Cytosine-rich oligonucleotides incorporating a non-nucleotide loop: A further step towards the obtainment of physiologically stable i-motif DNA

i-Motifs, also known as i-tetraplexes, are secondary structures of DNA occurring in cytosine-rich oligonucleotides (CROs) that recall increasing interest in the scientific community for their relevance in various biological processes and DNA nanotechnology. This study reports the design of new structurally modified CROs, named Double-Ended-Linker-CROs (DEL-CROs), capable of forming stable i-motif structures. Here, two C-rich strands having sequences d(AC 4 A) and d(C 6 ) have been attached, in a parallel fashion, to the two linker's edges by their 3′ or 5′ ends. The resulting DEL-CROs have been investigated for their capability to form i-motif structures by circular dichroism, poly-acrylamide gel electrophoresis, HPLC–size-exclusion chromatography, and NMR studies. This investigation established that DEL-CROs could form more stable i-motif structures than the corresponding unmodified CROs. In particular, the i-motif formed by DEL-5′-d(C 6 ) 2 resulted stable enough to be detected even at near physiological conditions (37 °C, pH 7.0). The results open the way to developing pH-switchable nanocarriers and aptamers based on suitably functionalized DEL-CROs. • New i-motif DNA structures incorporating a non-nucleotide loop (DEL linker) are presented. • The DEL linker allowed to obtain very stable i-motif structures at pH 4.5. • One of the synthesized DEL-i-motif structures proved stable even at pH 7.0. • DEL-i-motif structures can form higher-order aggregates at pH 4.5.