Construction of Coumarin-Conjugated Macromolecular Probe for Photoactivatable Bioimaging

Fluorophore-conjugated imaging has emerged as a powerful technique for visualizing the subcellular localization and dynamic trafficking of proteins, yet its performance is often limited by high background noise. Herein, we rationally designed four coumarin-based bioorthogonal fluorescent probes by site-specifically conjugating photoactivatable caging groups and HaloTag ligands to coumarin fluorophore. Fluorescence characterization revealed that probes Y8 and Y9 exhibited a pronounced aggregation-induced emission (AIE) effect. While probes Y11 and Y12 displayed rapid photoresponse in aqueous environments, Y12 achieved stable conjugation to HaloTag-fused proteins of interest (POIs) with robust photoactivated fluorescence. We further evaluated the intracellular fluorescence labeling performance of probes 4, Y9, and Y12 targeting the STING protein and confirmed that the combination of Y12 with genetically encoded STING-Halo enabled high-efficiency photoactivated fluorescence labeling, with a 16.5-fold fluorescence enhancement upon light irradiation. This ultra-high signal-to-background ratio (S/N) facilitated the real-time monitoring of dynamic STING activation, during which Y12_STING translocated from diffuse staining to fluorescent foci in cells stimulated with the STING agonist MSA-2. This fluorescent dye/protein hybrid system holds great promise for advancing the mapping of protein dynamics in cell homeostasis and medicine research.