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

• US 9,944,972 B2
• Filed: 01/19/2017
• Issued: 04/17/2018
• 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:
  US15/410,700
• Publication Number:
  US 20170137864A1
• Time in Patent Office: 453 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 of the target-specific binding partners is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands to produce one or more targets bound to one or more target-specific binding partners;

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

(3) contacting the sample with labeled imager strands that bind to docking strands to produce labeled imager strands bound to docking strands;

(4) optionally removing unbound labeled imager strands;

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

(6) removing the bound labeled imager strands from the docking strands, wherein the labeled imager strands are removed from the docking strands by enzymatically cleaving, modifying, or degrading the labeled imager strands; 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 of step (3).

2. The method of claim 1, wherein the labeled imager strands are removed enzymatically.

3. The method of claim 2, wherein the labeled imager strands are labeled imager nucleic acid strands and the docking strands are docking nucleic acid strands, and wherein the labeled imager nucleic acid strands and the docking nucleic acid strands are complementary nucleic acids relative to each other.

4. The method of claim 3, wherein the labeled imager strands are cleaved by a nucleic acid glycosylase, and wherein optionally the nucleic acid glycosylase is a uracil-DNA glycosylase.

5. The method of claim 3, wherein the labeled imager strands are removed by at least one nicking enzyme or restriction enzyme.

6. The method of claim 3, wherein the labeled imager strands are removed by cleavage at one or more ribonucleotide by RNase.

7. The method of claim 3, wherein the labeled imager strands are removed by cleavage at one or more deoxyuridine base.

8. The method of claim 7, wherein the cleavage at one or more deoxyuridine base is accomplished by a uracil-specific excision reagent enzyme.

9. The method of claim 8, wherein the sample is contacted with more than one target-specific binding partner in step (1).

10. The method of claim 8, wherein the target-specific binding partner is an antibody or an antibody fragment.

11. The method of claim 8, wherein the target-specific binding partner is a ligand, a small molecule, an aptamer, a peptide or an oligonucleotide.

12. The method of claim 8, wherein the labeled imager strands are labeled identically.

13. The method of claim 8, wherein the labeled imager strands comprise a distinct label.

14. The method of claim 8, wherein the labeled imager strands are fluorescently labeled imager strands.

15. The method of claim 8, wherein the one or more targets are proteins and/or the sample is a cell or tissue sample, a cell lysate or a tissue lysate, or a bodily fluid.

16. The method of claim 8, wherein the sample is imaged in step (5) using confocal or epi-fluorescence microscopy.

17. The method of claim 1, wherein the labeled imager strands comprise a photocleavable moiety that can be cleaved by UV exposure.

18. The method of claim 1, wherein removing the labeled imager strands comprises:

(a) enzymatic cleavage and photochemical modification;

or

(b) enzymatic cleavage and chemical modification/degradation.

19. The method of claim 1, wherein the labeled imager strands and/or the docking strands have a toehold sequence.

20. The method of claim 19, wherein the imager strands have a toehold sequence.

21. The method of claim 20, wherein the sample is contacted with more than one target-specific binding partner in step (1).

22. The method of claim 20, wherein the target-specific binding partner is an antibody or an antibody fragment.

23. The method of claim 20, wherein the target-specific binding partner is a ligand, a small molecule, an aptamer, a peptide or an oligonucleotide.

24. The method of claim 20, wherein the labeled imager strands are labeled identically relative to each other.

25. The method of claim 20, wherein each of the labeled imager strands comprises a distinct label.

26. The method of claim 20, wherein the labeled imager strands are fluorescently labeled imager strands.

27. The method of claim 20, wherein the one or more targets are proteins and/or the sample is a cell or tissue sample, a cell lysate or a tissue lysate, or a bodily fluid.

28. The method of claim 20, wherein the sample is imaged in step (5) using confocal or epi-fluorescence microscopy.