Time-resolved fluorescence of ANS dye as a sensor of proteins LLPS

The explosive growth in the number of works addressing the phase separation of intrinsically disordered proteins has driven both the development of new approaches and the optimization of existing methods for biomolecular condensate visualization. In this work, we studied the potential use of the fluorescent dye ANS as a sensor for liquid-liquid phase separation (LLPS), focusing on visualizing condensates formed by the stress-granules scaffold protein G3BP1. Using fluorescence lifetime imaging microscopy (FLIM), we demonstrated that ANS can accumulate in RNA-induced G3BP1 condensates in aqueous solutions, but not in G3BP1 condensates formed under macromolecular crowding conditions in highly concentrated PEG solutions. We showed that the experimentally determined limiting fluorescence anisotropy (r₀'), which characterizes the amplitude of high-frequency intramolecular mobility of ANS in aqueous solutions containing RNA-induced G3BP1 condensates, is half the value observed for ANS in aqueous G3BP1 solutions. Our results demonstrate the feasibility of using time-resolved fluorescence spectroscopy and microscopy of ANS for detecting LLPS of intrinsically disordered proteins in aqueous solutions.