Contrasting effects of different molecular crowding environments on base-pair opening/closing dynamics of DNA triplex...

DNA triplexes contribute to genomic instability and gene regulation. To understand how the molecular crowding environment (MCE) inside cells influences the dynamics of DNA triplex structures, we investigated base-pair opening and closing (BPOC) dynamics using crowding agents that mimic intracellular MCE. We report, for the first time, quantitative rate constants for opening ( $k_{\text{open}}$ ) and closing ( $k_{\text{close}}$ ) of individual base pairs under MCE. While MCE preserved the overall triplex structure, it significantly altered the lifetimes of the closed ( ${\tau }_{\text{closed}}=1/k_{\text{open}}$ ) and open ( ${\tau }_{\text{open}}=1/k_{\text{close}}$ ) states, and the standard Gibbs energy difference ( ${\Delta G}_{\text{open}}^{○}=-\mathrm{RT}\ln K_{\text{open}}$ , $K_{\text{open}}=\frac{k_{\text{open}}}{k_{\text{close}}}=\frac{{\tau }_{\text{open}}}{{\tau }_{\text{closed}}}$ ). Notably, we observed distinct changes in BPOC dynamics of the third strand. Ficoll PM 70, which enhances excluded-volume effects, lengthened both ${\tau }_{\text{closed}}$ and ${\tau }_{\text{open}}$ across most base pairs, with a larger increase in ${\tau }_{\text{closed}}$ , resulting in an increased ${\Delta G}_{\text{open}}^{○}$ . In contrast, PEG 200, which reduces water activity, increased ${\tau }_{\text{open}}$ for most base pairs except for those near the helix center, while PEG 200 either decreased or increased ${\tau }_{\text{closed}}$ only slightly. As a result, the $\frac{{\tau }_{\text{open}}}{{\tau }_{\text{closed}}}$ ratio increased, resulting in a decreased ${\Delta G}_{\text{open}}^{○}$ . These findings deepen mechanistic understanding of molecular-crowding effects on DNA-triplex stability and dynamics, providing new cellular regulatory perspectives.