Compositions and methods for analyte detection

Patent metadata

Field	Value
Publication number	US-11293052-B2
Application number	US-202117238682-A
Country	US
Kind code	B2
Filing date	Apr 23, 2021
Priority date	Dec 22, 2011
Publication date	Apr 5, 2022
Grant date	Apr 5, 2022

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 identifying an analyte at a location in a biological sample, comprising:
(a) binding a detection reagent to said analyte at said location of said biological sample, wherein said detection reagent comprises (i) a probe that binds to said analyte and (ii) a nucleic acid label that identifies said probe, and wherein said detection reagent is identifiable by a temporal order of signal signatures associated with said nucleic acid label;
(b) performing three or more readout cycles to generate said temporal order of signal signatures, wherein said three or more readout cycles comprise:
(i) a first readout cycle, comprising: (1) imaging said biological sample and detecting a first optical signal at said location, wherein said first optical signal is associated with said nucleic acid label of said detection reagent bound to said analyte at said location, thereby obtaining a first signal signature of said temporal order of signal signatures, and (2) removing said first optical signal from said location;
(ii) a second readout cycle, comprising imaging said biological sample and detecting an absence of an optical signal at said location, thereby obtaining a second signal signature of said temporal order of signal signatures; and
(iii) a third readout cycle, comprising imaging said biological sample and detecting a second optical signal at said location, wherein said second optical signal is associated with said nucleic acid label of said detection reagent bound to said analyte at said location, thereby obtaining a third signal signature of said temporal order of signal signatures; and

(c) using said temporal order of signal signatures to identify said analyte at said location of said biological sample.

2. The method of claim 1 , wherein said nucleic acid label comprises a predetermined sequence and wherein said first readout cycle further comprises, prior to said imaging of (b)(i)(1), using a first decoder probe to associate a first optical label with said predetermined sequence.

3. The method of claim 2 , wherein said third readout cycle further comprises, prior to said imaging of (b)(iii), using a second decoder probe to associate a second optical label with said predetermined sequence.

4. The method of claim 1 , wherein said nucleic acid label comprises two or more predetermined sequences, wherein said first readout cycle further comprises, prior to said imaging of (b)(i)(1), using a first decoder probe to associate a first optical label with a first predetermined sequence of said two or more predetermined sequences, wherein said first decoder probe is complementary to and hybridizes with said first predetermined sequence, and wherein said imaging of (b)(i)(1) comprises detecting said first optical label at said location, thereby detecting said first optical signal at said location.

5. The method of claim 4 , wherein said third readout cycle further comprises, prior to said imaging of (b)(iii), using a second decoder probe to associate a second optical label with a second predetermined sequence of said two or more predetermined sequences, wherein said first decoder probe is complementary to and hybridizes with said second predetermined sequence, and wherein said imaging of (b)(iii) comprises detecting said second optical label at said location, thereby detecting said second optical signal.

6. The method of claim 5 , wherein, in (b)(i)(2), removing said first optical signal from said location comprises photo-bleaching, quenching, chemically degrading, bleaching, or oxidizing said first optical label.

7. The method of claim 5 , wherein, in (b)(i)(2), removing said first optical signal from said location comprises washing or displacing said first decoder probe from said first predetermined sequence.

8. The method of claim 5 , wherein, in (b)(i)(2), removing said first optical signal from said location comprises washing said first decoder probe from said first predetermined sequence, wherein said washing comprises heating said biological sample, varying a salt concentration of said biological sample, applying a detergent to said biological sample, applying a denaturant to said biological sample, or a combination thereof.

9. The method of claim 5 , wherein, in (b)(i)(2), removing said first optical signal from said location comprises cleaving or enzymatically digesting said first decoder probe.

10. The method of claim 5 , wherein said first decoder probe comprises a cleavable linker and wherein removing said first optical signal from said location comprises cleaving said cleavable linker.

11. The method of claim 5 , wherein said first optical label is directly conjugated to said first decoder probe and wherein said second optical label is directly conjugated to said second decoder probe.

12. The method of claim 5 , wherein said first optical label is indirectly attached to said first decoder probe and wherein said second optical label is indirectly attached to said second decoder probe.

13. The method of claim 5 , wherein said first optical label is indirectly attached to said first decoder probe via a first intermediary molecule and said second optical label is indirectly attached to said second decoder probe via a second intermediary molecule.

14. The method of claim 5 , wherein said first optical 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 optical label is indirectly attached to said second decoder probe via a second binding molecule that hybridizes to said second decoder probe.

15. The method of claim 14 , wherein said first binding molecule is directly conjugated to said first optical label and wherein said second binding molecule is directly conjugated to said second optical label.

16. The method of claim 1 , wherein said biological sample is a cell or tissue sample, wherein said analyte is a cellular nucleic acid analyte, wherein said detection reagent is a linear nucleic acid molecule, wherein said probe comprises a first sequence complementary to a second sequence of said cellular nucleic acid analyte, and wherein binding said detection reagent to said analyte comprises hybridizing said first sequence to said second sequence.

17. The method of claim 16 , further comprising, prior to (b), circularizing said linear nucleic acid molecule to generate a circularized detection reagent and performing rolling circle amplification using said circularized detection reagent as a template.

18. The method of claim 17 , wherein circularizing said linear nucleic acid molecule comprises use of a ligase.

19. The method of claim 1 , wherein said biological sample is immobilized on a solid support.

20. The method of claim 19 , wherein said solid support does not comprise discrete sites configured to attach to beads.

21. The method of claim 19 , wherein said solid support comprises discrete sites configured to attach to or associate with beads.

22. The method of claim 19 , wherein said solid support is a planar substrate.

23. The method of claim 1 , wherein said biological sample is a tissue section on a microscopy slide.

24. The method of claim 23 , further comprising, prior to (a), permeabilizing said tissue section.

25. The method of claim 1 , wherein said first readout cycle, said second readout cycle, and said third readout cycle are performed sequentially.

26. A method for nucleic

Assignees

• Harvard College

Inventors

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

Classifications

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q1/6816

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

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title