Kinetic and Mechanistic Characterization of RSpFAST, A Non-Covalent Versatile Chemogenetic Platform for Fluorescence Imaging

Reversibly photoswitchable fluorophores have enabled a broad range of applications in advanced fluorescence bioimaging. Here, we introduce RSpFAST , a new class of reversibly photoswitchable fluorescent labels that combine a biomolecular host ( pFAST protein tag) with a reversibly photoisomerizable guest (fluorogen), allowing fluorescence brightness to be modulated through illumination and molecular complexation. We combine thermokinetic, photochemical, and structural investigations to obtain a comprehensive mechanistic and kinetic understanding of RSpFAST . Building on this theoretical framework, we demonstrate in both live and fixed cells that RSpFAST exhibits an unprecedented dual behavior: a stable and wash-free fluorescent labeling tag turns into a negative reversible photoswitcher by lowering the fluorogen concentration and increasing light intensity. In this photoejection-driven kinetic regime, RSpFAST is shown to be an efficient marker for dynamic contrast and super-resolution microscopy.