We report here on microcalorimetric studies of DNA/Drug systems exhibiting multiple equilibria with respect to both DNA conformation and ligand binding. G-quadruplex structures are known to form in the telomere and the promoter regions of several oncogenes. This study focuses on an abbreviated portion of the promoter region of the c-MYC oncogene. We have conducted studies on the separate G and C-rich strands, as well as the complete c-MYC silencer element (NHE-III1). Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry were used to determine thermodynamic parameters for quadruplex or i-motif DNA/TMPyP4 interactions. Circular Dichroism was used to confirm quadruplex and i-motif structures in solution and observe structural changes during drug binding. ITC data demonstrate unusually complex binding between TMPyP4 and model c-MYC quadruplex and i-motif structures. A particularly interesting observation is that TMPyP4 binding to the c-MYC i-motif is comparable to TMPyP4 binding to the c-MYC G-quadruplex with respect to affinity and stoichiometry. Two or three distinct binding modes are observed depending on salt concentration and temperature. Dissecting the free energy change for each binding mode into enthalpic and entropic contributions yields thermodynamic profiles consistent with both exterior “groove like” binding and interior “intercalation like” binding. While previous studies have focused on the G-quadruplex as a target for cancer chemotherapy, this work supports the i-motif as an additional bio-target for new anti cancer drug compounds. It is even possible that i-motif drug binding is responsible for some of the therapeutic effect of a quadruplex interactive drug (QIA) entering clinical trials.
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