Compositions and methods for analyte detection

Patent metadata

Field	Value
Publication number	US-11566277-B2
Application number	US-202217671803-A
Country	US
Kind code	B2
Filing date	Feb 15, 2022
Priority date	Dec 22, 2011
Publication date	Jan 31, 2023
Grant date	Jan 31, 2023

Abstract

Methods of analyzing nucleic acids of a cell are provided.

First claim

What is claimed is:

1. A method, comprising:
(a) binding one or more detection reagents to an analyte at a spatial location in a cell or tissue sample, wherein a detection reagent of said one or more detection reagents comprises: (1) a probe that binds to said analyte and (2) one or more predetermined sequences;
(b) using imaging to detect, in a temporally sequential manner, said one or more detection reagents bound to said analyte at said spatial location, thereby obtaining a plurality of images comprising a plurality of detected signal signatures associated with said analyte at said spatial location;
(c) using at least one distinguishable feature common to images of said plurality of images to obtain a plurality of aligned images comprising said plurality of detected signal signatures; and
(d) using a temporal order of said plurality of detected signal signatures of said plurality of aligned images to identify said analyte at said spatial location.

2. The method of claim 1 , wherein said plurality of images comprises (i) a first image comprising a first detected signal signature of said plurality of detected signal signatures and (ii) a second image comprising a second detected signal signature of said plurality of detected signal signatures; and wherein (c) comprises determining a first registered location of said first detected signal signature; determining a second registered location of said second detected signal signature; and using said at least one distinguishable feature to align said first registered location and said second registered location, thereby obtaining aligned images of said plurality of aligned images.

3. The method of claim 1 , wherein said analyte is a nucleic acid analyte, wherein each of said one or more detection reagents is a nucleic acid molecule comprising said probe and said one or more predetermined sequences, and wherein said probe comprises a sequence complementary to at least a portion of said nucleic acid analyte.

4. The method of claim 3 , wherein said nucleic acid analyte is a ribonucleic acid analyte.

5. The method of claim 1 , wherein said analyte is a protein analyte, wherein said probe comprises an antibody, and wherein said antibody is conjugated to a nucleic acid molecule comprising said one or more predetermined sequences.

6. The method of claim 1 , wherein (b) comprises (i) using a decoder probe to associate at least one optical label with a predetermined sequence of a detection reagent of said one or more detection reagents and (ii) using imaging to detect said at least one optical label, thereby obtaining an image of said plurality of images, wherein said image comprises a detected signal signature of said plurality of detected signal signatures, said detected signal signature generated from said at least one optical label.

7. The method of claim 6 , wherein one or more of said plurality of detected signal signatures is characterized by an absence of an optical signal at said spatial location.

8. A method, comprising:
(a) binding one or more detection reagents to an analyte at a spatial location in a cell or tissue sample, wherein a detection reagent of said one or more detection reagents comprises: (1) a probe that binds to said analyte and (2) one or more predetermined sequences;
(b) using imaging to detect, in a temporally sequential manner, said one or more detection reagents bound to said analyte at said spatial location, thereby obtaining a plurality of images comprising a plurality of detected signal signatures associated with said analyte at said spatial location;
(c) using at least one distinguishable feature common to images of said plurality of images to correct for spatial shift in one or more detected signal signatures of said plurality of detected signal signatures, thereby obtaining a plurality of aligned signal signatures detected at said spatial location; and
(d) using a temporal order of said plurality of aligned signal signatures detected at said spatial location to identify said analyte at said spatial location.

9. The method of claim 8 , wherein said plurality of detected signal signatures comprises at least a first detected signal signature and a second detected signal signature, wherein said plurality of images comprises at least a first image comprising said first detected signal signature and a second image comprising said second detected signal signature, and wherein (c) comprises determining a first registered location of said first detected signal signature, determining a second registered location of said second detected signal signature, and using said at least one distinguishable feature common to said first image and said second image to correct for spatial shift in said first registered location with respect to said second registered location, thereby aligning said first detected signal signature with respect to said second detected signal signature.

10. The method of claim 8 , wherein said analyte is a nucleic acid analyte, wherein each of said one or more detection reagents is a nucleic acid molecule comprising said probe and said one or more predetermined sequences, and wherein said probe comprises a sequence complementary to at least a portion of said nucleic acid analyte.

11. The method of claim 10 , wherein said nucleic acid analyte is a ribonucleic acid analyte.

12. The method of claim 8 , wherein said analyte is a protein analyte, wherein said probe comprises an antibody, and wherein said antibody is conjugated to a nucleic acid molecule comprising said one or more predetermined sequences.

13. The method of claim 8 , wherein said plurality of detected signal signatures comprises at least a first detected signal signature and a second detected signal signature, wherein a first image of said plurality of images comprises said first detected signal signature, wherein a second image of said plurality of images comprises said second detected signal signature, and wherein (b) comprises:
(i) using a first decoder probe to associate at least one optical label with a first predetermined sequence of a detection reagent of said one or more detection reagents;
(ii) using imaging to detect said at least one optical label to obtain said first image comprising said first detected signal signature, said first detected signal signature generated from said at least one optical label;
(iii) removing signal from said first detected signal signature;
(iv) using a second decoder probe to associate at least one additional optical label with a second predetermined sequence of said detection reagent of said one or more detection reagents; and
(v) using imaging to detect said at least one additional optical label thereby obtaining said second image comprising said second detected signal signature, said second signal signature generated from said at least one additional optical label.

14. The method of claim 13 , wherein one or more of said plurality of detected signal signatures is characterized by an absence of an optical signal at said spatial location.

15. A method, comprising:
(a) providing a tissue sample on a solid support, wherein said tissue sample comprises a plurality of cells, wherein a cell of said plurality of cells comprises a RNA molecule, and wherein said RNA molecule of said cell is at a spatial location in said tissue sample;
(b) binding one or more detection reagents to said RNA molecule in said cell to associate a plurality of predetermined sequences with said RNA molecule, wherein said cell is permeable to said one or more detection reagents, and wherein each of said one or more detection reagents is a nucleic acid molecule comprisi

Assignees

• Harvard College

Inventors

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

Classifications

• C12Q1/6874

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

• C12Q1/6869

  Methods for sequencing · CPC title

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• C12Q1/6806 Primary

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

• C12Q1/6804

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