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Updated: Aug 21, 2026
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Published on: November 7, 2025
A chemigenetic endogenous Ras activity biosensor for live-cell pharmacology
Ryan Weeks1,2, Daojia R Zhou1,3, Michelle S Frei1
1Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.
Abstract:
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.
Insights
Researchers developed a new chemigenetic biosensor, HaloRasAR, to track Ras protein activity in real-time within cells. This tool enhances understanding of Ras signaling in cancer and drug development.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Ras GTPases are key regulators of cell growth and proliferation.
- Hyperactive Ras is implicated in various cancers, making it a significant therapeutic target.
- Current biosensors lack the quantitative capacity to measure endogenous Ras activity effectively.
Purpose of the Study:
- To develop a novel chemigenetic biosensor for detecting and tracking endogenous Ras activity.
- To overcome the limitations of existing biosensors in quantitative measurements.
- To visualize the spatiotemporal dynamics of Ras signaling in live cells.
Main Methods:
- Introduction of a chemigenetic biosensor design utilizing a circularly permuted HaloTag labeled with JF635.
- Development of the HaloTag-based Ras Activity Reporter (HaloRasAR).
- Live-cell imaging to monitor Ras activity dynamics and inhibitor effects.
Main Results:
- HaloRasAR successfully revealed spatiotemporal dynamics of Ras activity.
- The biosensor tracked Ras activity downstream of growth factor and protein kinase C signaling.
- Live-cell characterization identified subcellular-specific inhibition profiles for Ras inhibitors.
Conclusions:
- HaloRasAR represents a significant advancement in spatiotemporal interrogation of Ras signaling.
- The biosensor enables precise live-cell pharmacology studies.
- This tool facilitates a deeper understanding of Ras function in normal and cancerous cells.
