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

Yuriy Shpinov; Mrinal Mandal; Vincent M.L. Van Deuren; Aliénor Lahlou; Matthias Le Bec; Raja Chouket; Chaima Hadj Moussa; Clara Bonin; Hessam Sepasi Tehrani; Ian Coghill; Lina El Hajji; Karim Ounoughi; Jaime Franco; Marie‐Aude Plamont; Philippe Pelupessy; Isabel Ayala; Franck Perez; Isabelle Aujard; Thomas Le Saux; Arnaud Gautier; Peter Dedecker; Bernhard Brutscher; Ludovic Jullien

doi:10.1021/jacs.6c08277

BiochemistryJournal of the American Chemical SocietyAug 25, 2026

Peer-reviewed journal article

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

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

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 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.

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