Selection of aptamers based on geometry

• US 9,938,641 B2
• Filed: 12/18/2007
• Issued: 04/10/2018
• Est. Priority Date: 12/18/2006
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: West, Jason Andrew Appleton, Satterfield, Brent Coleman
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US11/959,435
• Publication Number:
  US 20080182759A1
• Time in Patent Office: 3,766 Days
• Field of Search: 424 91, 435 61, 435 612, 435 911, 435455, 435 9131, 435458, 514 44, 536 231, 536 245, 506 9
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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 2310/16 Aptamers
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C40B 20/08 Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Abstract

Disclosed are methods for performing aptamer preselection based on unique geometry and the content of stems or loops of the aptamer, which methods are capable of providing suitable binders and also permit selection of aptamers performed essentially entirely on a chip or other device. Also disclosed are kits for aptamer selection.

Claims

1. A method, comprising:

geometrically enriching a plurality of aptamers of a given length or a range of aptamer lengths, wherein said geometric enrichment comprises formulating a set of possible aptamer geometries, wherein the aptamer geometries in the set share a common format defined by the number of stems, loops, and pockets present in the aptamer structure, and wherein the format comprises one or more stems, one or more loops, and one or more pockets, and;

each loop in the common format has a size of no less than 3 nucleotides and no more than 21 nucleotides,

each pocket in the common format has a size of no less than 1 nucleotide and no more than 8 nucleotides, and

each stem in the common format has a size of no less than 2 nucleotides and no more than 7 nucleotides;

further enriching the set of possible aptamer geometries by formulating a library of aptamer sequences from the set of geometries, wherein the library is formulated by excluding sequences that do not satisfy one or more selected aptamer statistics relating to sequence(s) of stems, loops, and/or pockets, wherein the aptamer statistics are derived from data on pre-existing apatmers, and wherein the library comprises more than 1% of all possible geometries for a given aptamer length;

affixing at least one candidate aptamer from the library of aptamer sequences to a substrate.

2. The method of claim 1 wherein the library comprises more than 10% of all possible geometries for a given aptamer length.

3. The method of claim 1 in which the aptamer statistic relating to sequence comprises stem GC content, and the stem GC content in aptamer sequences in the library is greater than 50%.

4. The method of claim 2 in which the aptamer statistic relating to sequence comprises stem GC content, and the stem GC content in aptamer sequences in the library is greater than 50%.

5. The method of claim 1 wherein a plurality of candidate aptamers is affixed to the substrate to form a microarray.

6. The method of claim 1 wherein two or more candidate aptamers are linked directly or indirectly and then affixed to the substrate.

7. The method of claim 6 wherein a plurality of linked aptamers is affixed to the substrate to form a microarray.

8. The method of claim 4 wherein two or more candidate aptamers are linked directly or indirectly and then affixed to the substrate.

9. The method of claim 8 wherein a plurality of linked aptamers is affixed to the substrate to form a microarray.

10. The method of claim 1 wherein one or more candidate aptamers is linked with one or more ligands and then affixed to the substrate.

11. The method of claim 10 wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray.

12. The method of claim 4 wherein one or more candidate aptamers is linked with one or more ligands selected through other means and then affixed to the substrate.

13. The method of claim 12 wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray.

14. The method of claim 1 wherein the method additionally comprises:

contacting the at least one candidate aptamer with at least one target; and

assaying for binding between the at least one target and the at least one candidate aptamer.

15. The method of claim 14, wherein the assaying comprises monitoring a fluorescent signal related to binding between the at least one target and the at least one candidate aptamer.

16. The method of claim 15, comprising contacting the at least one candidate aptamer with a wild-type target and a variant target.

17. The method of claim 14, further comprising monitoring binding over time to determine binding kinetics of the at least one candidate aptamer.

18. The method of claim 14, further comprising monitoring binding over multiple concentrations of one or more candidate aptamers so as to determine aptamer affinities.

19. The method of claim 14, further comprising monitoring binding between the at least one target and one or more candidate aptamers to so as to determine the inhibition, the acceleration, or both of a process.

20. The method of claim 19, wherein the process is enzymatic in nature.

21. The method of claim 19, wherein binding is used to determine acceleration of a process.

22. The method of claim 21, wherein the process is enzymatic in nature.

23. The method of claim 16, wherein contrast between wild type and variant binding is used to select aptamers.

24. The method of claim 14, wherein a plurality of candidate aptamers is affixed to the substrate to form a microarray, and wherein binding over time is used to observe kinetics of individual aptamers.

25. The method of claim 14, wherein a plurality of candidate aptamers is affixed to the substrate to form a microarray, and wherein binding over multiple concentrations is used to determine aptamer affinities.

26. The method of claim 14, wherein a plurality of candidate aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine inhibition of a process.

27. The method of claim 26, wherein the process is enzymatic in nature.

28. The method of claim 14, wherein binding is used to determine acceleration of a process.

29. The method of claim 28, wherein the process is enzymatic in nature.

30. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein the assaying comprises monitoring binding to the microarray via fluorescence.

31. The method of claim 30, wherein contrast between wild type and variant binding is used to select linked aptamers.

32. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding over time is used to observe kinetics of individual linked aptamers.

33. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding over multiple concentrations is used to determine linked aptamer affinities.

34. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine inhibition of a process.

35. The method of claim 34, wherein the process is enzymatic in nature.

36. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine acceleration of a process.

37. The method of claim 36, wherein the process is enzymatic in nature.

38. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein the assaying comprises monitoring binding to the microarray via fluorescence.

39. The method of claim 38, wherein contrast between wild type and variant binding is used to select linked aptamers.

40. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding over time is used to observe kinetics of individual linked aptamers.

41. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding over multiple concentrations is used to determine linked aptamer affinities.

42. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine inhibition of a process.

43. The method of claim 42, wherein the process is enzymatic in nature.

44. The method of claim 14, wherein a plurality of linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine acceleration of a process.

45. The method of claim 44, wherein the process is enzymatic in nature.

46. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein the assaying comprises monitoring binding to the microarray via fluorescence.

47. The method of claim 46, wherein contrast between wild type and variant binding is used to select ligand-linked aptamers.

48. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding over time is used to observe kinetics of individual ligand-linked aptamers.

49. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding over multiple concentrations is used to determine ligand-linked aptamer affinities.

50. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine inhibition of a process.

51. The method of claim 50, wherein the process is enzymatic in nature.

52. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine acceleration of a process.

53. The method of claim 52, wherein the process is enzymatic in nature.

54. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein the assaying comprises monitoring binding to the microarray via fluorescence.

55. The method of claim 54, wherein contrast between wild type and variant binding is used to select ligand-linked aptamers.

56. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding over time is used to observe kinetics of individual ligand-linked aptamers.

57. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding over multiple concentrations is used to determine ligand-linked aptamer affinities.

58. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine inhibition of a process.

59. The method of claim 58, wherein the process is enzymatic in nature.

60. The method of claim 14, wherein a plurality of ligand-linked aptamers is affixed to the substrate to form a microarray, and wherein binding is used to determine acceleration of a process.

61. The method of claim 60, wherein the process is enzymatic in nature.