Single molecule tracking reveals nanodomains in biomolecular condensates

Protein-rich biomolecular condensates formed by phase separation ¹ play a crucial role in cellular RNA regulation by the selective recruitment and processing of RNA molecules ² . The functional impact of condensates on RNA biology relies on the residence time of individual RNA molecules within a condensate ³ , which is governed by intra-condensate diffusion ⁴ – the slower or more confined the diffusion, the longer the residence time. However, the spatiotemporal organization of RNA and protein diffusion within a single condensate remains largely unknown due to the challenge of accurately profiling intra-condensate diffusion behaviors down to the single-molecule level. Here we introduced a general condensate-tethering approach that allows single molecule tracking (SMT) of fluorescently labeled proteins and RNAs within non-wetted spherical 3D condensates without the interference of condensate motions. We found that a significant fraction of RNA and protein molecules are locally confined, rather than freely diffusive, within a model condensate formed by full-length, tag-free, RNA-binding protein Fused-in-Sarcoma (FUS), known for its critical roles in RNA biology under both physiological and pathological conditions ⁵ . Dynamic Point Accumulation for Imaging in Nanoscale Topography (PAINT) ⁶ reconstruction further revealed that RNA and proteins are confined to distinct slow-moving nanometer-scale regions, termed nanodomains, within a single condensate. Remarkably, nanodomains affect the diffusion but not the density of the confined biomolecules, supporting local percolation ^1,7 rather than a secondary phase separation within the condensate as their origin. Beyond their regulatory roles on RNA residence time, nanodomains engender both elevated local connectivity and altered chemical environment, a prerequisite for pathological liquid-to-solid transition of FUS during aging. In summary, our study uncovers a patterned spatial organization of both protein and RNA molecules within a single condensate, revealing distinct diffusion dynamics that affect molecular retention time and interactions underlying cellular function and pathology.