Charge of Nanoconfinement Modulates i-Motif Formation at Physiological pH

The role of nanoconfinement in regulating the folding of cytosine-rich DNA into i-motif structures under physiological pH conditions remains poorly understood. Here, we investigate the effect of confinement charge on i-motif formation and stability by using positively and negatively charged reverse micelles (RMs) with varying water contents at neutral pH. The data reveal that positively charged RMs markedly enhance both folding propensity and thermal stability, whereas negatively charged RMs disfavor the folding at the same physiological pH. The positively charged confinement preserves a local environment that is conducive to cytosine protonation. In contrast, negatively charged confinement significantly reduces the effective acidity, shifting the i-motif formation to more acidic conditions. The enhanced thermal stability of the i-motif in positively charged RM is attributed to restricted water motion and excluded-volume effects within the confined space, which diminish with an increasing water pool size. Overall, our findings highlight the critical role of the charge of confinement in modulating i-motif formation and stability, providing insight into the behavior of cytosine-rich nucleic acids in biologically relevant confined environments.