Destabilization of i-Motif DNA at Neutral pH by G-Quadruplex Ligands

Numerous studies have been published stressing the importance of finding ligands that can bind specifically to DNA secondary structures. Several have identified ligands that are presented as having specific binding to the G-quadruplex; however, these were not originally tested on the complementary i-motif structure. The i-motif was overlooked and presumed to be irrelevant due to the belief that the hemiprotonated (cytosine⁺-cytosine) base pair at the core of the structure required acidic pH. The pathophysiological relevance of i-motifs has since been documented, as well as the discovery of several genomic sequences, which can form i-motif at neutral pH. Using different biophysical methodologies, we provide experimental evidence to show that widely used G-quadruplex ligands interact with i-motif structures at neutral pH, generally leading to their destabilization. Crucially, this has implications both for the search for quadruplex binding compounds as well as for the effects of compounds reported to have G-quadruplex specificity without examining their effects on i-motif.