High-throughput and highly multiplexed imaging with programmable nucleic acid probes

• US 10,294,510 B2
• Filed: 03/11/2015
• Issued: 05/21/2019
• Est. Priority Date: 03/11/2014
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Yin, Peng, Agasti, Sarit, Chen, Xi, Jungmann, Ralf
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/108,911
• Publication Number:
  US 20160319328A1
• Time in Patent Office: 1,532 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present invention provides, inter alia, methods and compositions for imaging, at high spatial resolution, targets of interest.

Claims

1. A method comprising

(1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands,

(2) optionally removing unbound target-specific binding partners,

(3) contacting the sample with labeled imager strands bind to a docking strand,

(4) optionally removing unbound labeled imager strands,

(5) imaging the sample to detect bound labeled imager strands,

(6) extinguishing signal from the bound labeled imager strand, and

(7) repeating at least some of steps (3)-(6) at least once with a labeled imager strand having a unique composition relative to at least one other labeled imager strand.

2. The method of claim 1, wherein the sample is contacted with more than one target-specific binding partner in step (1).

3. The method of claim 1, wherein the target-specific binding partner is an antibody or an antibody fragment.

4. The method of claim 1, wherein the labeled imager strands are labeled identically.

5. The method of claim 1, wherein the labeled imager strands each comprise a distinct label.

6. The method of claim 1, wherein the labeled imager strands are fluorescently labeled imager strands.

7. The method of claim 1, wherein the one or more targets are proteins.

8. The method of claim 1, wherein the sample is a cell or tissue sample, a cell lysate or a tissue lysate, or bodily fluid.

9. The method of claim 1, wherein the sample is imaged in step (5) using confocal or epi-fluorescence microscopy.

10. The method of claim 1, wherein extinguishing signal in step (6) comprises photobleaching.

11. The method of claim 1, wherein the unbound docking strand is partially double-stranded.

12. The method of claim 1, wherein the unbound imager strand is partially double-stranded.

13. The method of claim 1, wherein the imager strand is a molecular beacon or comprises a hairpin secondary structure.

14. The method of claim 1, wherein the imager strand is a molecular beacon or comprises a hairpin secondary structure that is self-quenching.

15. The method of claim 1, wherein the imager strand is a hemiduplex.

16. The method of claim 15, wherein the hemiduplex is self-quenching.

17. The method of claim 1, wherein the docking strand comprises a hairpin secondary structure.

18. The method of claim 1, wherein the imager strand is bound to multiple signal-emitting moieties through a dendrimeric structure or a polymeric structure.

19. The method of claim 1, wherein the extinguishing step eliminates signal from the bound labeled imager strand.

20. The method of claim 1, wherein the extinguishing step reduces signal from the bound labeled imager strand.

21. The method of claim 1, wherein detecting comprises determining the presence or absence of bound labeled imager strands.

22. The method of claim 1, wherein detecting comprises detecting the number of bound labeled imager strands.

23. The method of claim 1, wherein detecting comprises detecting the location of bound labeled imager strands.

24. The method of claim 1, wherein the labeled imager strands are labeled with a signal-emitting molecule and multiple copies of the signal-emitting molecule are recruited by the binding of the imager strand to the docking strand.

25. The method of claim 24, wherein the imager strand is a branched imager strand.

26. The method of claim 1, wherein the docking strand may be part of a multi-strand complex.

27. The method of claim 1, wherein the imager strand and/or docking strand comprise secondary structure.

28. The method of claim 27, wherein the secondary structure is a hairpin loop, 2D nanostructure, or 3D nanostructure.

29. The method of claim 1, wherein at least one docking strand is stably bound to a labeled imager strand.

30. The method of claim 1, wherein the imager and docking strands remain bound to each other for at least 30, 35, 40, 45, 50, 55, or 60 minutes or at least 2 hours.

31. The method of claim 1, wherein the imager and docking strands remain bound to each other for at least 30 to 60 minutes, 30 to 120 minutes, 40 to 50 minutes, 40 to 120 minutes, or 60 to 120 minutes.

32. The method of claim 1, wherein all imager strands are labeled with the same fluorophore.

33. The method of claim 32, wherein the fluorophore is a red fluorophore.

34. The method of claim 1, wherein at least some labeled imager strands are labeled identically and at least some labeled imager strands comprise a different label.

35. The method of claim 1, wherein step (1) comprises contacting a sample with a plurality of distinct target specific binding partners linked to docking strands.

36. The method of claim 35, wherein the method comprises at least 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 distinct species.

37. The method of claim 1, wherein the imager strand and docking strand are complementary nucleotides.

38. A method comprising

(1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands,

(2) optionally removing unbound target-specific binding partners,

(3) contacting the sample with labeled imager strands that bind to a docking strand,

(4) optionally removing unbound labeled imager strands,

(5) imaging the sample to detect bound labeled imager strands,

(6) removing the bound labeled imager strands from the docking strands by altering temperature and/or buffer condition, and

(7) repeating at least some of steps (3)-(6) at least once with a labeled imager strand having a unique composition relative to at least one other labeled imager strand.

39. The method of claim 38, wherein the sample is contacted with more than one target-specific binding partner in step (1).

40. The method of claim 38, wherein the target-specific binding partner is an antibody or an antibody fragment.

41. The method of claim 38, wherein the target-specific binding partner is a natural or engineered ligand, a small molecule, an aptamer, a peptide or an oligonucleotide.

42. The method of claim 38, wherein the labeled imager strands are labeled identically.

43. The method of claim 38, wherein the labeled imager strands each comprise a distinct label.

44. The method of claim 38, wherein the labeled imager strands are fluorescently labeled imager strands.

45. The method of claim 38, wherein the one or more targets are proteins.

46. The method of claim 38, wherein the sample is a cell, a cell lysate or a tissue lysate.

47. The method of claim 38, wherein the sample is imaged in step (5) using confocal or epi-fluorescence microscopy.

48. The method of claim 38, wherein the labeled imager strands are removed from the docking strands by decreasing salt concentration, addition of a denaturant, or increasing temperature.

49. The method of claim 48, wherein the salt comprises Mg++.

50. The method of claim 48, wherein the denaturant is formamide, urea or DMSO.

51. The method of claim 38, wherein the labeled imager strands are not removed from the docking strands by strand displacement in the presence of a competing strand.

52. A method comprising

(1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands,

(2) optionally removing unbound target-specific binding partners,

(3) contacting the sample with labeled imager strands that bind to a docking strand,

(4) optionally removing unbound labeled imager strands,

(5) imaging the sample to detect bound labeled imager strands,

(6) inactivating the bound labeled imager strands, by removing or modifying their signal-emitting moieties without removing the imager strand in its entirety, and

(7) repeating at least some of steps (3)-(6) at least once with a labeled imager strand having a unique composition relative to at least one other labeled imager strand.

53. The method of claim 52, wherein the sample is contacted with more than one target-specific binding partner in step (1).

54. The method of claim 52, wherein the target-specific binding partner is an antibody or an antibody fragment.

55. The method of claim 52, wherein the target-specific binding partner is a natural or engineered ligand, a small molecule, an aptamer, a peptide or an oligonucleotide.

56. The method of claim 52, wherein the labeled imager strands are labeled identically.

57. The method of claim 52, wherein the labeled imager strands each comprise a distinct label.

58. The method of claim 52, wherein the labeled imager strands are fluorescently labeled imager strands.

59. The method of claim 52, wherein the one or more targets are proteins.

60. The method of claim 52, wherein the sample is a cell, a cell lysate or a tissue lysate.

61. The method of claim 52, wherein the sample is imaged in step (5) using confocal or epi-fluorescence microscopy.

62. The method of claim 52, wherein the unbound docking strand is partially double-stranded.

63. The method of claim 52, wherein the unbound imager strand is partially double-stranded.

64. The method of claim 52, wherein the imager strand is a molecular beacon or comprises a hairpin secondary structure.

65. The method of claim 52, wherein the imager strand is a molecular beacon or comprises a hairpin secondary structure that is self-quenching.

66. The method of claim 52, wherein the imager strand is a hemiduplex.

67. The method of claim 66, wherein the hemiduplex is self-quenching.

68. The method of claim 52, wherein the docking strand comprises a hairpin secondary structure.

69. The method of claim 52, wherein the imager strand is bound to multiple signal-emitting moieties through a dendrimeric structure or a polymeric structure.

70. A method comprising

(1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands,

(2) optionally removing unbound target-specific binding partners,

(3) contacting the sample with labeled imager strands having a nucleotide sequence

that is complementary to a docking strand and stably binding the imager strands to the complementary docking strands,

(4) optionally removing unbound labeled imager strands,

(5) imaging the sample to detect location and number of bound labeled imager strands,

(6) optionally extinguishing signal from the bound labeled imager strand, and

(7) optionally repeating at least some of steps (3)-(6) at least once with a labeled imager strand having a unique nucleotide sequence relative to at least one other labeled imager strand.

71. The method of claim 70, wherein the imager strand is bound to multiple signal-emitting moieties.