Destabilization of i-motif by submolar concentrations of a monovalent cation

Counterions are crucial for self-assembly of nucleic acids. Submolar monovalent cations are generally deemed to stabilize various types of base pairs in nucleic acids such as Watson-Crick and Hoogsteen base pairs via screening of electrostatic repulsion. Besides monovalent cations, acidic pH is required for i-motif formation because protons facilitate pairing between cytosines. Here we report that Li(+) ions destabilize i-motif, whereas other monovalent cations, Na(+) and K(+), have the usual stabilizing effect. The thermodynamics data alone, however, cannot reveal which mechanism, enhanced unfolding or suppressed folding or both, is responsible for the Li(+)-induced destabilization. To gain further insight, we examined the kinetics of i-motif. To deal with slow kinetics of i-motif, we developed a method dubbed HaRP to construct a long FRET time trace to observe a sufficient number of transitions. Our kinetics analysis shows clearly that Li(+) ions promote unfolding of i-motif but do not hinder its folding, lending strong support for our hypothesis on the origin of this unusual effect of Li(+). Although the subangstrom size of Li(+) ions allows them to infiltrate the space between cytosines in competition with protons, they cannot adequately fulfill the role of protons in mediating the hydrogen bonding of cytosine pairs.