Compositions and methods for analyte detection

Patent metadata

Field	Value
Publication number	US-11549136-B2
Application number	US-202117366127-A
Country	US
Kind code	B2
Filing date	Jul 2, 2021
Priority date	Dec 22, 2011
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

Methods of making a three-dimensional matrix of nucleic acids within a cell is provided.

First claim

What is claimed is:

1. A method for biological analysis, comprising:
(a) binding a plurality of detection reagents to an analyte in a cell or tissue sample, wherein said plurality of detection reagents comprises: (i) a first detection reagent comprising: (1) a first probe that specifically binds to said analyte; and (2) a first predetermined sequence; and (ii) a second detection reagent comprising: (1) a second probe that specifically binds to said analyte; and (2) a second predetermined sequence;
(b) generating a first hybridized complex comprising (i) a first decoder probe hybridized to said first predetermined sequence and (ii) a first detectable label;
(c) detecting said first detectable label to obtain a first signal signature from said first hybridized complex;
(d) removing said first signal signature from said first hybridized complex;
(e) generating a second hybridized complex comprising (i) a second decoder probe hybridized to said second predetermined sequence and (ii) a second detectable label;
(f) detecting said second detectable label to obtain a second signal signature from said second hybridized complex; and
(g) using at least said first signal signature and said second signal signature to identify said analyte.

2. The method of claim 1 , wherein said first detection reagent comprises a third predetermined sequence and wherein said method further comprises, prior to (g):
(i) generating a third hybridized complex comprising (1) a third decoder probe hybridized to said third predetermined sequence and (2) a third detectable label; and
(ii) detecting said third detectable label to obtain a third signal signature from said third hybridized complex;

wherein (g) comprises using at least said first signal signature, said second signal signature, and said third signal signature to identify said analyte.

3. The method of claim 2 , wherein said second detection reagent comprises a fourth predetermined sequence and wherein said method further comprises, prior to (g):
(i) generating a fourth hybridized complex comprising (1) a fourth decoder probe hybridized to said fourth predetermined sequence and (2) a fourth detectable label; and
(ii) detecting said fourth detectable label to obtain a fourth signal signature from said fourth hybridized complex;

wherein (g) comprises using at least said first signal signature, said second signal signature, said third signal signature, and said fourth signal signature to identify said analyte.

4. The method of claim 1 , wherein removing said first signal signature from said first hybridized complex comprises removing said first detectable label from said first hybridized complex.

5. The method of claim 1 , wherein removing said first signal signature from said first hybridized complex comprises inactivating said first detectable label.

6. The method of claim 1 , wherein said first detectable label is a first optical label and wherein said second detectable label is a second optical label.

7. The method of claim 6 , wherein removing said first signal signature from said first hybridized complex comprises photo-bleaching, quenching, chemically degrading, bleaching, or oxidizing said first optical label.

8. The method of claim 1 , wherein removing said first signal signature from said first hybridized complex comprises washing said first decoder probe from said first predetermined sequence.

9. The method of claim 1 , wherein removing said first signal signature from said first hybridized complex comprises use of heat, varying a salt concentration, use of a detergent, use of a denaturant, or any combination thereof.

10. The method of claim 1 , wherein removing said first signal signature from said first hybridized complex comprises cleaving or enzymatically digesting said first decoder probe.

11. The method of claim 1 , wherein said first detectable label is conjugated to said first decoder probe and wherein said second detectable label is conjugated to said second decoder probe.

12. The method of claim 11 , wherein said first detectable label and said second detectable label are conjugated to said first decoder probe and said second decoder probe, respectively, via a linker.

13. The method of claim 11 , wherein said first detectable label is conjugated to said first decoder probe via a cleavable linker and wherein (d) comprises cleaving said cleavable linker.

14. The method of claim 1 , wherein said first detectable label is indirectly attached to said first decoder probe and wherein said second detectable label is indirectly attached to said second decoder probe.

15. The method of claim 14 , wherein said first detectable label and said second detectable label are indirectly attached to said first decoder probe and said second decoder probe, respectively, via non-covalent bonds.

16. The method of claim 14 , wherein said first detectable label is indirectly attached to said first decoder probe via a first intermediary molecule and said second detectable label is indirectly attached to said second decoder probe via a second intermediary molecule.

17. The method of claim 14 , wherein said first detectable label is indirectly attached to said first decoder probe via a first binding molecule that hybridizes to said first decoder probe, and wherein said second detectable label is indirectly attached to said second decoder probe via a second binding molecule that hybridizes to said second decoder probe.

18. The method of claim 17 , wherein said first binding molecule is conjugated to said first detectable label and wherein said second binding molecule is conjugated to said second detectable label.

19. The method of claim 1 , further comprising using one or more additional signal signatures to identify said analyte, wherein said one or more additional signal signatures comprise detecting an absence of a signal associated with said analyte.

20. The method of claim 1 , wherein said analyte is a nucleic acid molecule.

21. The method of claim 20 , wherein said nucleic acid molecule is a ribonucleic acid (RNA) molecule.

22. The method of claim 21 , wherein said RNA molecule is a messenger RNA molecule.

23. The method of claim 1 , wherein said analyte is a protein and wherein said plurality of detection reagents comprise a plurality of antibodies.

24. A method for biological analysis, comprising:
(a) binding a plurality of detection reagents to a ribonucleic acid (RNA) molecule in a cell or tissue sample, wherein said cell or tissue sample is immobilized on a solid support, and wherein said plurality of detection reagents comprises:
(i) a first detection reagent comprising a first nucleic acid molecule comprising: (1) a first probe sequence that hybridizes to said RNA molecule; and (2) a first predetermined sequence; and
(ii) a second detection reagent comprising a second nucleic acid molecule comprising: (1) a second probe sequence that hybridizes to said RNA molecule; and (2) a second predetermined sequence, wherein said second predetermined sequence is different than said first predetermined sequence;

(b) performing a plurality of readout cycles, thereby obtaining a temporal order of signal signatures associated with said RNA molecule, said plurality of readout cycles comprising:
(i) a first readout cycle comprising: (1) generating a first hybridized complex comprising (A) a first decoder probe hybridized to said first predetermined sequence and (B) a first optical label; (2) detecting a

Assignees

• Harvard College

Inventors

• Church George M
• Lee Jehyuk
• Levner Daniel
• Super Michael

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title