We report here the solution structure of several repetitive DNA sequences containing d(TCGTTCCGT) and related repeats. At physiological pH, these sequences fold into i-motif like quadruplexes in which every two repeats a globular structure is stabilized by two hemiprotonated C:C base pairs, flanked by two minor groove tetrads resulting from the association of G:C or G:T base pairs. The interaction between the minor groove tetrads and the nearby C:C base pairs affords a strong stabilization, which results in effective pH values above 7.5. Longer sequences with more than two repeats are able to fold in tandem, forming a rosary bead-like structure. Bioinformatics analysis shows that these sequences are prevalent in the human genome, and are present in development-related genes.

We report here the three-dimensional structure of an i-motif/duplex junction, determined by NMR methods at neutral pH. By including a minor groove tetrad at one side of the C:C + stack of a monomeric i-motif, and a stem/loop hairpin at the other side, we have designed stable DNA constructs in which i-DNA and B-DNA regions coexist in a wide range of experimental conditions. This study demonstrates that i- and B-DNA are structurally compatible, giving rise to a distinctive fold with peculiar groove shapes. The effect of different residues at the i-motif/duplex interface has been explored. We also show that these constructs can be adapted to sequences of biological relevance, like that found in the promoter region of the KRAS oncogene.

We study here a DNA oligonucleotide having the ability to form two different i-motif structures whose relative stability depends on pH and temperature. The major species at neutral pH is stabilized by two C:C+ base pairs capped by two minor groove G:C:G:C tetrads. The high pH and thermal stability of this structure are mainly due to the favorable effect of the minor groove tetrads on their adjacent positively charged C:C+ base pairs. At pH 5, we observe a more elongated i-motif structure consisting of four C:C+ base pairs capped by two G:T:G:T tetrads. Molecular dynamics calculations show that the conformational transition between the two structures is driven by the protonation state of key cytosines. In spite of large conformational differences, the transition between the acidic and neutral structures can occur without unfolding of the i-motif. These results represent the first case of a conformational switch between two different i-motif structures and illustrate the dramatic pH-dependent plasticity of this fascinating DNA motif.