Spatiotemporal dynamics of protamine-DNA condensation revealed by high-speed atomic force microscopy

Abstract

Protamines (PRMs) play a crucial role in sperm chromatin condensation, replacing histones to form nucleo-PRM structures, specifically PRM-DNA complexes. Despite their importance in reproduction, the detailed mechanisms underlying PRM-mediated DNA condensation have remained elusive. In this study, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the real-time binding dynamics of PRM to DNA under physiological conditions. Our HS-AFM observations reveal that PRM insertion initiating the formation of DNA coils. Further, we observed a heterogeneous spatial distribution of PRM-induced DNA looping. With continuous PRM addition, DNA progresses through a series of folding transitions, forming coiled-like structures that evolve into clockwise spirals, rod-shaped intermediates, and ultimately toroid-like nanostructures. Based on these real-time observations, we propose the CARD (Coil-Assembly-Rod-Doughnut) model to describe the stepwise process of toroid formation during DNA condensation. Our findings underscore the versatility of HS-AFM in capturing the spatiotemporal dynamics of PRM-DNA interactions and provide critical insights into the molecular mechanisms driving PRM-induced chromatin compaction. This study advances our understanding of sperm chromatin architecture and offers a framework for future research into chromatin organization, reproductive biology, and nucleic acid therapeutics.