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 struc-tures called i-Motifs (iMs). The interest in these DNA secondary structures is increasing due to their therapeut-ical and technological applications. Still, limited knowl-edge of their folding requirements is currently available. We developed a novel step-by-step pipeline for the systematic screening of putative iM-forming model sequences. Focusing on structures comprising only three cytosine-cytosine+ base pairs, we investigated what the minimal lengths of the loops required for formation of an intra-molecular iM are. Our data indicate that two and three nucleotides are required to connect the strands through the minor and majorgrooves of the iM, 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 iMs with the same stability of those formed when the first and the third run comprise two cytosines. This knowledge represents a step forward toward the development of prediction tools for the proper identi-fication of biologically functional iMs, as well as for the rational design of these secondary structures as techno-logical devices.