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 open k₎₄₍) and closing (k close k₂₋₎ₒ₄) of individual base pairs under MCE. While MCE preserved the overall triplex structure, it significantly altered the lifetimes of the closed (τ closed = 1 / k open ₂₋₎ₒ₄₃=1/k₎₄₍) and open (τ open = 1 / k close ₎₄₍=1/k₂₋₎ₒ₄) states, and the standard Gibbs energy difference (Δ G open ○ = - RT ln K open G₎₄₍^ =- RT K₎₄₍, K open = k open k close = τ open τ closed K₎₄₍=k₎₄₍k₂₋₎ₒ₄=₎₄₍₂₋₎ₒ₄₃). Notably, we observed distinct changes in BPOC dynamics of the third strand. Ficoll PM 70, which enhances excluded-volume effects, lengthened both τ closed ₂₋₎ₒ₄₃ and τ open ₎₄₍ across most base pairs, with a larger increase in τ closed ₂₋₎ₒ₄₃, resulting in an increased Δ G open ○ G₎₄₍^. In contrast, PEG 200, which reduces water activity, increased τ open ₎₄₍ for most base pairs except for those near the helix center, while PEG 200 either decreased or increased τ closed ₂₋₎ₒ₄₃ only slightly. As a result, the τ open τ closed ₎₄₍₂₋₎ₒ₄₃ ratio increased, resulting in a decreased Δ G open ○ G₎₄₍^. These findings deepen mechanistic understanding of molecular-crowding effects on DNA-triplex stability and dynamics, providing new cellular regulatory perspectives.
Sakamoto et al. (Wed,) studied this question.