QuasAr6 GEVI Red-Shifted Voltage Sensing
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Solution Overview
Problem
Current methods for measuring membrane potential are limited by slow setup times, inability to measure deeply buried tissues, inaccessibility to small or hard-walled cells, and phototoxicity issues with existing genetically encoded voltage indicators (GEVIs), which hinder precise and simultaneous optical perturbation and measurement of neural activity across spatial and temporal scales.
Innovation Solution
Development of fluorescent polypeptides like QuasAr6a and QuasAr6b, based on Archaerhodopsin, which provide improved brightness, sensitivity, and response kinetics, allowing for sub-millisecond temporal and sub-micron spatial resolution of membrane potential in eukaryotic cells, including neurons and cardiomyocytes, with reduced ion pumping activity and minimal spectral overlap with optogenetic actuators.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If GFP-based GEVIs are used for voltage sensing, then genetic targeting is achieved, but severe optical crosstalk occurs with channelrhodopsins during simultaneous stimulation and measurement
Solution Approach 1:
The patent employs spectral shifting by developing red-shifted GEVIs (Archon1, QuasAr6) that emit at longer wavelengths (600-680 nm) compared to traditional GFP-based indicators. This color change separates the emission spectrum from the excitation spectrum of channelrhodopsins, eliminating optical crosstalk and enabling simultaneous all-optical electrophysiology.
Solution Approach 2:
The patent creates composite fluorescent polypeptides by fusing archaerhodopsin-based voltage-sensing domains with fluorescent protein domains. This composite structure combines the voltage-sensing capability of archaerhodopsin with the optical properties of fluorescent proteins, achieving both genetic targeting and reduced optical crosstalk through spectral separation.
2Object-affected harmful factors
If Arch 3 D95N is used as a voltage indicator, then phototoxicity is reduced, but response time becomes too slow (40 ms) for detecting neuronal action potentials
Solution Approach 1:
The patent optimizes multiple parameters of the archaerhodopsin-based GEVIs simultaneously: reducing ion pumping activity to minimize phototoxicity, enhancing fluorescence brightness to improve signal-to-noise ratio, and engineering faster response kinetics through mutagenesis. The resulting QuasAr6 indicators achieve sub-millisecond response times while maintaining low phototoxicity.
Solution Approach 2:
The patent replaces the proton pumping mechanism of wild-type archaerhodopsin with a fluorescence-based reporting mechanism. By mutating the ion pumping function and enhancing voltage-dependent fluorescence, the system substitutes a slow ion transport mechanism with a faster optical signaling mechanism that maintains low phototoxicity.
3Measurement precision
If electrophysiological methods are used for measuring membrane potential, then direct voltage recording is achieved, but cell damage or death occurs and long-term measurements are impossible
Solution Approach 1:
The patent replaces invasive electrical measurement methods with non-invasive optical measurement using genetically encoded fluorescent indicators. The GEVIs report membrane voltage through changes in fluorescence intensity or wavelength, eliminating the need for physical electrodes that damage cells and enabling long-term, repeated measurements in living tissues.
Solution Approach 2:
The patent implements a self-reporting system where the GEVIs are expressed within the cells themselves and autonomously report membrane voltage through fluorescence changes. This self-service mechanism eliminates the need for external electrodes and invasive procedures, allowing continuous monitoring without cell damage.
4Extent of automation
If existing GEVIs are used for simultaneous optical stimulation and measurement, then all-optical electrophysiology is achieved, but spectral overlap between GEVIs and optogenetic actuators prevents robust implementation
Solution Approach 1:
The patent achieves spectral separation by developing red-shifted GEVIs with emission maxima at 600-680 nm, which are distinct from the excitation spectra of blue- and green-light actuating channelrhodopsins. This color change enables independent optical control of stimulation and measurement channels without crosstalk,实现ing robust all-optical electrophysiology.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These polypeptides enable sensitive and fast optical detection of membrane voltage with improved signal-to-noise ratios and linearity, facilitating robust all-optical electrophysiology without perturbing cellular dynamics, thus overcoming the limitations of previous GEVIs.
Implementation Method 1
The inventive polypeptides...show voltage-dependent fluorescence. By optically measuring the membrane potential of cells and sub-cellular compartments, the inventive polypeptides are capable of indicating electrical dynamics
Implementation Method 2
It was previously demonstrated that the membrane potential in a membrane containing Archaerhodopsin 3 (Arch 3) can alter the optical properties of the protein, thereby making Arch 3 a voltage sensor
Data Source
AI summary
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.