Unified Mechanistic Landscape of Protein and Protein-RNA Phase Separation: Implications in p53 cancer and Viral Replication

Liquid–liquid phase separation (LLPS) lies behind biomolecular condensation, the molecular basis for  membraneless organelles.   Condensates orchestrate essential biochemistry yet can evolve into pathogenic species.  By combining time-resolved kinetics, single-droplet imaging, dynamic light scattering and, for the viral system, cryo-electron microscopy, we mapped mechanistic landscapes for two archetypal condensation systems: full-length p53 (homotypic-protein) and a SARS CoV2 nucleoprotein (heterotypic, protein-RNA).  At sub-micromolar concentrations, p53 nucleates homotypic droplets within seconds; sequential tetramer addition yields uniform ~200 nm proto-droplets that fuse into micron-scale coacervates, which in over 10³ s evolve into hydrogel-like clusters with strong thioflavin-T reactivity, and eventually branch into fiber-like structures.  DNA dissolves these condensates stoichiometrically, whereas the HPV oncoprotein E2 remodels late-stage aggregates back into fluid droplets, revealing druggable kinetic checkpoints for tumor-suppressor rescue.  In the case of the viral nucleocapsid, sub-second binding of two RNAs to a dimeric RNP protomer initiates crystallization-like primary nucleation that outpaces secondary events; weaker RNA contacts then drive multistep growth into filaments and ~600 nm nanoclusters within minutes.  Cryo-EM unveils concentric layers of RNP filaments reminiscent of nucleocapsid packing, and the nanoclusters liquefy into droplets that partially fuse and age into protein-RNA hydrogels, putative scaffolds of viral factories in infected cells.  Despite distinct molecular grammars (tetramer stacking versus RNA-templated filamentation) both systems display rapid primary nucleation, hierarchical growth and time-dependent maturation. Mapping their rate-limiting steps yields a unified kinetic framework for condensate biology and pinpoints intervention nodes for aggregation rescuing cancer therapeutics and antiviral strategies that disrupt viral factory assembly.