Effect of Potassium Concentration on Triplex Stability under Molecular Crowding Conditions

The properties of non-canonical DNA structures, like G-quadruplexes and triplexes, change under cell-mimicking molecular crowding conditions relative to dilute aqueous solutions. The analysis of environmental effects on their stability is crucial since they play important roles in gene expression and regulation. In this study, three intramolecular and intermolecular triplex-forming sequences of different C⁺_*G-C triplet content (_*: Hoogsteen base pair; - : Watson-Crick base pair) were designed and their stability measured in the absence and presence of a crowding agent with different K⁺ concentrations. In dilute solution, the stability of the triplexes was reduced by decreasing the concentration of KCl. This reduction became smaller as the number of C⁺_*G-C triplets increased. Under molecular crowding conditions, Watson-Crick base pairs and Hoogsteen base pairs were destabilized and stabilized, respectively. Interestingly, with lower KCl concentrations (≤1 M), the destabilization of the triplexes due to reduction of KCl concentration was significantly smaller than in dilute solutions. In addition, the C⁺_*G-C content had greater influence on triplex stability under molecular crowding conditions. Our work provides quantitative information about the effects of K⁺ concentration on triplex stability under molecular crowding conditions and should further our understanding of the function and regulation of triplexes in bioprocesses.