Methods for high-throughput labelling and detection of biological features in situ using microscopy

• US 10,179,932 B2
• Filed: 07/10/2015
• Issued: 01/15/2019
• Est. Priority Date: 07/11/2014
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Lee, Je-Hyuk, Daugharthy, Evan R.
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US15/325,577
• Publication Number:
  US 20170159136A1
• Time in Patent Office: 1,285 Days
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2543/101 in situ amplification

Abstract

Methods of labelling one or more subcellular components (e.g., an organelle and/or subcellular region) in vivo are provided. Methods of labelling a protein in vivo are provided. Methods of determining a nucleic acid sequence in situ are also provided.

Claims

1. A method for identifying a protein of interest in a cell, comprising:

(a) providing a cell comprising (i) a ribonucleic acid (RNA) molecule comprising a barcode and (ii) said protein of interest fused in frame to an RNA binding domain, wherein said RNA molecule binds to said RNA binding domain such that said RNA molecule localizes to said protein of interest in the cell;

(b) within said cell, (i) reverse transcribing the RNA molecule to generate a deoxyribonucleic acid (DNA) molecule from said RNA molecule, wherein said DNA molecule comprises said barcode, and (ii) amplifying the DNA molecule to produce one or more amplicons, wherein said one or more amplicons of said DNA molecule co-localize with said protein of interest and comprise said barcode; and

(c) identifying said barcode within said cell, thereby identifying said protein of interest.

2. The method of claim 1, wherein amplifying the DNA molecule includes circularizing the DNA molecule to generate a circular DNA molecule;

and performing rolling circle amplification (RCA) on said circular DNA molecule to produce said one or more amplicons.

3. The method of claim 1, wherein identifying the barcode comprises detecting the one or more amplicons.

4. The method of claim 1, wherein the protein of interest is localized in or to a subceullar component.

5. The method of claim 1, further comprising, prior to (a), expressing the RNA molecule within the cell, wherein expression of the RNA molecule within the cell is controlled by one or more members selected from the group consisting of an inducible promoter, a cell type-specific promoter and a signal-specific promoter.

6. The method of claim 1, further comprising, prior to (a), delivering the RNA molecule directly to the cell.

7. The method of claim 1, wherein said RNA binding domain is a MS2 domain, a lambda phage N peptide, or a P22 phage N peptide.

8. The method of claim 1, further comprising, prior to (a), contacting said cell with a matrix-forming material to form a matrix.

9. The method of claim 8, wherein said matrix-forming material permeates throughout said cell.

10. The method of claim 8, wherein said matrix-forming material comprises polyacrylamide, cellulose, alginate, polyamide, agarose, dextran, or polyethylene glycol.

11. The method of claim 8, wherein said DNA molecule or one or more amplicons are attached to said matrix.

12. The method of claim 8, wherein said DNA molecule, or said one or more amplicons are modified with a functional moiety.

13. The method of claim 12, wherein said functional moiety is crosslinked, copolymerized, or non-covalently attached to said matrix.

14. The method of claim 13, wherein said functional moiety is an amine, an acrydite, an alkyne, a biotin, an azide, or a thiol functional moiety.

15. The method of claim 4, wherein said subcellular component is a cell membrane, a nucleus, or a synapse.

16. The method of claim 1, wherein said barcode comprises a nucleic acid sequence that is specific to said protein of interest, and wherein (c) comprises sequencing said one or more amplicons to identify said protein of interest.

17. The method of claim 1, wherein said RNA binding domain is fused in frame to said protein of interest internally, at an N-terminus, or at a C-terminus of said protein of interest.

18. The method of claim 1, wherein said RNA binding domain is an RNA recognition motif, a zinc finger domain, a KH domain, or a double-stranded RNA binding motif.

19. The method of claim 1, further comprising, in said step (b), removing said RNA molecule by a nuclease or a restriction enzyme.