Fusion dynamics and size-dependent droplet microstructure in ssDNA mediated protein phase separation

Biomolecular cocondensation involving proteins and nucleic acids has been recognized to play crucial roles in genome organization and transcriptional regulation. However, the biophysical mechanisms underlying the fusion dynamics and microstructure evolution of the droplets during the early stage of liquid-liquid phase separation (LLPS) remain elusive. In this work, we study the phase separation of linker histone H1, which is among the most abundant chromatin proteins, in the presence of single-stranded DNA (ssDNA) capable of forming G-quadruplex structures by using residue-resolved molecular dynamics simulations. Firstly, we uncovered a kinetic bottleneck step in the droplet fusion. Productive fusion events are triggered by the formation of ssDNA mediated electrostatic bridge within the contacting zone of two droplets. Secondly, the simulations revealed a size-dependence of the droplet microstructure and stoichiometry. With droplet growth, its microstructure evolves as driven by the maximization of the electrostatic contacts between ssDNA and the highly charged segment of H1. Finally, we showed that the folding of ssDNA to G-quadruplex promotes LLPS by increasing the multivalency and strength of protein-DNA interactions. These findings provided new mechanistic insights into the microstructure and growth dynamics of the biomolecular droplets formed during the early stage of the ssDNA-protein cocondensation.