Fluorescence monitoring of STING using a coumarin-based chemigenetic probe

Abstract

Protein condensate, a fundamental biological structure in biomolecular research, plays an important role in cell fate decision and cellular homeostasis. However, the lack of specific, stimulus-responsive tools to track the dynamics of proteins has hindered the in-depth investigation into their regulatory functions in cells. Herein, we report a fluorescent chemigenetic probe Y6_STING, where coumarin derivative Y6 is specifically conjugated onto the stimulator of interferon genes (STING) protein, enabling H₂O₂-mediated fluorescence monitoring of the dynamics of STING in living cells. Through structural modification of the coumarin backbone, substituent effects reveal that electron density at the C7 position of coumarin dictates the initial fluorescence intensity of Y1–Y3. By introducing a boron-cage onto the hydroxyl group of coumarin, Y4–Y6 exhibit a dual fluorescence response upon H₂O₂ stimulation. Upon reaching the proteins of interest (POIs), the combination of Y6 containing a PEG-linked HaloTag ligand and a genetically encoded STING protein allows for H₂O₂-triggered STING-labeling and fluorescence activation in both gels and aqueous solution. This stimulus-mediated response of Y6_STING enables monitoring the dynamics of STING in living cells. We evaluate the intracellular tracing ability of Y6 and demonstrate that Y6 could be retained in cells for long-term tracking of STING. Hence, this novel strategy would greatly facilitate the advancement of monitoring protein dynamics in proteostasis and medicine research.