Real-Time Visualization of Isoform-Specific RAF-KRAS Interactions in Living Cells Using FRET-BRET Hybrid Biosensors

Area of Science:

• Molecular Biology
• Cell Signaling
• Biochemistry

Background:

• The RAS-RAF-MEK-ERK pathway (MAPK pathway) is vital for cell functions and often dysregulated in cancer.
• Directly observing KRAS interactions with RAF isoforms in live cells is challenging.

Purpose of the Study:

• To develop and validate a dual-mode biosensor for real-time monitoring of RAF-KRAS interactions in living cells.
• To analyze isoform-specific binding dynamics and evaluate oncogenic KRAS mutants.

Main Methods:

• A dual-mode biosensor platform using FRET and BRET was engineered.
• NanoLuc luciferase was incorporated for enhanced sensitivity and high-throughput applications.
• Oncogenic KRAS mutants and targeted KRAS inhibitors were evaluated.

Main Results:

• Isoform-specific biosensors revealed distinct RAF-KRAS interaction dynamics.
• ARAF-based sensors showed strong and reversible FRET responses.
• Oncogenic KRAS mutants displayed altered basal signals and differential binding; targeted inhibitors showed allele-selective effects.

Conclusions:

• The biosensor platform allows live-cell, quantitative monitoring of RAF-KRAS interactions.
• It facilitates analysis of oncogenic signaling and evaluation of mutation-specific responses to targeted therapies.