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

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.


Email Address of Submitting Author


University of California, Berkeley


United States

ORCID For Submitting Author


Declaration of Conflict of Interest

MKS and TAK are employees of Promega Corp.