Aptamers and antiaptamers

• US 20050176940A1
• Filed: 06/28/2002
• Published: 08/11/2005
• Est. Priority Date: 06/29/2001
• Status: Abandoned Application

• Current Assignee: Unisearch Limited (University Of New South Wales)
• Original Assignee: Unisearch Limited (University Of New South Wales)
• Inventors: King, Garry Charles
• Application Number:
  US10/482,006
• Publication Number:
  US 20050176940A1
• Time in Patent Office: Days
• US Class Current: 536/23.100
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• 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 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/151 more than 3 strands, e.g. tetrads, H-DNA
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12Y 304/21005 Thrombin (3.4.21.5)

Abstract

The present invention relates to: An aptamer comprising a circular oligonucleotide defining one to four target binding regions; An aptamer comprising an oligonucleotide defining two, three or four thrombin binding quadruplex regions separated by at least partially duplex regions, wherein the quadruplex regions comprise a GGTMGGXGGTTGG sequence wherein M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues; An aptamer represented by formula (I): 5′D₁, wQxD₁D₂yQzD₂,3′—the variables are as defined in the specification; and Aptamers selected from specific sequences.

Claims

1. An aptamer comprising a circular oligonucleotide defining one to four target binding regions.

2. The aptamer of claim 1 which defines two, three or four target binding regions wherein said binding regions are separated by at least partially duplex regions.

3. The aptamer of claim 1 which defines one or more protein, cellular, cell component or material binding region.

4. The aptamer of claim 3 wherein the protein binding region is a thrombin binding region

5. The aptamer of claim 3 wherein the cellular binding region is an L-selectin binding domain.

6. The aptamer of claim 1 consisting of nucleotides.

7. The aptamer of claim 1 consisting of RNA.

8. The aptamer of claim 1 consisting of DNA.

40. A method of treatment of thrombosis in a patient requiring such treatment which comprises administering to said patient an effective amount of an aptamer according to claim 1.

41. A method of preventing or reducing coagulation of blood or blood derived products which comprises contacting the blood or blood derived product with an effective amount of an aptamer according to claim 1.

42. Use of a compound according to claim 1 in preparation of a medicament for the treatment of thrombosis.

43. A method for capturing leukocytes from a physiological fluid comprising contacting the physiological fluid with an effective amount of an aptamer according to claim 1.

44. A composition comprising an aptamer according to claim 1 or its antisense antidote together with one or more pharmaceutically acceptable carriers or excipients.

45. A composition according to claim 41 in oral dosage form.

46. A method for counteracting the effect of an aptamer according to claim 1 comprising contacting the aptamer with a counteracting effective amount of an antidote aptamer thereof.

9. An aptamer comprising an oligonucleotide defining two, three or four thrombin binding quadruplex regions separated by at least partially duplex regions, wherein the quadruplex regions comprise a GGTWGGXGGTTGG (SEQ ID NO:

3) sequence wherein M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues.

10. The aptamer of claim 9 ligated at its termini to form a circular oligonucleotide.

11. The aptamer of claim 10 wherein the termini have been chemically ligated.

12. The aptamer of claim 10 wherein the termini have been enzymatically ligated.

13. The aptamer of claim 9 consisting of nucleotides.

14. The aptamer of claim 9 consisting of RNA.

15. The aptamer of claim 9 consisting of DNA.

16. The aptamer of claim 15 wherein X represents a sequence selected from TGT, GCA and TGA.

17. An aptamer represented by formula I:

5N D₁NwQxD₁D₂yQzD₂N 3N  

 

Formula I

wherein

Q represents a sequence GGTWGGXGGTTGG (SEQ ID NO;

3) where M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues;

w, x, y and z are the same or different and represent a sequence of zero to ten nucleotides and/or nucleotide analogues;

D₁ and D₂ are the same or different and each represent a sequence of zero to twenty-five nucleotides and/or nucleotide analogues with the proviso that D₁ and D₂ together comprise at least two nucleotides or nucleotide analogues;

D₁N and D₂N are the same or different and each represent a sequence of zero to fifty nucleotides and/or nucleotide analogues, wherein at least two consecutive nucleotides or nucleotide analogues of D₁N and/or D₂N are complimentary to at least two consecutive nucleotides or nucleotide analogues of D₁ and/or D₂, so as to allow duplex formation between complimentary nucleotides or nucleotide analogues.

18. The aptamer of claim 17 wherein the 5N terminus is phosphorylated.

19. The aptamer of claim 17 wherein w, x, y and z are the same or different and each represent zero, one or two nucleotides and/or nucleotide analogues.

20. The aptamer of claim 17 wherein D₁ and D₂ in total represent two to twenty nucleotides and/or nucleotide analogues.

21. The aptamer of claim 20 wherein D₁ and D₂ in total represent four to twelve nucleotides and/or nucleotide analogues.

22. The aptamer of claim 17 wherein D₁N and D₂N in total represent two to twenty nucleotides and/or nucleotide analogues.

23. The aptamer of claim 22 wherein D₁N and D₂N in total represent four to twelve nucleotides and/or nucleotide analogues.

24. The aptamer of claim 17 ligated at its termini to form a circular sequence of nucleotides and/or nucleotide analogues.

25. The aptamer of claim 24 wherein the termini have been chemically ligated.

26. The aptamer of claim 24 wherein the termini have been enzymatically ligated.

27. The aptamer of claim 17 consisting of nucleotides.

28. The aptamer of claim 17 consisting of RNA.

29. The aptamer of claim 17 consisting of DNA.

30. The aptamer of claim 17 wherein X represents a sequence selected from TGT, GCA and TGA.

31. The aptamer of claim 17 wherein D₁ and D₁N are selected from the following respective pairs:

CAG and CTG;

CAGC and GCTG;

CATGC and GCATG;

CATCGC and GCGATG.

32. The aptamer of claim 17 wherein D₂ and D₂N are selected from the following respective pairs:

CAC and GTG;

GCAC and GTGC;

GCTAC and GTAGC;

GACTAC and GTAGTC.

34. An antidote aptamer comprising at least ten nucleotides and/or nucleotide analogues complimentary to a sequence of at least ten nucleotides and/or nucleotide analogues from an aptamer according to claim 17.

35. An antisense oligonucleotide to an aptamer of claim 17.

36. An antidote aptamer according to claim 34 ligated at its termini to form a circular oligonucleotide.

37. The antidote aptamer or the antisense oligonucleotide of claim 36 wherein the termini have been chemically ligated.

38. The antidote aptamer or the antisense oligonucleotide of claim 37 wherein the termini have been enzymatically ligated.

39. An aptamer according to claim 34 having the following sequence:

47. An antisense oligonucleotide according to claim 35 ligated at its termini to form a circular oligonucleotide.

33. Aptamers selected from those with the following sequences: