Genetically encoded voltage indicators and uses thereof
AssigneePresident and Fellows of Harvard College
InventorsAdam Ezra Cohen, He Tian
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Abstract
Provided herein are genetically encoded voltage indicator (GEVI) variants (e.g., QuasArba, QuasArbb) of Archaerhodopsin 3 useful for applications, such as optical measurement of membrane potential. Described herein are also polynucleotides encoding the variants, nucleic acid constructs, vectors (e.g., expression vectors), cells comprising the polynucleotides, nucleic acid constructs, and vectors, and cells comprising the polypeptides; and methods of using the variants.
Claims40
A polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:
- 1, wherein the amino acid sequence comprises at least one mutation at a position selected from the group consisting of positions 42, 85, 98, 124, and 148 of SEQ ID NO:
- 1.
A polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:
- 1, wherein the amino acid sequence comprises at least one mutation selected from the group consisting of W42G, V124G, M85I, F98L, and W148C in SEQ ID NO:
- 1.
The polypeptide of any one of the preceding claims, wherein the polypeptide is fluorescent and has reduced ion pumping activity compared to a natural member of the archaerhodopsin family of proteins from which it is derived.
The polypeptide of any one of the preceding claims, wherein the polypeptide is activated by contact with light having a non-blue light wavelength.
The polypeptide of any one of the preceding claims, wherein the polypeptide is activated by contact with at least one of yellow light, orange light, or red light.
The polypeptide of any one of the preceding claims wherein the polypeptide is activated by contact with red light having a wavelength of about 600 nm to about 690 nm.
A polynucleotide encoding the polypeptide of any one of the preceding claims.
A kit comprising the polypeptide or variant thereof, polynucleotide or variant thereof, nucleic acid construct, expression vector, or cell of any one of the preceding claims.
A method for measuring membrane potential in a cell expressing a nucleic acid encoding a microbial rhodopsin protein comprising the polypeptide of any one of the preceding claims, the method comprising the steps of:
- exciting, in vitro, at least one cell comprising a nucleic acid encoding a microbial rhodopsin protein with light of at least one wave length;
- and
- detecting, in vitro, at least one optical 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.
A method for characterizing a cell, the method comprising:
- incorporating into an electrically excitable cell an optical actuator of, and an optical reporter of, electrical activity;
- wherein the polypeptide of any one of the preceding claims is used as the optical reporter;
- obtaining a signal from the optical reporter in response to a stimulation of the cell;
- and evaluating the signal, thereby characterizing the cell.
A method for characterizing an interaction between cells, the method comprising:
- incorporating into a first electrically excitable cell an optical actuator of electrical activity;
- incorporating into a second electrically excitable cell an optical reporter of electrical activity;
- wherein the polypeptide of any one of the preceding claims is used as the optical reporter;
- culturing the first electrically excitable cell and the second electrically excitable cell in proximity to one another;
- obtaining a signal from the optical reporter in response to a stimulation of the first electrically excitable cell;
- and
- evaluating the signal,
- thereby characterizing an interaction between the first electrically excitable cell and the second electrically excitable cell.
Dependencies are inferred from the claim text.