Genetically encoded fluorescent indicators under optogenetic control and uses thereof

Patent metadata

Field	Value
Publication number	US-2022017976-A1
Application number	US-201917289946-A
Country	US
Kind code	A1
Filing date	Oct 31, 2019
Priority date	Oct 31, 2018
Publication date	Jan 20, 2022
Grant date	—

Abstract

Disclosed are methods and compositions for real-time monitoring cellular calcium ion dynamics by red-shifted genetically encoded indicators under optogenetic control, in which millisecond-timescale of temporal control of optical activation or inactivation and signal recording can be achieved. The methods include artifact-free functional imaging in conjunction with optogenetic tools for studying cellular physiology, signal transduction and neuronal activity. Thus, all-optical and non-invasive approaches for drug screening, toxicity testing and assessment of cell functions may be provided.

First claim

1 . A method for examining a function of a cell, the method comprising:
(a) expressing a red-shifted genetically encoded Ca 2+ indicator (GECI) in the cell; (b) obtaining an optical signal from the red-shifted GECI in response to a stimulation; and (c) characterizing the sample by analyzing the optical signal responses in order to determine functional properties of the sample.

2 . The method of claim 1 comprising the further steps of:
a. expressing an optical actuator which is activated by light in the wavelength range of about 400 nm to about 570 nm, together with the GECI, which has an excitation wavelength greater than the activation wavelength of the optical actuator, in the cell; and
b. obtaining an optical signal from the red-shifted GECI in response to a stimulation imposed by the optical actuator caused by exposure to activation light.

3 . The method of claim 1 , wherein the sample is a two- or three-dimensional (2D or 3D) cell culture, cellular organelles, stem cells or a mammalian blood plasma.

4 . The method of claim 1 , wherein the red-shifted GECI is K-GECO1, NIR-GECO1 or NIR-GECO2, or a substantially similar red-shifted GECI.

5 . The method of claim 2 , wherein the optical actuator is a microbial light-sensitive protein.

6 . The method of claim 5 , wherein the microbial light-sensitive protein is a bacteriorhodopsin, or halorhodopsin, or channelrhodopsin optionally with one or more mutations.

7 . The method of claim 2 , wherein the optical actuator is a genetically encoded calcium actuator, which can activate or inactivate calcium signaling directly or indirectly.

8 . The method of claim 7 , wherein the genetically encoded calcium actuator is a fusion protein that includes a phytochrome, or a cryptochome, or a light-oxygen-voltage-sensing domain.

9 . The method of claim 1 , wherein the sample characterization step comprises determining cell response to exposure to an agent.

10 . The method of claim 1 , wherein the signal analysis step comprises characterization of calcium influx of the sample.

11 . The method of claim 4 wherein the cell is transformed or transfected with a nucleic acid encoding a GECI having an amino acid sequence selected from SEQ ID NO. 5 (K-GECO1), SEQ ID NO. 6 (NIR-GECO1) or SEQ ID NO. 7 (NIR-GECO2), or a substantially similar red-shifted GECI.

12 . The method of claim 11 wherein the nucleic acid has a nucleotide sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or a substantially similar nucleotide sequence.

Assignees

• Lumistar Biotechnology Inc
• Univ Alberta

Inventors

• Chang yu-fen
• Shen Yi
• Qian Yong
• Chung Min-Wen
• Campbell Robert

Classifications

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · 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

• C07K14/415 Primary

  from plants · CPC title