Early Folding Dynamics\nof i‑Motif DNA Revealed\nby pH-Jump Time-Resolved X‑ray Solution Scattering

The i-motif is a pH-responsive cytosine-rich oligonucleotide\nsequence\nthat forms, under acidic conditions, a quadruplex structure. This\ntunable structural switching has made the i-motif a useful platform\nfor designing pH-responsive nanomaterials. Despite the widespread\napplication of i-motif DNA constructs as biomolecular switches, the\nmechanism of i-motif folding on the atomic scale has yet to be established.\nWe investigate the early folding structural dynamics of i-motif oligonucleotides\nwith laser-pulse-induced pH-jump time-resolved X-ray solution scattering.\nFollowing the pH-jump, we observe that the initial random coil ensemble\nconverts into a contracted intermediate state within 113 ns followed\nby further folding on the 10 ms time scale. We reveal the representative\nstructures of these transient species, hitherto unknown, with molecular\ndynamics simulations and ensemble fitting. These results pave the\nway for understanding metastable conformations of i-motif folding\nand for benchmarking emerging theoretical models for simulating noncanonical\nnucleic acid structures.