An ultra-low background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain.
red
FenixS/Ash1
CHO-K1
HEK293T
HeLa
in vitro
Control of intracellular / vesicular transport
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 use in mammalian cells all rely on phytochrome based photoreceptors. These are large (70 kDa) proteins that act as dimers, thereby enforce dimerization on attached proteins. Naturally occurring or engineered binding partners can function effectively in certain cases, but large size, complex mode of interaction, background binding, relatively weak affinity and/or low fold changes between on and off states are significant limitations. Using structure-based design and directed evolution we developed a small (17 kDa) monomeric bilverdin binding photoreceptor FenixS, and a highly selective, high-affinity binder, Ash1 (6 kDa). Negligible off-state binding and a >1200-fold increase in binding affinity upon 700 nm illumination result in a high performance, ultra-low background, light responsive core for a diverse range of applications. An optogenetic tool for red light activation of transcription in mammalian cells based on the FenixS-Ash1 core exhibits robust performance without the need for biliverdin supplementation.
