<p>We report a small molecule enzyme pair for optical voltage
sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent
V<u>olt</u>age 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.</p>
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