In situ spatiotemporal tracking of endogenous protein dynamics with allosteric genetically encoded biosensor in living cells

Proteins' functions are dictated by their abundance and unique subcellular localization, necessitating analysis under native conditions. Current imaging approaches often rely on overexpression or genome editing, which work for exogenous or modified proteins, risk introducing artifacts, and fail to reveal the true roles of native proteins in situ. Imaging of endogenous, unmanipulated proteins in living cells remains extremely scarce. Herein, an allosteric biosensor comprising a protein of interest (POI) recognizing aptamer and a dual-locked fluorogenic aptamer module, termed the spatiotemporal tracking endogenous proteins (Step), is developed. The intracellular circular Step (circStep) system translates a single POI-aptamer binding event into an allosteric switch that simultaneously unlocks two caged fluorogenic Aptamers (M18), which restore their active conformations and yield a robust fluorescent signal, enabling high-fidelity, real-time tracking of native POI localization and abundance fluctuations. By remodeling the aptamer into an allosteric switch, the design lowers background, curbs off-target binding and sharpens POI specificity. Importantly, it also enables spatiotemporal dynamic tracking of protein translocation by directly visualizing the process of the nucleocytoplasmic transport of POI, establishing a robust, imaging-based high-throughput platform that accurately screens drugs by artificially manipulating protein relocalization. Therefore, this plug-and-play circStep, which monitors the native protein dynamics without perturbing its expression or localization and leaves POI function intact, expands the current toolbox for investigating the authentic functions of proteins and accelerate drug discovery, especially for therapies that reprogram protein localization.