An ultralow-background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain.
blue
red
FenixS/Ash1
iLID
iLight
MagRed
nanoReD
CHO-K1
HEK293T
HeLa
in vitro
Transgene expression
Organelle manipulation
Benchmarking
Abstract:
The robustness and broad applicability of an optogenetic tool depends heavily on the properties of the underlying photoreceptor protein and its cognate binding partner-the light-responsive 'core'. Current red light optogenetic systems for mammalian cells rely on phytochrome-based photoreceptors: large (70-kDa) proteins that act as dimers, enforcing dimerization on attached proteins. Naturally occurring or engineered binding partners can function effectively, but large size, complex interaction, background binding, weak affinity and modest dynamic range remain limiting. Here we developed a small (17-kDa) monomeric biliverdin-binding photoreceptor, FenixS, and a highly selective, high-affinity binder, Ash1 (6 kDa) using structure-based design and directed evolution. Negligible OFF-state binding and a >1,200-fold increase in binding affinity upon 700-nm illumination yield a high-performance, ultralow background core for diverse applications. A FenixS-Ash1-based optogenetic tool for red light activation of transcription in mammalian cells performs robustly without biliverdin supplementation, with head-to-head comparisons confirming its control of gene expression versus established tools.
