Kinetic and mechanistic characterization of RSpFAST, anon-covalent versatile chemogenetic platform for fluorescence　imaging

Author

Shpinov, Yuriy and Mandal, Mrinal and van Deuren, Vincent and Lahlou, Alienor and Le Bec, Matthias and Chouket, Raja and Hadj Moussa, Chaima and Bonin, Clara and Sepasi Tehrani, Hessam and Coghill, Ian and El Hajji, Lina and Ounoughi, Karim and Franco Pinto, Jaime and Plamont, Marie-Aude and Pelupessy, Philippe and Ayala, Isabel and Perez, Franck and Aujard, Isabelle and Saux, Thomas Le and Gautier, Arnaud and Dedecker, Peter and Brutscher, Bernhard and Jullien, Ludovic

Abstract

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 decreasing 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.