene glycol.

14. The antisense oligonucleotide of claim 1, wherein said plurality of adjacent universal bases comprises four universal bases.

15. The antisense oligonucleotide of claim 1, wherein said target molecule comprises RNA.

16. The antisense oligonucleotide of claim 15, wherein said target molecule comprises messenger RNA.

17. The antisense oligonucleotide of claim 1, wherein said universal bases comprise an unnatural universal base moiety comprising hypoxanthine.

18. The antisense oligonucleotide of claim 1, wherein said universal bases comprise an unnatural universal base moiety comprising nitroindole.

19. The antisense oligonucleotide of claim 1, wherein said universal bases comprise an unnatural universal base moiety comprising 4-nitrobenzimidazole.

20. A composition for activation of RNase cleavage of a target RNA sequence comprising:

a first oligonucleotide analog comprising the antisense oligonucleotide of claim 1 comprising a first binding domain that binds to a target sequence and further comprising a first coupling moiety; and

a second oligonucleotide analog comprising a second binding domain that binds to said target sequence, and a second coupling moiety which binds to said first coupling moiety;

wherein said first and second coupling moieties join spontaneously in the absence of said target sequence.

21. The composition of claim 20, wherein said first or second binding domain comprises from about 3 to about 24 bases.

22. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising phosphorothioate.

23. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising 2′

-O-hydrocarbyl.

24. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising 2′

-F.

25. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising 2′

-O-methoxyethyl.

26. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising MMI.

27. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising methylphosphonate.

28. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising morpholino.

29. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising peptide nucleic acid.

30. The composition of claim 20, wherein said first or second binding domain comprises an oligonucleotide analog moiety comprising 3′

-amidate.

31. The composition of claim 20, wherein said first binding domain and said first coupling moiety are joined together by a flexible linker.

32. The composition of claim 20, wherein said second binding domain and said second coupling moiety are joined together by a flexible linker.

33. The composition of claim 32, wherein said first binding domain and said first coupling moiety are joined together by a flexible linker.

34. The composition of claim 31, wherein said flexible linker comprises polyethylene glycol.

35. The composition of claim 32, wherein said flexible linker comprises polyethylene glycol.

36. The composition of claim 34, wherein said flexible linker comprises polyethylene glycol having from 1 to about 10 ethylene glycol monomers.

37. The composition of claim 35, wherein said flexible linker comprises polyethylene glycol having from 1 to about 10 ethylene glycol monomers.

38. The composition of claim 20, wherein said first and second coupling moieties are selected from the group consisting of oligonucleotide duplexes, oligonucleotide analog duplexes, and protein-ligand pairs.

39. The composition of claim 20, wherein said first and second coupling moieties are selected to participate in a non-covalent binding interaction.

40. The composition of claim 20, further comprising a third oligonucleotide analog, comprising a third binding domain capable of binding said target sequence, and a third coupling moiety capable of binding a fourth coupling moiety;

wherein said first oligonucleotide analog or said second oligonucleotide analog further comprises a fourth coupling moiety.

41. The antisense oligonucleotide of claim 1, wherein said RNase activation region comprises DNA.

42. The antisense oligonucleotide of claim 1, wherein said RNase is RNase H or RNase L.

43. The antisense oligonucleotide of claim 1, wherein said RNase is RNase H.

44. The antisense oligonucleotide of claim 1, wherein said RNase is RNase L.

45. An antisense oligonucleotide comprising an RNase activation region and a plurality of adjacent universal bases, wherein each said universal base comprises an unnatural nucleobase analog;

wherein said oligonucleotide acts as a substrate for an RNase when hybridized to a target nucleic acid, wherein said RNase recognizes double stranded RNA or RNA/DNA hybrids; and

wherein said plurality of adjacent universal bases increase the activity of said RNase to cleave a target molecule compared to an antisense oligonucleotide without said plurality of adjacent universal bases.

46. The antisense oligonucleotide of claim 45, wherein said RNase is RNase H or RNase L.

47. The antisense oligonucleotide of claim 45, wherein said RNase is RNase H.

48. The antisense oligonucleotide of claim 45, wherein said RNase is RNase L.

49. An antisense oligonucleotide comprising a plurality of adjacent universal bases, wherein said antisense oligonucleotide recruits an RNase when hybridized to a target polynucleotide;

wherein each said universal base comprises an unnatural nucleobase analog, and wherein said plurality of adjacent universal bases increase the activity of said RNase to cleave a target molecule compared to an antisense oligonucleotide without said plurality of adjacent universal bases.

50. An antisense oligonucleotide comprising a binding domain and a plurality of adjacent universal bases, wherein said antisense oligonucleotide recruits an RNase when hybridized to a target polynucleotide;

wherein each said universal base comprises an unnatural nucleobase analog, and wherein said plurality of adjacent universal bases increase the activity of said RNase to cleave a target molecule compared to an antisense oligonucleotide without said plurality of adjacent universal bases.