DNA Local-Flexibility-Dependent Assembly of Phase-Separated Liquid Droplets

TLDR

Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid‑liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. We find that DNA local flexibility—not merely charge density—drives LLPS, that free nucleotide triphosphates enhance LLPS of polypeptide–dsDNA complexes that would otherwise precipitate, and that dsDNA can undergo a secondary liquid‑crystalline phase separation within droplets, underscoring the sequence‑encoded role of DNA flexibility in regulating multicomponent LLPS.

Abstract

Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid-liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. Comparing the phase behavior of different single-stranded DNA as well as double-stranded DNA (dsDNA) sequences that differ in persistence lengths, we find that DNA local flexibility, not simply charge density, determines the LLPS. Furthermore, in a nucleotide- and DNA-dependent manner, free nucleotide triphosphates promote LLPS of polypeptide-dsDNA complexes that are otherwise prone to precipitation. Under these conditions, dsDNA undergoes a secondary phase separation forming liquid-crystalline subcompartments inside the droplets. These results point toward a role of local DNA flexibility, encoded in the sequence, in the regulation and selectivity of multicomponent LLPS in membraneless intracellular organization.