Optogenetic regulation of endogenous cell cycle proteins through synthetic library screening of nanobodies

Abstract

Low kinetics, intrinsic toxicity and poor selectivity of conventional biochemical techniques made it challenging to study how integrators and cyclin dependent kinase families involve in cell fate decision. In this work, we set up a synthetic nanobody library platform for screening cell cycle-related proteins. We successfully screened nanobody candidates that bind to CDK2 proteins. We then combined screened nanobody with protein clustering optogenetic tool, light-activated reversible inhibition by assembled trap (LARIAT), to modulate the target protein upon light excitation. By applying intracellular antibodies and optogenetic tools, we aimed to selectively track endogenous proteins in highly temporal manner. Nanobody-based optogenetic protein modulation provides a powerful tool to identify how cell cycle proteins involve in proliferation-quiescence decision.