Optogenetic probes for measuring membrane potential

Patent metadata

Field	Value
Publication number	US-9518103-B2
Application number	US-201514742648-A
Country	US
Kind code	B2
Filing date	Jun 17, 2015
Priority date	Jun 18, 2014
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

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.

First claim

What is claimed is:

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.

Assignees

• Harvard College
• Univ Alberta

Inventors

• Cohen Adam E
• Hochbaum Daniel
• Zou Peng
• Farhi Samouil Leon
• Campbell Robert Earl
• Zhao Yongxin
• Harrison Daniel Jed

Classifications

• C07K14/215

  from Halobacteriaceae (F) · CPC title

• G01N33/566 Primary

  using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} · CPC title

• G01N2333/726

  G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH · CPC title

• G01N21/6428

  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 title

• C07K14/705 Primary

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title