Chemigenetic voltage indicators

Patent metadata

Field	Value
Publication number	US-12474345-B2
Application number	US-202217835535-A
Country	US
Kind code	B2
Filing date	Jun 8, 2022
Priority date	Nov 30, 2016
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

Provided herein are a voltage indicator and a method of measuring voltage. The voltage indicator includes a membrane-localized voltage-sensitive protein coupled to a capture protein. The method of measuring voltage includes administering a voltage indicator including a membrane-localized voltage-sensitive protein coupled to a capture protein, and determining changes in fluorescence of a small-molecule fluorescent dye captured by the capture protein.

First claim

What is claimed is:

1 . A voltage indicator comprising:
a polypeptide comprising a membrane-localized voltage-sensitive protein and a capture protein, and a cell-permeable fluorescent dye, wherein the voltage-sensitive protein is coupled to the capture protein and the capture protein is covalently or noncovalently labeled with the cell-permeable fluorescent dye.

2 . The voltage indicator of claim 1 , wherein the membrane-localized voltage sensitive protein is selected from the group consisting of QuasAr2, Ace2N, Ciona intestinalis voltage-sensing domain (CiVSD), Danio rerio voltage-sensing domain (DrVSD), Gallus voltage-sensing domain (GgVSD), and a combination thereof.

3 . The voltage indicator of claim 2 , wherein the voltage-sensitive protein and the capture protein are coupled through a linker.

4 . The voltage indicator of claim 2 , wherein the capture protein is a self-labeling protein.

5 . The voltage indicator of claim 4 , wherein the membrane-localized voltage sensitive protein comprises QuasAr2.

6 . The voltage indicator of claim 5 , wherein the polypeptide comprises an amino acid having the sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 16, 18, 20, and 22.

7 . The voltage indicator of claim 4 , wherein the membrane-localized voltage sensitive protein comprises Ace2N.

8 . The voltage indicator of claim 7 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 24.

9 . The voltage indicator of claim 4 , wherein the membrane-localized voltage sensitive protein comprises CiVSD.

10 . The voltage indicator of claim 9 , wherein the polypeptide comprises an amino acid having the sequence selected from the group consisting of: SEQ ID NOS: 26 and 28.

11 . The voltage indicator of claim 4 , wherein the membrane-localized voltage sensitive protein comprises DrVSD.

12 . The voltage indicator of claim 11 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 30.

13 . The voltage indicator of claim 4 , wherein the membrane-localized voltage sensitive protein comprises GgVSD.

14 . The voltage indicator of claim 13 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 32.

15 . The voltage indicator of claim 2 , wherein the capture protein is biotin-avidin.

16 . The voltage indicator of claim 1 , wherein the cell permeable fluorescent dyes is an azetidine-containing dye.

17 . The voltage indicator of claim 16 , wherein the azetidine-containing dye is selected from the group consisting of

and combinations thereof.

18 . A method of measuring voltage, the method comprising
administering
(a) a polypeptide comprising a membrane-localized voltage-sensitive protein coupled to a capture protein, and
(b) a cell-permeable fluorescent dye, wherein the capture protein is capable of capturing the cell-permeable fluorescent dye; and
measuring changes in fluorescence when the cell-permeable fluorescent dye is captured by the capture protein of the polypeptide.

19 . The method of claim 18 , wherein changes in fluorescence are observed with a microscope.

20 . The method of claim 18 , further comprising measuring changes in voltage based upon changes in fluorescence.

Assignees

• Hughes Howard Med Inst

Inventors

• Schreiter Eric R
• Lavis Luke D
• Abdelfattah Ahmed

Classifications

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · 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

• 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

• C12Y308/01005

  Haloalkane dehalogenase (3.8.1.5) · CPC title

• C12N9/14

  Hydrolases (3) · CPC title