HS-AFM visualizes stepwise condensation dynamics in RNA-driven LLPS

Liquid-liquid phase separation (LLPS) is a fundamental mechanism of intracellular biomolecular condensation formation, involving both proteins and RNAs. While RNA self-assembly has been implicated in condensate formation, the molecular mechanisms of RNA-driven condensation remain unclear. To investigate RNA’s role in LLPS, we employed high-speed AFM (HS-AFM) to observe real-time, single-molecule dynamics of RNA condensation in near-physiological conditions. HS-AFM imaging revealed that RNA-driven condensation occurs through a multistep process: intramolecular ( cis ) interactions first promote intermolecular RNA clustering, and these small clusters subsequently assemble into larger condensates, ultimately forming mature RNA condensates. Our data provides the first single-molecule visualization of RNA-driven LLPS formation, demonstrating its stepwise progression from the folding of single RNA molecules to condensate assembly. This study highlights HS-AFM as a powerful tool for visualizing the molecular transitions underlying RNA-driven phase separation, allowing real-time observation of RNA folding events and condensate formation dynamics.