Optogenetic probes for measuring membrane potential

• US 10,457,715 B2
• Filed: 11/28/2016
• Issued: 10/29/2019
• Est. Priority Date: 06/18/2014
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), The Governors of the University of Alberta (University of Alberta)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Cohen, Adam E., Hochbaum, Daniel, Zou, Peng, Farhi, Samouil Leon, Campbell, Robert Earl, Zhao, Yongxin, Harrison, Daniel Jed
• Primary Examiner(s): Noakes, Suzanne M
• Application Number:
  US15/362,594
• Publication Number:
  US 20170313757A1
• Time in Patent Office: 1,065 Days
• Field of Search: None
• CPC Class Codes: C07K 14/215 from Halobacteriaceae (F)
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• 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

Abstract

Provided herein are variants of an archaerhodopsin useful for application such as optical measurement of membrane potential. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, cells comprising the polynucleotides, and cells comprising the polypeptides; and methods of using the variants.

Claims

1. A polynucleotide encoding a polypeptide having at least 80% identity to SEQ ID NO:

1, wherein the polypeptide comprises, relative to SEQ ID NO;

1, at least

a first substitution mutation selected from the group consisting of D95Q and D106H; and

a second substitution mutation selected from the group consisting of P60S, T80S, and F161V.

2. A nucleic acid construct comprising the polynucleotide of claim 1.

3. An expression vector comprising the polynucleotide of claim 1.

4. A cell comprising the polynucleotide of claim 1.

5. A cell comprising the vector of claim 3.

6. A kit comprising the polypeptide or variant thereof, polynucleotide or variant, nucleic acid construct, vector, or cell of any one of the preceding claims.

7. A method for measuring membrane potential in a cell expressing a nucleic acid encoding a microbial rhodopsin protein, the method comprising the steps of:

exciting, in vitro, at least one cell comprising the polynucleotide of claim 1 with light of at least one wave length; and

detecting, in vitro, at least one optical signal from the at least one cell, wherein the level of fluorescence emitted by the at least one cell compared to a reference is indicative of the membrane potential of the cell.

8. An expression vector comprising a polynucleotide encoding:

a polypeptide having at least 80% identity to SEQ ID NO;

1, wherein the polypeptide comprises, relative to SEQ ID NO;

1, at least the following mutations;

D95H or D95Q,

D106H, and

one mutation selected from the group consisting of P60S, T80S, and F161V; and

a promoter sequence to control the expression of the polypeptide.

9. The expression vector of claim 8, wherein the polypeptide comprises, relative to SEQ ID NO:

1, at least two mutations selected from the group consisting of P60S, T80S, and F161V.

10. The expression vector of claim 9, wherein the polypeptide comprises, relative to SEQ ID NO:

1, all three mutations of P60S, T80S, and F161V.

11. The expression vector of claim 10, wherein the polypeptide comprises an amino sequence of SEQ ID NO:

3.

12. The expression vector of claim 9, wherein the polypeptide comprises an amino sequence of SEQ ID NO:

2.

13. The expression vector of claim 9, wherein the polynucleotide further encodes a second polypeptide.

14. The expression vector of claim 13, wherein the second polypeptide is fluorescent.

15. The expression vector of claim 14, wherein the second polypeptide is mOrange2.

16. The expression vector of claim 13, wherein the second polypeptide is capable of indicating ion concentration.