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

The RAS-RAF-MEK-ERK signaling cascade is a central component of the mitogen-activated protein kinase (MAPK) pathway, regulating cell proliferation, differentiation, and survival, and is frequently dysregulated in Cancer. Despite extensive biochemical characterization, direct observation of KRAS interactions with Raf isoforms in living cells remains limited. To overcome this limitation, a dual-mode biosensor platform is presented that enables real-time monitoring of RAF-KRAS interactions through both fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET). Isoform-specific biosensors reveal distinct interaction dynamics, with ARAF-based sensors exhibiting the strongest and most reversible FRET responses. Importantly, incorporation of NanoLuc luciferase into the hybrid biosensor preserves FRET sensitivity while introducing a luminescent BRET mode suitable for high-throughput and low-background applications. Evaluation of oncogenic KRAS mutants indicates elevated basal FRET signals and differential binding profiles across Raf isoforms. Pharmacologic profiling further demonstrates allele-selective inhibition, with mutant-specific FRET and BRET responses observed upon treatment with targeted KRAS inhibitors. This biosensor platform enables live-cell, real-time, and quantitative monitoring of RAF-KRAS interactions, facilitating the analysis of oncogenic signaling dynamics and the evaluation of mutation-specific responses to targeted therapies under physiologically relevant conditions.

BRET; FRET; KRAS; RAF; biosensor; live‐cell imaging.