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

• US 10,190,151 B2
• Filed: 03/23/2018
• Issued: 01/29/2019
• 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:
  US15/934,031
• Publication Number:
  US 20180216159A1
• Time in Patent Office: 312 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 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,

(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 of step (3).

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 target-specific binding partner is a ligand, a small molecule, an aptamer, a peptide or an oligonucleotide.

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

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

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

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

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

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

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 removal or modification of the signal-emitting moieties in step (6) comprises cleaving a linker between the imager strand and the signal-emitting moiety.

20. The method of claim 1, wherein the removal or modification of the signal-emitting moieties in step (6) comprises chemically or photochemically modifying the signal-emitting moiety.

21. The method of claim 1, wherein the removal or modification of the signal-emitting moieties in step (6) comprises bleaching by chemical agents.

22. The method of claim 1, wherein the removal or modification of the signal-emitting moieties in step (6) comprises photobleaching.