HS‐AFM Reveals Hierarchical RNA Folding Driving Condensate Assembly and Material States

Biomolecular condensation is a fundamental mechanism for organizing intracellular components without membranes. 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 high-speed atomic force microscopy (HS-AFM), this study characterizes RNA condensation dynamics with nanoscale 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 liquid-like poly A condensates from solid-like condensates 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.