Combined All-Atom Simulations and Biophysical Assays Uncover Loop-Driven Stabilization in the HRAS i-motif

I-motifs are noncanonical DNA secondary structures stabilized by hemiprotonated C +:C base pairs. Their intrinsic flexibility, conformational heterogeneity, and sensitivity to environmental conditions often hinder structural characterization. Here, all-atom simulations, combined with biophysical experiments, were used to characterize the structure of the i-motif monomer formed by the HRAS gene promoter (iHRAS), a member of the RAS proto-oncogene family. Our results reveal that iHRAS exhibits intricate conformational behavior characterized by multiple interconverting states. The core i-motif is stabilized by a protective G:G cap, a recurrent i-motif-stabilizing factor, on one side, while the C +:C base pairs content on the other side is variable. Structural heterogeneity is most pronounced in loops, which sample several base-exposed states aided by K + ion binding. These findings contribute to a deeper understanding of the i-motif structure and dynamics.