HS-AFM visualizes stepwise condensation dynamics in RNA-driven LLPS
Liquid-liquid phase separation (LLPS) is a fundamental mechanism of intracellular biomolecular condensation formation. While RNA self-assembly has been implicated in condensate formation, it remains unclear how RNA condensation translates into distinct physical behaviors at the nanoscale. Utilizing HS-AFM, this study characterizes RNA condensation dynamics with single-molecule resolution. Imaging captured the transition from individual RNA folding to intermolecular clustering, ultimately leading to progressive condensate assembly. Beyond morphological description, condensate behavior was further examined through fusion dynamics and mechanical response. Post-fusion shape evolution quantified how merged condensates recover circular morphology over time, providing a dynamic readout of material behavior. Nanomechanical properties were independently assessed through force-curve measurements. Together, these analyses consistently distinguish the droplet-like poly A condensates from fractal-shaped assemblies of total RNA. Taking together, these findings directly link RNA folding dynamics, condensate assembly, and emergent physical properties, establishing a quantitative framework for defining condensate material states at the nanoscale.