DNA order/disorder regulates phase separation and dynamics of peptide/DNA condensates

Synthetic biomolecular condensates, formed through liquid-liquid phase separation (LLPS), are increasingly studied using RNA and DNA polymers as fundamental building blocks. While RNA/peptide LLPS is well characterized, the phase behavior of DNA and its interactions with peptides remain largely unexplored. Inspired by the viral machinery that allows the packaging of large nucleic acid polymers into nanometer-sized compartments—facilitated by specific nucleic acid-amino acid interactions–we investigated how minimalistic peptides modulate the structural order of single-stranded DNA (ssDNA) to regulate condensate formation. We engineered two ssDNA variants—an ordered ssDNA with tRNA-like structure and another largely disordered—and combined experimental and computational approaches to analyze their phase behavior. Our results show that ordered DNA restricts LLPS, whereas disordered DNA enhances condensate density and slows molecular diffusivity. These findings establish a direct link between DNA order/disorder and mesoscale phase separation, expanding our understanding of nucleic acid-driven LLPS and offering new strategies for designing programmable DNA-peptide condensates with tunable properties.