Curated Optogenetic Publication Database

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.

Abstract: Multiple display techniques, including phage display, mRNA display, and ribosome display, have been used to expand the optogenetic toolbox beyond what nature provides. These techniques are most often applied to the development of binding partners that selectively recognize different conformational states of photoswitchable proteins. However, for some targets, in particular the spectrally diverse cyanobacteriochrome (CBCR) GAF domain family, the subtle differences between conformational states pose a significant challenge to discovering highly selective binders. We present an optimized phage display-based protocol designed to effectively capture these subtle changes. This optimized protocol applies high selection pressure by changing the elution method and tightening negative selection, leading to the enrichment of selective binders. Through multiple selection campaigns, we demonstrate the utility of this protocol for identifying highly selective binders.