Multiscale Conformational Dynamics of DNA Holliday Junctions Modulated by Ion Concentration: Insights from Fluorescence Correlation Spectroscopy and Time‐Resolved Anisotropy

The DNA Holliday junction (HJ) is a central recombination intermediate formed by the association of two double‐stranded DNA molecules during DNA repair and genetic recombination, with divalent cations such as Mg 2+ playing a critical role in stabilizing its structure. However, the role of Mg 2+ in governing single‐molecule conformational dynamics of DNA Holliday junctions remains unresolved. Here, we use fluorescence correlation spectroscopy (FCS) to quantify microsecond‐timescale conformational fluctuations and diffusion of DNA HJ. From the FCS measurements, it has been observed that increasing Mg 2+ concentration gives rise to three resolvable microsecond‐scale dynamic modes in DNA Holliday junctions, attributable to fast Cy3 dye motion (~1 µs), intermediate branch flexibility (~50 µs), and slower large‐scale conformational rearrangements (~120 µs). These dynamic changes are accompanied by a 2.2‐fold reduction in the diffusion coefficient and a decreased hydrodynamic radius, indicating enhanced structural compactness. Single‐molecule time‐resolved fluorescence anisotropy shows no significant change in rotational correlation times from 100 mM to 1.5 M Mg 2+ , ruling out oligomerization, while MSD analysis confirms reduced displacement consistent with monomeric Brownian diffusion. By directly linking Mg 2+ ‐dependent structural compaction to microsecond‐scale dynamics, this work reveals fundamental physical principles governing the behavior of recombination intermediates at the single‐molecule level.