Noninvasive Imaging of Ras Activity by Monomolecular Biosensor Based on Split-Luciferase Complementary Assay

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Ras GTPases play a crucial role in human cancer development.
• Monitoring Ras activity in vivo is essential for understanding tumorigenesis and developing targeted therapies.
• Existing methods for Ras activity assessment are often invasive and lack sensitivity.

Purpose of the Study:

• To develop a novel monomolecular bioluminescent biosensor for imaging endogenous Ras activity in living subjects.
• To establish a sensitive and noninvasive tool for real-time monitoring of Ras signaling.
• To facilitate high-throughput screening of Ras-targeted therapeutics.

Main Methods:

• Utilized a split firefly luciferase complementation strategy.
• Engineered a biosensor where luciferase fragments are linked by Raf-1, which changes conformation upon GTP-Ras binding.
• Validated the biosensor's performance in living colorectal cancer cells and in vivo models.

Main Results:

• The developed biosensor demonstrated robust and sensitive reporting of dynamic Ras activity.
• Bioluminescence intensity directly correlated with endogenous Ras activity levels.
• The sensor provided a faster, simpler, and more sensitive alternative to traditional pull-down assays.

Conclusions:

• The novel bioluminescent biosensor enables effective in vivo imaging of Ras activity.
• This technology facilitates preclinical evaluation of cancer therapies targeting the Ras pathway.
• The biosensor is a valuable tool for drug discovery and personalized medicine in oncology.