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.