Red genetically encoded calcium indicators and methods of use

Patent metadata

Field	Value
Publication number	US-2016176931-A1
Application number	US-201514974483-A
Country	US
Kind code	A1
Filing date	Dec 18, 2015
Priority date	Dec 23, 2014
Publication date	Jun 23, 2016
Grant date	—

Abstract

Protein indicators useful for calcium imaging, in particular, red genetically-encoded calcium indicators (GECIs) disclosed herein rival best-of-class green GECIs in terms of sensitivity for detecting neural activity, and can be monitored in vivo. The presently-disclosed subject matter further includes a method of monitoring cell activity comprising stimulating a cell comprising a red GECI polypeptide; and detecting fluorescence emitted by the cell.

First claim

We claim:

1 . A nucleic acid molecule comprising a sequence that encodes a genetically encoded calcium indicator (GECI) polypeptide having at least 95% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

2 . The nucleic acid molecule of claim 1 , comprising a sequence that encodes a GECI polypeptide having at least 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

3 . The nucleic acid molecule of claim 1 , comprising a sequence that encodes a GECI polypeptide consisting of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

4 . A vector comprising the nucleic acid molecule of claim 1 .

5 . A cell comprising the vector of claim 4 .

6 . A cell comprising the nucleic acid molecule of claim 1 .

7 . A nucleic acid molecule comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3.

8 . A vector comprising the nucleic acid molecule of claim 7 .

9 . A polypeptide comprising a GECI polypeptide sequence having at least 95% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

10 . The polypeptide of claim 9 , wherein the polypeptide comprises a GECI polypeptide sequence having at least 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

11 . The polypeptide of claim 9 , wherein the polypeptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4.

12 . A cell comprising the polypeptide of claim 9 .

13 . A method of monitoring the activity of a cell, comprising:
(i) stimulating a cell comprising a polypeptide of claim 9 ; and (ii) detecting fluorescence emitted by the cell.

14 . The method of claim 13 , wherein the cell is in a biological sample from a subject.

15 . The method of claim 14 , wherein the steps are performed in vivo.

16 . The method of claim 15 , wherein the subject is a mouse, a fish, a worm or a fly.

17 . The method of claim 16 , wherein the subject is a mouse and the cell is in the primary visual cortex.

18 . The method of claim 16 , wherein the cell is a motor neuron with terminals in the neuro-muscular junction (NW), a trigeminal neuron, or an ASH neuron.

19 . The method of claim 13 , wherein the detecting step comprises imaging.

20 . The method of claim 13 , wherein the cell is a neuronal cell, a muscle cell or a cardiomyocyte.

21 . A nucleic acid molecule comprising a sequence that encodes a genetically encoded calcium indicator (GECI) polypeptide having at least 95% sequence identity to SEQ ID NO:6.

22 . The nucleic acid molecule of claim 21 , comprising a sequence that encodes a GECI polypeptide having at least 99% sequence identity to SEQ ID NO:6.

23 . The nucleic acid molecule of claim 1 , comprising a sequence that encodes a GECI polypeptide consisting of the amino acid sequence of SEQ ID NO:6.

24 . A vector comprising the nucleic acid molecule of claim 21 .

25 . A cell comprising the vector of claim 24 .

26 . A cell comprising the nucleic acid molecule of claim 21 .

27 . A nucleic acid molecule comprising the sequence of SEQ ID NO:5.

28 . A vector comprising the nucleic acid molecule of claim 27 .

29 . A polypeptide comprising a GECI polypeptide sequence having at least 95% sequence identity to SEQ ID NO:6.

30 . The polypeptide of claim 29 , wherein the polypeptide comprises a GECI polypeptide sequence having at least 99% sequence identity to SEQ ID NO:6.

31 . The polypeptide of claim 29 , wherein the polypeptide comprises the sequence of SEQ ID NO:6.

32 . A cell comprising the polypeptide of claim 29 .

33 . A method of monitoring the activity of a cell, comprising:
(i) stimulating a cell comprising a polypeptide of claim 29 ; and (ii) detecting fluorescence emitted by the cell.

Assignees

• Hughes Howard Med Inst

Inventors

• Kim Douglas S
• Looger Loren L
• Schreiter Eric R
• Svoboda Karol

Classifications

• G01N2333/4727

  Calcium binding proteins, e.g. calmodulin · CPC title

• A61K49/0045

  the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo · CPC title

• C07K14/001 Primary

  by chemical synthesis · CPC title

• G01N33/5041 Primary

  involving analysis of members of signalling pathways · CPC title

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title