Optogenetic probes for measuring membrane potential

• US 9,791,455 B2
• Filed: 06/15/2015
• Issued: 10/17/2017
• Est. Priority Date: 08/23/2010
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Cohen, Adam E., Kralj, Joel, Douglass, Adam D.
• Primary Examiner(s): NAVARRO, ALBERT MARK
• Application Number:
  US14/739,908
• Publication Number:
  US 20150285820A1
• Time in Patent Office: 855 Days
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2810/00 Vectors comprising a targeting moiety
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The invention provides methods, cells and constructs for optical measurement of membrane potential. These methods can be used in cells that are not accessible to presently available methods using electrodes. The methods can be directed to, for example, high-throughput drug screening assays to determine agents that can affect membrane potential of a target cell.

Claims

1. An isolated and purified nucleic acid encoding Archaerhodopsin 3 with a mutation of D95 to reduce ion pumping activity compared to Arch 3 WT, the isolated and purified nucleic acid being operably linked to a nucleic acid encoding at least one additional fluorescent protein capable of indicating ion concentration in a cell.

2. The isolated and purified nucleic acid of claim 1, wherein the mutation comprises D95N.

3. The isolated and purified nucleic acid of claim 1 operably linked to a nucleic acid sequence encoding a membrane-targeting nucleic acid sequence.

4. The isolated and purified nucleic acid of claim 3, wherein the membrane-targeting nucleic acid sequence is a plasma membrane targeting nucleic acid sequence.

5. The isolated and purified nucleic acid of claim 3, wherein the membrane-targeting nucleic acid sequence is a subcellular membrane-targeting nucleic acid sequence.

6. The isolated and purified nucleic acid of claim 5, wherein the subcellular membrane is a mitochondrial membrane, an endoplasmic reticulum, a sarcoplastic reticulum, a synaptic vesicle, an endosome or a phagosome.

7. The isolated and purified nucleic acid of claim 1 operably linked to a cell-type specific promoter.

8. The isolated and purified nucleic acid of claim 1, wherein the at least one additional fluorescent protein is a green fluorescent protein or a homolog thereof.

9. The isolated and purified nucleic acid of claim 1, wherein the fluorescent protein capable for indicating ion concentration is a calcium indicator.

10. The isolated and purified nucleic acid of claim 1, wherein the fluorescent protein capable for indicating ion concentration is a pH indicator.

11. The isolated and purified nucleic acid of claim 1, further comprising a vector.

12. The isolated and purified nucleic acid of claim 11, wherein the vector is a viral vector.

13. The isolated and purified nucleic acid of claim 12, wherein the viral vector is a lentiviral vector.

14. The isolated and purified nucleic acid of claim 12, wherein the viral vector is an adeno-associated viral vector.

15. An isolated cell comprising a nucleic acid encoding Archaerhodopsin 3 with a mutation at D95 to reduce ion pumping activity compared to Arch 3 WT and at least one additional fluorescent protein capable of indicating an ion concentration in the cell.

16. An isolated and purified nucleic acid encoding Archaerhodopsin 3 with a mutation of D95 to reduce ion pumping activity compared to Arch 3 WT, the isolated and purified nucleic acid being operably linked to a nucleic acid encoding at least one green fluorescent protein or a homolog thereof.

17. The isolated and purified nucleic acid of claim 16, wherein the mutation comprises D95N.

18. The isolated and purified nucleic acid of claim 16 operably linked to a nucleic acid sequence encoding a membrane-targeting nucleic acid sequence.

19. The isolated and purified nucleic acid of claim 17, wherein the membrane-targeting nucleic acid sequence is a plasma membrane targeting nucleic acid sequence.

20. The isolated and purified nucleic acid of claim 17, wherein the membrane-targeting nucleic acid sequence is a subcellular membrane-targeting nucleic acid sequence, wherein the subcellular membrane is selected from the group consisting of a mitochondrial membrane, an endoplasmic reticulum, a sarcoplastic reticulum, a synaptic vesicle, an endosome, and a phagosome.

21. The isolated and purified nucleic acid of claim 16 operably linked to a cell-type specific promoter.

22. The isolated and purified nucleic acid of claim 16, wherein the green fluorescent protein is capable of undergoing nonradiative fluorescence resonance energy transfer to the microbial rhodopsin, with a rate of energy transfer dependent on membrane potential.