Nucleic acids induce protein condensation through charge-charge interaction in Escherichia coli

Understanding how nucleic acids contribute to the physicochemical mechanisms of intracellular protein condensation remains important for elucidating how biomolecular assemblies form in cells. Here, we examined how polyanionic nucleic acids influence charge-charge interaction-driven protein condensation using supercharged proteins with defined net charges. Positively and negatively charged GFP variants (+28 and - 27) were expressed in Escherichia coli, and diverse nucleic acids, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), RNA, and peptide nucleic acid (PNA), were introduced via electroporation. ssDNA oligomers of different lengths (L1-L40) were synthesized to probe charge- and length-dependent effects. ssDNA, dsDNA, and RNA consistently increased condensation of the cationic GFP (+28) by approximately 10%, whereas neither the anionic GFP (-27) nor charge-neutral PNA showed measurable changes. Extending this approach to a natural system, RNA enhanced condensation of the disease-relevant, positively charged hnRNPA1. Molecular dynamics simulations reveal ssDNA mediate interactions between two GFP (+28) moieties through phosphate-lysine/arginine charge-charge interactions, reducing intermolecular distances and stabilizing compact conformations. In contrast, GFP (-27) formed only transient, repulsive complexes with ssDNA. Together, these findings suggest that direct charge-charge complementarity governs nucleic acid-protein co-condensation and identify nucleic acids as regulators of charge-mediated condensation.