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 (iMs). 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 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 identification of biologically functional iMs, as well as for the rational design of these secondary structures as technological devices.

Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications.

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

pH-driven conformational switch between non-canonical DNA structures in a C-rich domain of EGFR promoter.

EGFR is an oncogene that encodes for a trans-membrane tyrosine kinase receptor. Its mis-regulation is associated to several human cancers that, consistently, can be treated by selective tyrosine kinase inhibitors. The proximal promoter of EGFR contains a G-rich domain located at 272 bases upstream the transcription start site. We previously proved it folds into two main interchanging G-quadruplex structures, one of parallel and one of hybrid topology. Here we present the first evidences supporting the ability of the complementary C-rich strand (EGFR-272_C) to assume an intramolecular i-Motif (iM) structure that, according to the experimental conditions (pH, presence of co-solvent and salts), can coexist with a different arrangement we referred to as a hairpin. The herein identified iM efficiently competes with the canonical pairing of the two complementary strands, indicating it as a potential novel target for anticancer therapies. A preliminary screening for potential binders identified some phenanthroline derivatives as able to target EGFR-272_C at multiple binding sites when it is folded into an iM.