Chemigenetic voltage indicators

Patent metadata

Field	Value
Publication number	US-11385236-B2
Application number	US-201716347765-A
Country	US
Kind code	B2
Filing date	Nov 30, 2017
Priority date	Nov 30, 2016
Publication date	Jul 12, 2022
Grant date	Jul 12, 2022

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 membrane-localized voltage sensitive protein coupled to a capture protein, wherein the capture protein is covalently or noncovalently labeled with one or more cell permeable fluorescent dyes.

2. The voltage indicator of claim 1 , where the one or more cell permeable fluorescent dyes are azetidine-containing dyes.

3. The voltage indicator of claim 2 , wherein the one or more azetidine-containing dyes are selected from the group consisting of

and combinations thereof.

4. The voltage indicator of claim 1 , wherein the voltage sensitive protein is an opsin.

5. The voltage indicator of claim 4 , wherein the opsin is a microbial opsin.

6. The voltage indicator of claim 5 , wherein the microbial opsin is selected from the group consisting of QuasAr2, Ace2N, or a combination thereof.

7. The voltage indicator of claim 1 , wherein the capture protein is covalently labeled by the one or more fluorescent dyes.

8. The voltage indicator of claim 7 , wherein the covalent capture protein is a self-labeling protein tag.

9. The voltage indicator of claim 1 , wherein the capture protein is non-covalently labeled by the one or more fluorescent dyes.

10. The voltage indicator of claim 9 , where in the capture protein is biotin-avidin.

11. The voltage indicator of claim 1 , wherein the membrane-localized voltage sensitive protein comprises at least one voltage sensitive domain selected from the group consisting of an opsin, Ciona intestinalis voltage-sensing domain (CiVSD), Dario rerio voltage-sensing domain (DrVSD), Gallus voltage-sensing domain (GgVSD), and a combination thereof.

12. A method of measuring voltage, the method comprising administering one or more cell permeable fluorescent dyes and a voltage indicator comprising a membrane-localized voltage sensitive protein coupled to a capture protein and determining measuring changes in fluorescence when the one or more cell permeable fluorescent dye are captured by the covalent-capture protein of the voltage.

13. The method of claim 12 , wherein changes in fluorescence are observed with a microscope.

14. The method of claim 12 , wherein the voltage indicator further comprises a linker between the voltage-sensitive protein and the capture protein.

15. The method of claim 14 , further comprising modifying a length of the linker.

16. The method of claim 15 , wherein modifying the length of the linker comprises removing at least one amino acid residue.

17. The method of claim 16 , wherein removing at least one amino acid residue comprises removing between 1 and 22 amino acid residues.

18. The method of claim 15 , wherein modifying the length of the linker modifies the size of a fluorescence response.

19. The method of claim 12 , further comprising measuring changes in voltage based upon changes in fluorescence.

20. The method of claim 12 , wherein the membrane-localized voltage sensitive protein comprises at least one voltage sensitive domain selected from the group consisting of an opsin, Ciona intestinalis voltage-sensing domain (CiVSD), Dario rerio voltage-sensing domain (DrVSD), Gallus voltage-sensing domain (GgVSD), and a combination thereof.

Assignees

• Hughes Howard Med Inst

Inventors

• Schreiter Eric R
• Lavis Luke D
• Abdelfattah Ahmed

Classifications

• C07K14/461

  from fish · CPC title

• C07K14/47

  from mammals · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K14/43504

  from invertebrates · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title