Characterization of the Interaction of Known G‐quadruplex Ligands With a Minimal i‐Motif Structure

Peculiar genomic regions are prone to evolve transiently from double-helix DNA to noncanonical secondary structures in response to physiological stress. Particularly interesting are G-rich genomic sites that are known to fold into G-quadruplexes (G4s) while their complementary C-rich strands can potentially arrange into i-Motifs (iMs). Indeed, both may interfere with the regulation of biological processes, including the expression of oncogenes. While G4s have been extensively studied as potential therapeutic targets, iMs were long neglected due to the acidic conditions thought necessary for their stability. Nevertheless, their proven existence within cells has prompted interest in dual G4/iM ligands. Although recent studies suggest that G4 ligands modulate iM stability, elucidating the underlying structural interfaces requires more in-depth investigations. In this work, we employed NMR to screen four common G4 ligands against a resolved minimal intramolecular iM. By combining UV-vis, CD, and NMR, we characterized the interaction with the most promising ligand, TMPyP4. Our findings highlight a specific structural domain as the preferential binding site for TMPyP4, with the ligand-DNA interactions coupled to a stepwise unfolding of the iM. These results provide structural insights for the rational design of ligands that modulate iM stability and related biological outcomes.