Structural characterization of a cytosine-rich potential quadruplex forming sequence in the EGFR promoter

I-motifs are tetra-helixes that may form in cytosine-rich strands. They are based on cytosine-cytosine(+) base pairs that require the N3 hemi-protonation of the nucleobases, and therefore, the stability of these non-canonical DNA arrangements depends on pH. These structures are promising targets for the development of new cancer therapies since they are enriched in the promoters of oncogenes where they can play a role in the regulation of transcription. The proximal promoter of the EGFR oncogene has multiple regions with a significant potential to form such a tetra-helix arrangement. Here, we present the thermodynamic characterization of a C-rich sequence located 37 nucleotides upstream of the transcription starting site of EGFR. We confirmed the ability of this sequence to fold into an I-motif. By applying a global analysis of calorimetric and spectroscopic data, we derived the dependency of the apparent standard Gibbs free energy change associated with the I-motif folding upon temperature and pH. The results showed that, in contrast to in silico prediction, only 4 CC+ base pairs formed while additional GC and TT base pairings were detected in the I-motif. Noteworthy, a single residue mutation at G14 largely shifts the equilibrium toward the formation of multimeric species.