Protamine Drives Liquid and Solid Condensation of DNA and Glycosaminoglycans

Abstract

Phase-separated biomolecular condensates play a crucial role in cellular organization. In the nucleus, phase separation regulates the assembly and function of nuclear proteins and the genome. Here, we demonstrate that protamine, an arginine-rich nuclear protein responsible for promoting double-stranded DNA (dsDNA) compaction in sperm, undergoes phase separation with single-stranded DNA (ssDNA) and induces dsDNA aggregation. The protamine-ssDNA condensates behave as viscoelastic liquid droplets and can be modulated by varying salt concentrations or by treatment with heparin, a glycosaminoglycan polyanion, which we show displaces ssDNA. Moreover, protamine-dsDNA aggregates dissolve upon exposure to heparin, leading to the formation of condensates with a distinct morphology. This observation provides a compelling example of an aggregate-to-condensate transition in polyelectrolyte systems. Notably, direct combination of protamine with heparin results in the formation of similar phase-separated liquid-like droplets, suggesting that heparin can compete with both ssDNA and dsDNA for protamine binding. We performed a comparative analysis using other positively charged proteins and negatively charged glycosaminoglycans to gain insights into the condensation behavior of protamine and heparin. Finally, we leveraged these findings to conduct a proof-of-concept analysis aimed at developing programmable biomolecular condensates for protamine-assisted nucleic acid delivery. Given that polyanionic glycosaminoglycans have been used for sperm capacitation and that DNA fragmentation is a biomarker for infertility in males, our findings offer new insights into the mechanisms of protamine-driven DNA compaction in sperm and its potential implications for reproductive medicine.