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bioRxiv · 10.1101/2020.07.31.230128

Improving the flexibility of Genetically Encoded Voltage Indicators via intermolecular FRET

Leong, L. M.·Kang, B. E.·Baker, B. J.
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Abstract

A new family of Genetically Encoded Voltage Indicators (GEVIs) has been developed based on inter-molecular Forster Resonance Energy Transfer (FRET). To test the hypothesis that the GEVI, ArcLight, functions via interactions between the fluorescent protein (FP) domain of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were co-transfected with both the FRET donor and acceptor GEVIs. Using a CFP donor and an RFP acceptor, we were able to observe a voltage dependent signal with a Stokes shift of over 200 nm. The intermolecular FRET strategy also works for rhodopsin-based probes potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. First, the signals are larger. Apparently the voltage-induced conformational change moves the two FPs independently thereby increasing the dynamic range. Second, the expression of the FRET donor and acceptor can be restricted independently enabling greater cell type specificity as well as refined subcellular voltage reporting.

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Leong, L. M., Kang, B. E., Baker, B. J.. 2020-08-02. Improving the flexibility of Genetically Encoded Voltage Indicators via intermolecular FRET. https://doi.org/10.1101/2020.07.31.230128

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