A chemigenetic endogenous Ras activity biosensor for live-cell pharmacology

Ras is a small GTPase that regulates cell growth and proliferation. Hyperactive Ras is prevalent in cancer and has been a therapeutic target for decades. Many lines of evidence have demonstrated that Ras signals from the plasma membrane as well as noncanonical compartments such as the Golgi, making live-cell biosensors for tracking the spatiotemporal dynamics of Ras activity a valuable approach for Ras studies. However, current biosensors are limited in their quantitative capacity for measuring endogenous Ras activity. Here we introduce a chemigenetic biosensor design, making use of circularly permuted HaloTag labeled with the fluorophore JF₆₃₅, for detecting endogenous Ras activity. This HaloTag-based Ras Activity Reporter (HaloRasAR) revealed the spatiotemporal dynamics of Ras activity downstream of either growth factor signaling or protein kinase C activation. In addition, live-cell characterization of a Ras(G12C) inhibitor and a Ras GEF inhibitor revealed subcellular-specific inhibition profiles. HaloRasAR represents a major advance in spatiotemporal interrogation of Ras signaling and live-cell pharmacology.