SELECTION OF APTAMERS BASED ON GEOMETRY

• US 20080182759A1
• Filed: 12/18/2007
• Published: 07/31/2008
• Est. Priority Date: 12/18/2006
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Arcxis Biotechnologies, Inc. (ARCXIS)
• Inventors: West, Jason A. A., Satterfield, Brent Coleman
• Granted Patent: US 9,938,641 B2
• Time in Patent Office: Days
• US Class Current: 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 for selecting one or more aptamers by geometric enrichment, comprising consideration of one or more characteristics of the one or more aptamers to so as to formulate one or more possible geometries of the one or more aptamers.

2. The method of claim 1 wherein said method includes more than 1% of all possible geometries for a given aptamer length.

3. The method of claim 1 wherein said method includes more than 10% of all possible geometries for a given aptamer length.

4. The method of claim 1 in which stem GC content is greater than 50% on average.

5. The method of claim 3 in which stem GC content is greater than 50% on average.

6. The method of claim 1 in which additional geometric enrichment is performed on a substrate.

7. The method of claim 6 wherein attachment to the substrate forms a microarray.

8. The method of claim 7 wherein enrichment further comprises monitoring binding to the microarray via fluorescence.

9. The method of claim 8 wherein contrast between wild type and variant binding is used to select aptamers.

10. The method of claim 7 wherein binding over time is used to observe kinetics of individual aptamers.

11. The method of claim 7 wherein binding over multiple concentrations of one or more aptamers is used to determine aptamer affinities.

12. The method of claim 7 wherein binding is used to determine inhibition of a process.

13. The method of claim 12 wherein the process is enzymatic in nature.

14. The method of claim 7 wherein binding is used to determine acceleration of a process

15. The method of claim 14 wherein the process is enzymatic in nature.

16. The method of claim 5 in which further enrichment is performed on a substrate.

17. The method of claim 16 wherein attachment to the substrate forms a microarray.

18. The method of claim 17 wherein enrichment is performed by monitoring binding to the microarray via fluorescence.

19. The method of claim 18 wherein contrast between wild type and variant binding is used to select aptamers.

20. The method of claim 17 wherein binding over time is used to observe kinetics of individual aptamers.

21. The method of claim 17 wherein binding over multiple concentrations is used to determine aptamer affinities.

22. The method of claim 17 wherein binding is used to determine inhibition of a process.

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

24. The method of claim 17 wherein binding is used to determine acceleration of a process

25. The method of claim 24 wherein the process is enzymatic in nature.

26. The method of claim 1 wherein two or more geometric enrichment selected aptamers are linked directly or indirectly for further enrichment.

27. The method of claim 26 wherein attachment to the substrate forms a microarray.

28. The method of claim 27 wherein enrichment is performed by monitoring binding to the microarray via fluorescence.

29. The method of claim 28 wherein contrast between wild type and variant binding is used to select aptamers.

30. The method of claim 27 wherein binding over time is used to observe kinetics of individual aptamers.

31. The method of claim 27 wherein binding over multiple concentrations is used to determine aptamer affinities.

32. The method of claim 27 wherein binding is used to determine inhibition of a process.

33. The method of claim 32 wherein the process is enzymatic in nature.

34. The method of claim 27 wherein binding is used to determine acceleration of a process

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

36. The method of claim 5 wherein two or more geometric enrichment selected aptamers are linked directly or indirectly for further enrichment.

37. The method of claim 36 wherein attachment to the substrate forms a microarray.

38. The method of claim 37 wherein enrichment is performed by 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 aptamers.

40. The method of claim 37 wherein binding over time is used to observe kinetics of individual aptamers.

41. The method of claim 37 wherein binding over multiple concentrations is used to determine aptamer affinities.

42. The method of claim 37 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 37 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 1 wherein one or more geometric enrichment selected aptamer is linked with one or more ligands for further enrichment.

47. The method of claim 46 wherein attachment to the substrate forms a microarray.

48. The method of claim 47 wherein enrichment is performed by monitoring binding to the microarray via fluorescence.

49. The method of claim 48 wherein contrast between wild type and variant binding is used to select aptamers.

50. The method of claim 47 wherein binding over time is used to observe kinetics of individual aptamers.

51. The method of claim 47 wherein binding over multiple concentrations is used to determine aptamer affinities.

52. The method of claim 47 wherein binding is used to determine inhibition of a process.

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

54. The method of claim 47 wherein binding is used to determine acceleration of a process.

55. The method of claim 54 wherein the process is enzymatic in nature.

56. The method of claim 5 wherein one or more geometric enrichment selected aptamers is linked with one or more ligands selected through other means for further enrichment.

57. The method of claim 56 wherein attachment to the substrate forms a microarray.

58. The method of claim 57 wherein enrichment is performed by monitoring binding to the microarray via fluorescence.

59. The method of claim 58 wherein contrast between wild type and variant binding is used to select aptamers.

60. The method of claim 57 wherein binding over time is used to observe kinetics of individual aptamers.

61. The method of claim 57 wherein binding over multiple concentrations is used to determine aptamer affinities.

62. The method of claim 57 wherein binding is used to determine inhibition of a process.

63. The method of claim 62 wherein the process is enzymatic in nature.

64. The method of claim 57 wherein binding is used to determine acceleration of a process

65. The method of claim 64 wherein the process is enzymatic in nature.

66. A kit comprising one or more geometrically enriched aptamers developed through the method of claim 1 and instructions for using them to select the appropriate aptamer.

67. A kit comprising one or more geometrically enriched aptamers developed through the method of claim 5 and instructions for using them to select the appropriate aptamer.

68. A kit comprising one or more pairs of geometrically enriched aptamers developed through the method of claim 26 and instructions for using them to select the appropriate aptamer.