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Details for Patent: 5,843,653

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Details for Patent: 5,843,653

Title: Method for detecting a target molecule in a sample using a nucleic acid ligand
Abstract:This invention discloses a method of detecting the presence or absence of a target molecule in a sample and a method of measuring the amount of a target molecule in a sample using nucleic acid ligands. In a preferred embodiment the nucleic acid ligands are identified by the method of the invention referred to as the Systematic Evolution of Ligands by EXponential Enrichment (SELEX), wherein a candidate mixture of nucleic acids are iteratively enriched in high affinity nucleic acids and amplified for further partitioning.
Inventor(s): Gold; Larry (Boulder, CO), Tuerk; Craig (Morehead, KY)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:Jun 06, 1995
Application Number:08/469,609
Claims:1. A method for detecting the presence or absence of a target molecule in a sample which may contain said target molecule comprising:

a) exposing said sample which may contain said target molecule to a non-naturally occurring nucleic acid ligand of said target molecule, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by said target molecule, under conditions wherein a target molecule; nucleic acid ligand binding pair is formed if said target molecule is present, wherein said binding is not due to Watson/Crick base pairing; and

b) determining whether said target molecule:nucleic acid ligand binding pair is formed, thereby detecting the presence or absence of the target molecule in the sample.

2. The method of claim 1 wherein said nucleic acid ligand is single-stranded.

3. The method of claim 2 wherein said single-stranded nucleic acid ligand is ribonucleic acid.

4. The method of claim 2 wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid.

5. The method of claim 1 wherein said nucleic acid ligand is labeled.

6. The method of claim 5 wherein said label is a radiolabel.

7. The method of claim 1 wherein said target molecule is a protein.

8. The method of claim 7 wherein said protein is not known to bind nucleic acids as part of its biological function.

9. The method of claim 1 wherein said target molecule is a small molecule.

10. The method of claim 1 wherein said target molecule is a controlled substance.

11. The method of claim 1 wherein said target molecule is a metabolite.

12. The method of claim 1 wherein said sample is a biological substance.

13. The method of claim 1 wherein said detection is achieved by PCR amplification of said nucleic acid ligand.

14. A method for detecting the presence or absence of a target molecule in a sample which may contain said target molecule comprising:

a) identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of said target molecule, by a method comprising:

i) contacting the candidate mixture with said target molecule, wherein nucleic acids having an increased affinity to said target molecule relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

ii) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby an increased affinity nucleic acid ligand may be identified;

b) exposing said sample which may contain said target molecule to said increased affinity nucleic acid ligand of said target molecule under conditions wherein a target molecule; nucleic acid ligand binding pair is formed if said target molecule is present; and

c) determining whether said target molecule:nucleic acid ligand binding pair is formed, thereby detecting the presence or absence of the target molecule in the sample.

15. The method of claim 14 further comprising:

iv) repeating steps i), ii) and iii).

16. The method of claim 14 wherein said nucleic acid ligand is single-stranded.

17. The method of claim 16 wherein said single-stranded nucleic acid ligand is ribonucleic acid.

18. The method of claim 16 wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid.

19. The method of claim 14 wherein said target molecule is a protein.

20. The method of claim 19 wherein said protein is not known to bind nucleic acids as part of its biological function.

21. The method of claim 14 wherein said target molecule is a controlled substance.

22. The method of claim 14 wherein said target molecule is a metabolite.

23. The method of claim 14 wherein said target molecule is a small molecule.

24. The method of claim 14 wherein said sample is a biological substance.

25. The method of claim 14 wherein said detection is achieved by PCR amplification of said nucleic acid ligand.

26. The method of claim 14 wherein said candidate mixture is prepared by synthesis from a template comprising a region of conserved sequence and a region of randomized and/or biased sequence.

27. The method of claim 14 wherein said candidate mixture comprises nucleic acids each comprising a region of conserved sequence and a region of randomized sequence.

28. A method for measuring the amount of a target molecule in a sample which may contain said target molecule comprising:

a) exposing said sample which may contain said target molecule to a nucleic acid ligand of said target molecule, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule, under conditions wherein a target molecule; nucleic acid ligand binding pair is formed;

b) determining whether said target molecule:nucleic acid ligand binding pair is formed wherein said binding is not due to Watson/Crick base pairing; and

c) measuring the amount of the target molecule in the sample by quantitatively detecting the nucleic acid ligand.

29. The method of claim 28 wherein said nucleic acid ligand is single-stranded.

30. The method of claim 29 wherein said single-stranded nucleic acid ligand is ribonucleic acid.

31. The method of claim 29 wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid.

32. The method of claim 28 wherein said nucleic acid ligand is labeled.

33. The method of claim 32 wherein said label is a radiolabel.

34. The method of claim 28 wherein said target molecule is a protein.

35. The method of claim 34 wherein said protein is not known to bind nucleic acids as part of its biological function.

36. The method of claim 28 wherein said target molecule is a small molecule.

37. The method of claim 28 wherein said target molecule is a controlled substance.

38. The method of claim 28 wherein said target molecule is a metabolite.

39. The method of claim 28 wherein said sample is a biological substance.

40. The method of claim 28 wherein said detection is achieved by PCR amplification of said nucleic acid ligand.

41. The method of claim 28 wherein said measurement is qualitative.

42. The method of claim 28 wherein said measurement is quantitative.

43. A method for measuring the amount of a target molecule in a sample which may contain said target molecule comprising:

a) identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of said target molecule, by a method comprising:

i) contacting the candidate mixture with said target molecule, wherein nucleic acids having an increased affinity to said target molecule relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

ii) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby an increased affinity nucleic acid ligand may be identified;

b) exposing said sample which may contain said target molecule to said increased affinity nucleic acid ligand of said target molecule under conditions wherein a target molecule; nucleic acid ligand binding pair is formed;

c) determining whether said target molecule:nucleic acid ligand binding pair is formed; and

d measuring the amount of the target molecule in the sample, by quantitatively detecting the nucleic acid ligand.

44. The method of claim 43 further comprising:

iv) repeating steps i), ii) and iii).

45. The method of claim 43 wherein said nucleic acid ligand is single-stranded.

46. The method of claim 45 wherein said single-stranded nucleic acid ligand is ribonucleic acid.

47. The method of claim 45 wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid.

48. The method of claim 43 wherein said target molecule is a protein.

49. The method of claim 48 wherein said protein is not known to bind nucleic acids as part of its biological function.

50. The method of claim 43 wherein said target molecule is a controlled substance.

51. The method of claim 43 wherein said target molecule is a metabolite.

52. The method of claim 43 wherein said target molecule is a small molecule.

53. The method of claim 43 wherein said sample is a biological substance.

54. The method of claim 43 wherein said measurement is qualitative.

55. The method of claim 43 wherein said measurement is quantitative.

56. The method of claim 43 wherein said candidate mixture is prepared by synthesis from a template comprising a region of conserved sequence and a region of randomized and/or biased sequence.

57. The method of claim 43 wherein said candidate mixture comprises nucleic acids each comprising a region of conserved sequence and a region of randomized sequence.
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