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Kinetic and Mechanistic Characterization of RSpFAST, a Non-Covalent Versatile Chemogenetic Platform for Fluorescence
Yuriy Shpinov1, Mrinal Mandal1, Vincent van Deuren2
1Chimie Physique et Chimie du Vivant, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, Paris75005, France.
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.