Single-fluorophore Biosensors for Sensitive and Multiplexed Detection of Signaling Activities

Unraveling the dynamic molecular interplay behind complex physiological processes such as neuronal plasticity requires the ability to both detect minute changes in biochemical states in response to physiological signals and track multiple signaling activities simultaneously. Fluorescent protein-based biosensors have enabled the real-time monitoring of dynamic signaling processes within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (e.g., dynamic range) and are limited to imaging signaling activities in isolation. Here, we address this challenge by developing a suite of excitation ratiometric kinase activity biosensors that offer the highest reported dynamic range and enable the detection of subtle changes in signaling activity that could not be reliably detected previously, as well as a suite of single-fluorophore biosensors that enable the simultaneous tracking of as many as six distinct signaling activities in single living cells.