Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization

Given the increasing interest in i-motif structures, obtaining such structures as well as detailed structural information under physiological conditions have become hot topics in the structural biology field. In this context, the main objectives of this thesis are focused on the design and detailed characterization of several oligonucleotide sequences that may form stable i-motif structures at neutral pH. The starting point is the mini i-motif structure, extensively studied in the research group that exhibits unusual high pH and thermal stability. These are dimeric structures stabilized by the formation of two hemiprotonated C:C+ base pairs capped at both ends by minor groove G:T:G:T tetrads. With the aim of getting deeper insights in this type of structures and enhance their stability at physiological conditions, different approaches were followed. A first strategy consisted in the incorporation of a neutral analogue of protonated cytidine (pseudoisocytidine, psC) occupying specific positions of the motif. The 3H-tautomer of psC, thanks to the extra hydrogen-bond donor, can form neutral base pairs completely isomorphic to hemiprotonated C:C+ pairs. psC was incorporated in different positions of dimeric mini i-motifs and in the telomeric sequence (HT0). The effect of the incorporation of psC depends on its position in the structure, being in most cases destabilizing. Neutral psC:C base pairs stabilize i-motifs at neutral pH, but the stabilization only occurs when psC:C base pairs are located at the ends of intercalated C:C+ stacks. Structural and stability data on the incorporation of pseudocytidine in i-motifs suggest that positively charged base pairs in the core of the structure are necessary to stabilize this non-canonical DNA structure. A second approach focused on exploring the compatibility of i-motif structures with other reported minor groove tetrads. The effect of different minor groove tetrads in i-motifs was studied in the context of short linear and cyclic oligonucleotides, affording dimeric mini i-motifs, but also in longer sequences that may form monomeric mini i-motif structures. The results show that the mini i-motif is compatible with different type of minor groove tetrads and a stability ranking could be established: G:C:G:T ≥ G:C:G:C >> G:T:G:T, exhibiting monomeric structures enhanced stability. Interestingly, a consensus sequence was outlined based on the results obtained for the set of mini i-motif-forming sequences. The mapping of t