Photoejection turns non-covalent fluorescent tags into negative reversible photoswitchers

Reversibly photoswitchable fluorophores have enabled a broad range of applications in advanced fluorescence bioimaging. Here, we provide an entirely new class of representatives based on a pair: a biomolecular host and a dark photoisomerizable guest, which becomes bright upon complexation. Hence, we introduce RSpFAST, which delivers the first non-covalent chemogenetic reversibly photoswitchable fluorescent proteins from combining photoisomerizable fluorogens with the FAST protein tag. Our experimental strategy involving thermokinetic, photochemical, and structural investigations provides 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 simply 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.