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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

preprint
submitted on 18.12.2020, 23:34 and posted on 21.12.2020, 13:18 by Brittany Benlian, Pavel Klier, Kayli Martinez, Marie Schwinn, Thomas Kirkland, Evan Miller

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

History

Email Address of Submitting Author

evanwmiller@berkeley.edu

Institution

University of California, Berkeley

Country

United States

ORCID For Submitting Author

0000-0002-6556-7679

Declaration of Conflict of Interest

MKS and TAK are employees of Promega Corp.

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