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International audience ; i-Motifs of DNA (hereafter, i-DNA), known in vitro for nearly three decades, are unusual, four-stranded structures, in which cytosines are intercalated via a stack of hemi-protonated C–C base pairs (CH+:C) (Fig. 1A, B). Some of these structures have been well characterized in vitro and, because i-DNA may mirror other four-stranded G-rich structures (G-quadruplexes) present in gene promoters or at telomeres, their biological relevance is being investigated.<br>However, our knowledge about i-DNA biology is still limited: the main challenges in this regard being the strong pH dependency, flexibility, and polymorphism of i-DNA, that introduce potential bias into studies. In particular, low-pH conditions that are required for the formation of i-DNA can lead to the protonation of many ligands (including small molecules or proteins), strongly increasing their non-specific nucleic acid binding. In this context, we have developed a peptide-DNA conjugate (Fig. 1C) being able to fold into a stable i-motif at room temperature and, most importantly, at near-neutral pH.1<br>The stabilized mimic of the i-motif adopted by the telomeric sequence was used to study the interactions with already reported ligands (TMPyP4, mitoxantrone, IMC-48, berberine, etc) at physiologically relevant pH by Bio-Layer Interferometry (BLI) and CD. We demonstrated that none of the reported ligands were shown to discriminate between folded and unfolded i-motif structures.2<br>In conclusion, the constrained i-motif reveals to be a powerful tool for studying i-motif structure.
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