Fusion Dynamics and\nSize-Dependence of Droplet Microstructure\nin ssDNA-Mediated Protein Phase Separation

Biomolecular condensation involving proteins and nucleic\nacids\nhas been recognized to play crucial roles in genome organization and\ntranscriptional regulation. However, the biophysical mechanisms underlying\nthe droplet fusion dynamics and microstructure evolution during the\nearly stage of liquid–liquid phase separation (LLPS) remain\nelusive. In this work, we study the phase separation of linker histone\nH1, which is among the most abundant chromatin proteins, in the presence\nof single-stranded DNA (ssDNA) capable of forming a G-quadruplex by\nusing molecular simulations and experimental characterization. We\nfound that droplet fusion is a rather stochastic and kinetically controlled\nprocess. Productive fusion events are triggered by the formation of\nssDNA-mediated electrostatic bridges within the droplet contacting\nzone. The droplet microstructure is size-dependent and evolves driven\nby maximizing the number of electrostatic contacts. We also showed\nthat the folding of ssDNA to the G-quadruplex promotes LLPS by increasing\nthe multivalency and strength of protein–DNA interactions.\nThese findings provide deep mechanistic insights into the growth dynamics\nof biomolecular droplets and highlight the key role of kinetic control\nduring the early stage of ssDNA–protein condensation.