Active Protein–RNA Polyelectrolyte Condensates: Structure, Rheology, and Non-Equilibrium States

Biomolecular condensates formed by protein–RNA assemblies behave as soft polyelectrolyte materials whose structure and dynamics emerge from interactions encoded in both folded domains and intrinsically disordered regions (IDRs). Yet the non-equilibrium remodeling that defines condensates in cells remains challenging to isolate, control, or engineer in vitro. To address this gap, we developed a minimal peptide–RNA–ubiquitin fusion system that disentangles the contributions of folded domains, charge topology, and enzymatic energy input. Using this platform, we show that mutations confined to the folded ubiquitin domain, while leaving the R/G-rich IDR sequence unchanged, can reprogram condensate phase behavior, viscoelasticity, and internal flow patterns. Electrostatic changes in the folded domain reorganize long-range interactions within the condensed network, demonstrating that condensate properties are not dictated solely by IDR sequence grammar—folded-domain charge architecture can also modulate the emergent material state of protein–RNA condensates. Introducing enzymatic activity within these condensates produces sustained non-equilibrium structures, directional flows, and spatiotemporal patterning, creating a synthetic active-matter system in which chemical fuel flux drives dynamic reorganization. Overall, our work establishes a quantitative, tunable model that bridges living, energy-dissipating condensates with reconstituted systems. These findings outline emerging design principles for active protein–RNA polyelectrolyte materials, providing a framework for probing life-like phase transitions and engineering programmable condensate-based technologies.