Reversible Nucleolar Complex Coacervation by Short Cationic Peptides.

Biomolecular condensates formed through liquid-liquid phase separation play central roles in intracellular organization and regulation. Replicating such dynamic compartmentalization using minimal synthetic components remains exceptionally challenging inside living cells. Here, we report short cationic peptides that undergo directed complex coacervation in living cells through preferential interactions with endogenous polyanionic biomolecules. Although the peptides were designed to contain a mitochondrial targeting motif, the Arg residues and cellular RNA guide the supramolecular interactions toward selective enrichment of liquid-like coacervates in nucleolar regions. In vitro studies reveal that polymeric RNA mimics promote coacervation far more efficiently than ATP, establishing RNA-peptide interactions as the principal driving force. In cells, nucleolar complex coacervates form rapidly and exhibit liquid-like behavior with fast molecular exchange. Importantly, the assemblies are transient and reversible: sustained peptide supply maintains the condensed state, whereas substrate depletion triggers droplet dissolution and recovery of cellular function. These findings demonstrate that endogenous biopolymer distributions can guide and participate in the formation of synthetic coacervates with minimalistic peptides, achieving reversible reorganization of intracellular components. More broadly, this work provides a framework for engineering synthetic coacervates with nonequilibrium, life-like features that operate in direct exchange with living cellular environments.