Summary/Abstract The ability to control protein function with light provides excellent temporal and spatial resolution for precise investigation in situ, and thus is having significant impact on neuroscience. There are two major barriers imposed by existing optogenetic methods: one being that they cannot be readily applied on any protein of choice, and the other being lack of high specificity and flexibility in site selection for photo-modulation. These limitations significantly restrain the scope, precision, and depth of investigations on neuronal processes. To overcome these challenges, we propose here a nano-switch technology for optical control of neuronal proteins in their native settings with general applicability and ultra-specificity. Through the expansion of the genetic code, we will site-specifically incorporate photo-reversible unnatural amino acids (Uaas) into proteins to modulate a single site, and to build novel nano-bridges able to modulate secondary structures and domains, so as to photo-regulate protein activities in a reversible manner. Compared with existing methods using large proteins and domains, our method uses only a single Uaa for light sensitivity. Our method thus has minimal perturbation to proteins under study, and can be generally applied to any protein without limitations to protein type, function, or cellular localization. In addition, rather than relying on protein function or interaction for photo- regulation as in current methods, our method is able to photo-modulate a protein without knowing its function in advance. Using genetically encoded Uaas also enables our method compatible with a broad range of neural cells and model animals. More importantly, our method will confer photo-responsiveness on target neuronal proteins with unprecedented resolution that is specific for desired subunits, domains, and even single residues. This unparalleled specificity will open vast new opportunities for investigation of neuronal processes with pinpoint accuracy. The success of this project will afford a novel nano-switch platform technology for optical modulation of any neuronal protein both in vitro and in vivo, laying a foundation for the emerging molecular opto-neurobiology to uncover fine molecular insights previously inaccessible for neural signaling.

Relevance Statement We will develop an innovative method to enable the study of neural signaling and circuits on the molecular level with ultra-specificity using light. Our method will be generally useful in various neural cells and model organisms. The success of this project will afford in depth molecular understanding of neuronal processes previously inaccessible with existing optogenetic methods.