Twisting tetraplex DNA: A strand dynamics regulating i-motif function in diverse molecular crowding environments

Intercalated motif (i-motif) tetraplex DNA plays a crucial role in gene expression and diseases. However, due to the limited number of i-motif binding proteins in human cells, the chemical mechanisms regulating i-motifs within cell remain currently unknown. Thus, molecular environment should have a main factor to control i-motif formation and functions in cells. Here, we systematically investigated the stability and functions of i-motif DNAs by using various polyethylene glycols (PEGs) and oligoethylene glycols (OEGs) that mimicked diverse cellular crowding environments. We found that the human telomere i-motif was significantly stabilized by PEGs and OEGs having six or more ethylene glycol units, whereas it was destabilized by those having less than six units. As these stabilization effects coincided with the drastic changes in hypochromicity by i-motif helixes, we quantitatively validated these effects through changes in solution properties and by assessing the twisting of the tetraplex structure using nuclear magnetic resonance (NMR) and molecular dynamics simulations. Furthermore, cosolute-induced twisting dynamics controlled by different cosolutes changed the activation energy barrier of replication by a twofold magnitude along the i-motif-forming DNAs. Our findings indicate that regulatory mechanisms underlying the biological roles of i-motifs across different cellular phases may exist by molecular environments.