Methods for High-Throughput Labelling and Detection of Biological Features in situ Using Microscopy

• US 20170159136A1
• Filed: 07/10/2015
• Published: 06/08/2017
• 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: Lee, Je-Hyuk, Daugharthy, Evan R., Church, George M.
• Granted Patent: US 10,179,932 B2
• Time in Patent Office: 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 of labelling a subcellular component in a cell comprising the steps of:

providing the cell with an RNA comprising a barcode and a localization sequence that targets the RNA to the subcellular component wherein the localization sequence localizes to the subcellular component;

reverse transcribing the RNA to produce DNA; and

amplifying the DNA to produce amplicons.

2. The method of claim 1 wherein amplifying the DNA includes circularizing the DNA;

and performing rolling circle amplification (RCA) to produce an amplicon.

3. The method of claim 1, further comprising the step of detecting the amplicon.

4. The method of claim 2, wherein the subcellular component is an organelle or a subcellular region.

5. The method of claim 4, wherein the organelle is selected from the group consisting of one or any combination of a nucleus, a nucleolus, a mitochondria, a Golgi apparatus, an endoplasmic reticulum, a ribosome, a lysosome, a vacuole, an endocytic vesicle, an exocytic vesicle, a cytoskeleton and a chloroplast.

6. The method of claim 4, wherein the subcellular region is selected from the group consisting of one or any combination of a plasma membrane, a cell wall and a ribosomal subunit.

7. The method of claim 1, wherein the RNA is provided by expression of the RNA by the cell which is controlled by a promoter selected from the group consisting of one or any combination of an inducible promoter, a cell type-specific promoter and a signal-specific promoter.

8. The method of claim 1 wherein the RNA is provided by being delivered directly to the cell.

9. A method of labelling a protein in a cell comprising the steps of:

providing the cell with an RNA comprising a barcode and a protein comprising an RNA binding domain and a localization sequence wherein the localization sequence localizes to the subcellular component and the RNA binds to the RNA binding domain;

reverse transcribing the RNA to produce DNA;

circularizing the DNA; and

performing RCA to produce an amplicon.

10. The method of claim 9, further comprising the step of detecting the amplicon.

11. The method of claim 9, wherein the subcellular component is an organelle or a subcellular region.

12. The method of claim 11, wherein the organelle is selected from the group consisting of one or any combination of a nucleus, a nucleolus, a mitochondria, a Golgi apparatus, an endoplasmic reticulum, a ribosome, a lysosome, a vacuole, an endocytic vesicle, an exocytic vesicle, a cytoskeleton and a chloroplast.

13. The method of claim 11, wherein the subcellular region is selected from the group consisting of one or any combination of a plasma membrane, a cell wall and a ribosomal subunit.

14. The method of claim 9, wherein the RNA is provided by expression of the RNA by the cell which is controlled by a promoter selected from the group consisting of one or any combination of an inducible promoter, a cell type-specific promoter and a signal-specific promoter.

15. A method of labelling a protein in a cell comprising the steps of:

providing the cell with an RNA comprising a barcode, wherein the RNA binds to an RNA binding domain of a protein within the cell;

reverse transcribing the RNA to produce DNA;

circularizing the DNA;

performing RCA to produce an amplicon; and

sequencing the amplicon.

16. A method of labelling a subcellular component in vivo comprising the steps of:

providing a cell expressing an RNA comprising a barcode;

reverse transcribing the RNA to produce DNA;

circularizing the DNA; and

performing rolling circle amplification (RCA) to produce an amplicon.

17. The method of claim 16, further comprising the step of detecting the amplicon.

18. The method of claim 16, wherein the RNA comprises a localization sequence that targets the RNA to the subcellular component.

19. The method of claim 16, wherein the subcellular component is an organelle or a subcellular region.

20. The method of claim 19, wherein the organelle is selected from the group consisting of one or any combination of a nucleus, a nucleolus, a mitochondria, a Golgi apparatus, an endoplasmic reticulum, a ribosome, a lysosome, a vacuole, an endocytic vesicle, an exocytic vesicle, a cytoskeleton and a chloroplast.

21. The method of claim 19, wherein the subcellular region is selected from the group consisting of one or any combination of a plasma membrane, a cell wall and a ribosomal subunit.

22. The method of claim 16, wherein expression of the RNA is controlled by a promoter selected from the group consisting of one or any combination of an inducible promoter, a cell type-specific promoter and a signal-specific promoter.

23. A method of labelling a protein in vivo comprising the steps of:

providing a cell expressing an RNA comprising a barcode and expressing a protein comprising an RNA binding domain;

allowing the RNA and the protein to interact;

reverse transcribing the RNA to produce DNA;

circularizing the DNA; and

performing RCA to produce an amplicon.

24. The method of claim 23, further comprising the step of detecting the amplicon.

25. The method of claim 23, wherein the protein further comprises a domain that localizes it to a subcellular component.

26. The method of claim 23, wherein the subcellular component is an organelle or a subcellular region.

27. The method of claim 26, wherein the organelle is selected from the group consisting of one or any combination of a nucleus, a nucleolus, a mitochondria, a Golgi apparatus, an endoplasmic reticulum, a ribosome, a lysosome, a vacuole, an endocytic vesicle, an exocytic vesicle, a cytoskeleton and a chloroplast.

28. The method of claim 26, wherein the subcellular region is selected from the group consisting of one or any combination of a plasma membrane, a cell wall and a ribosomal subunit.

29. The method of claim 23, wherein expression of the RNA is controlled by a promoter selected from the group consisting of one or any combination of an inducible promoter, a cell type-specific promoter and a signal-specific promoter.

30. A method of determining a nucleic acid sequence in situ comprising the steps of:

providing a cell expressing an RNA comprising a barcode;

reverse transcribing the RNA to produce DNA;

circularizing the DNA;

performing RCA to produce an amplicon; and

sequencing the amplicon.

31. The method of claim 30, wherein the cell further expresses a protein comprising an RNA binding domain.