A Screening Protocol for Exploring Loop Length Requirements for the Formation of a Three Cytosine‐Cytosine+ Base‐Paired i‐Motif

DNA sequences containing at least four runs of repetitive cytosines can fold into tetra‐helical structures called i‐Motifs. The interest in these DNA secondary structures is increasing due to their therapeutical and technological applications. Still, limited knowledge of their folding requirements is currently available. We developed a novel step‐by‐step pipeline for the systematic screening of putative i‐Motif forming model sequences. Focusing on structures comprising only three cytosine‐cytosine+ base pairs, we investigated what are the minimal lengths of the loops required for formation of an intra‐molecular i‐Motif. Our data indicate that two and three nucleotides are required to connect the strands through the major and the minor grooves of the i‐Motif, respectively. Additionally, they highlight an asymmetric behavior according to the distribution of the cytosines. Specifically, no sequence containing a single cytosine in the first and third run was able to fold into intra‐molecular i‐Motifs with the same stability of those formed when the first and the third run comprise two cytosines. This knowledge represents a step forward to the development of prediction tools for the proper identification of biologically functional i‐Motif as well as for the rational design of these secondary structures as technological devices.