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Impact of a Snap-Back Loop on Stability and Ligand Binding to a Parallel G‑Quadruplex

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journal contribution
posted on 25.03.2020, 18:34 by Lena Schnarr, Jagannath Jana, Pit Preckwinkel, Klaus Weisz
Various genomic DNA sequences including a MYC promoter sequence are amenable to the formation of a G-quadruplex featuring a snap-back loop with the incorporation of a 3′-terminal guanine into the quadruplex core. To evaluate relative stabilities and ligand binding in more detail, optical, microcalorimetric, and NMR structural studies were performed on both a minimal mutant sequence Pu22T that exclusively folds into a snap-back loop quadruplex and a parallel MYC quadruplex proposed to be the most relevant fold of the MYC promoter in a cellular environment. Similar thermal stabilities for Pu22T and MYC suggest the coexistence of both quadruplexes when derived from a sequence able to fold into both topologies. Isothermal titration calorimetry indicates a mostly identical enthalpy-driven strong binding of an indoloquinoline ligand but with a reduced number of high-affinity binding sites in Pu22T in line with a novel modified FRET competitive melting assay. Corroborated by fluorescence titrations using 2-aminopurine as a fluorescent probe, NMR chemical shift footprints show binding of the ligand at the Pu22T 5′-outer tetrad with the formation of a binding pocket. On the other hand, steric restrictions due to the snap-back loop severely restrict ligand stacking on the 3′-outer tetrad of Pu22T.