Homotypic RNA clustering accompanies a liquid-to-solid transition inside the core of multi-component biomolecular condensates

RNA-driven condensation plays a central role in organizing and regulating ribonucleoprotein granules within cells. Disruptions to this process, such as the aberrant aggregation of repeat-expanded RNA, are associated with numerous neurological disorders. Here, we report that irreversible RNA aggregation is facilitated within multicomponent protein–nucleic acid condensates. Using time-lapse confocal fluorescence microscopy, we tracked the growth of RNA clusters as a function of condensate age and found that RNA aggregation is driven by RNA percolation, proceeding via a transition from intramolecular to intermolecular RNA–RNA interactions. For GC-rich repeat RNAs, the size of RNA clusters increases, and the timescale of cluster formation decreases with the number of repeat units. Utilizing nanorheology, optical tweezer-based droplet fusion assays, and high-resolution confocal fluorescence imaging, we uncovered that RNA clustering drives a liquid-to-solid phase transition in the condensate core, leading to the emergence of multiphasic condensate structures. Multivalent RNA-binding cofactors, such as antisense oligonucleotides and RNA-binding proteins, that compete with homotypic RNA–RNA interactions can raise the activation energy barrier for RNA clustering, thereby acting as inhibitors of intra-condensate RNA aggregation. The insights gained from this study provide a complementary perspective on the role of multicomponent biomolecular condensates in regulating aberrant RNA self-assembly in living cells.