Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA

Background: Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in> 80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence. Methods: UV–Vis, FRET melting Assay, Isothermal Titration Calorimetry, Time-resolved Fluorescence lifetime, T-Jump and Molecular Dynamics. Results: TMPyP4 yields two different complexes with two Tel22 telomeric conformations in the presence of Na+ or K+. T-Jump kinetic experiments show that the rates of formation and dissociation of these complexes in the ms time scale differ by one order of magnitude. MD simulations reveal that, in K+ buffer, “hybrid 1” conformation yields kinetic constants on interaction with TMPyP4 one order lower than “hybrid 2”. The binding involves π–π stacking with external loop bases. Conclusions: For the first time we show that for a particular buffer TMPyP4 interacts in a kinetically different way with the two Tel22 conformations even if the complexes formed are thermodynamically indistinguishable. General significance: G-quadruplexes, endowed with technological applications and potential impact on regulation mechanisms, define a new research field. The possibility of building different conformations from same sequence is a complex issue that confers G-quadruplexes very interesting features. The obtaining of reliable kinetic data constitutes an efficient tool to determine reaction mechanisms between conformations and small molecules.
“la Caixa” Foundation (project OSLC-2012-007), MINECO, (CTQ2014- 58812-C2-2-R) and Junta de Castilla y León, (BU042U16), FEDER Funds Spain