Optogenetic probes for measuring membrane potential

• US 9,518,103 B2
• Filed: 06/17/2015
• Issued: 12/13/2016
• 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), The Governors of the University of Alberta (University of Alberta)
• Inventors: Cohen, Adam E., Hochbaum, Daniel, Zou, Peng, Farhi, Samouil Leon, Campbell, Robert Earl, Zhao, Yongxin, Harrison, Daniel Jed
• Primary Examiner(s): NASHED, NASHAAT T
• Application Number:
  US14/742,648
• Publication Number:
  US 20150369740A1
• Time in Patent Office: 545 Days
• Field of Search: None
• US Class Current: 1/1
• 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 polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises at least one substitution mutation selected from the group consisting of P60S, T80S, D95H, D95Q, D106H, and F161V, wherein the position of the at least one substitution mutation in the amino acid sequence corresponds to SEQ ID NO;

1.

2. The polypeptide of claim 1, wherein the amino acid sequence comprises at least two mutations selected from the group consisting of P60S, T80S, D95N, D95H, D95Q, D106H, and F161V.

3. The polypeptide of claim 1 comprising an amino acid sequence of SEQ ID NO:

2 or SEQ ID NO;

3.

4. A FRET pair comprising the polypeptide of claim 1 serving as a fluorescence acceptor and a fluorescent protein serving as a fluorescence donor.

5. The polypeptide of claim 1, wherein the polypeptide comprises a membrane-targeting sequence.

6. The polypeptide of claim 5, wherein the membrane-targeting sequence is a subcellular compartment-targeting nucleic acid sequence.

7. The polypeptide of claim 1, wherein the polypeptide is linked to at least one additional protein or a chromophore.

8. The polypeptide of claim 1, wherein the variant polypeptide is fluorescent and has reduced ion pumping activity compared to a natural member of the archaerhodopsin family of proteins from which the polypeptide is derived.

9. A method for measuring a change in membrane potential in a cell expressing a variant microbial rhodopsin polypeptide, the method comprising the steps of:

irradiating, in vitro, at least one cell expressing the polypeptide of claim 1 with light having a wave length suitable to produce fluorescence signal from the variant polypeptide;

detecting, in vitro, a fluorescent 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;

exposing, in vitro, the at least one cell to a stimulus capable of changing the membrane potential of the at least one cell;

irradiating, in vitro, the at least one cell expressing the polypeptide of claim 1 with light having a wave length suitable to produce fluorescence signal from the variant polypeptide;

detecting, in vitro, a second fluorescent 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; and

comparing the first fluorescent signal with the second fluorescent signal to determine the change in membrane potential of the cell.

10. The method of claim 9, wherein the cell is irradiated with yellow light, red light, or combinations thereof.

11. A polynucleotide comprising a nucleic acid sequence encoding a variant polypeptide having at least 80% identity to SEQ ID NO:

1, wherein the variant polypeptide comprises at least one substitution mutation selected from the group consisting of P60S, T80S, D95H, D95Q, D106H, and F161V, wherein the position of the at least one substitution mutation in the amino acid sequence corresponds to SEQ ID NO;

1.

12. A polynucleotide comprising a nucleic acid sequence of SEQ ID NO:

5 or SEQ ID NO;

6.

13. A polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises a first substitution mutation at position 95, and at least one second substitution mutation selected from the group consisting of P60S, T80S, D106H, and F161V, wherein the positions of the substitution mutations in the amino acid sequence corresponds to SEQ ID NO;

1.

14. A polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises a substitution mutation at position 95, a D106H mutation, and at least one substitution mutation selected from the group consisting of P60S, T80S, and F161V, wherein the positions of the substitution mutations in the amino acid sequence corresponds to SEQ ID NO;

1.

15. A polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises a substitution mutation at position 95 of D to Q, C, or N, and

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

wherein the positions of the substitution mutations in the amino acid sequence corresponds to SEQ ID NO;

1.

16. A polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:

2 or SEQ ID NO;

3.

17. A fusion protein comprising an amino acid sequence of SEQ ID NO:

2 or SEQ ID NO;

3 and an amino acid sequence of a second fluorescent protein.

18. A polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises a D to H or D to Q substitution mutation at position 95, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO;

1.

19. A polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises a P to S substitution mutation at position 60, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO;

1.

20. A polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises a T to S substitution mutation at position 80, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO;

1.

21. A polypeptide comprising a variant having at least 80% identity to SEQ ID NO:

1, wherein the amino acid sequence of the variant comprises a D to H substitution mutation at position 106, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO;

1.