Membraneless organelles play a key role in homeostasis and regulation of multiple cellular processes. Understanding the molecular mechanisms that lead to biological liquid-liquid phase separation is central to elucidate how biomolecular condensates assemble inside cells. In this study we investigate an archetypal system composed of homopolymeric polyuridylic acid chains (PolyU) and short cationic peptides of varying lengths and charges. Using both static and dynamic light scattering, complemented with confocal microscopy, we probed increasing peptide concentrations in a dilute RNA solution, mapping the condensation transition, quantifying the molecular partition in the two phases, and characterizing the behavior of the polyelectrolytes mixtures as a function of the total PolyU/peptide ratio across the phase boundary. Our experiments reveal a threshold mechanism in which below a critical peptide concentration the system remains homogeneous and composed of independent peptide-decorated PolyU coils. In such complexes, peptides form intra-RNA contacts, inducing a compaction of the polymer. Once the critical peptide concentration is exceeded, phase separation occurs, producing a dense phase where peptide-mediated inter-RNA contacts are possible, allow a relaxation of the conformational constraint imposed in the dilute phase, as indicated by the experimentally observed sticky reptation dynamics. We also present a phenomenological numerical model reproducing the experimentally observed phase transition and molecular partitioning, interpreting the early stages of biomolecular condensate nucleation as regulated primarily by an entropic balance of polyelectrolytes interactions, conformations and distribution. Our results show that the loss in RNA conformational entropy induced by peptide decoration is large enough to drive the phase transition, suggesting a mechanism that might be of general relevance in cellular complex coacervation.
Inzani et al. (2026) studied this question.