Biophysical Studies of the Structure, Stability, and Ligand Binding Properties of G-Quadruplex DNA: Thoughts and Comparisons of the K-ras, c-MYC, and Bcl-2 Oncogene Promoter Sequence Quadruplexes

摘要

Expression of the K-ras proto-oncogene is a well known hallmark of pancreatic and other cancers. We report here the results of a biophysical characterization of model human K-ras promoter sequence constructs having either the wild type, WT, 30-mer sequence or four mutant 26-mer sequences in which three G→T mutations were introduced in the second, third, forth, or fifth G-run starting from the 5' end of the WT construct. The model WT and mutant K-ras constructs were studied using analytical ultracentrifugation (AUC), circular dichroism spectroscopy (CD), differential scanning calorimetry (DSC), isothermal titration Calorimetry (ITC), and DMS footprinting. Analytical ultracentrifugation experiments demonstrated that the WT K-ras promoter sequence folds into a compact structure with a sedimentation coefficient of 2.4 S. This result is consistent with the calculated sedimentation coefficient for the proposed K-ras G-quadruplex structure and similar to the sedimentation coefficients found for c-MYC and Bcl-2 quadruplex structures. The CD and DMS footprinting results indicate that the WT and two mutant (Mut 1-2-3-4 and Mutl-2-3-5) K-ras promoter sequences fold into intramolecular, parallel-stranded, G-quadruplexes. The DMS cleavage patterns observed for the WT sequence are most similar to those for Mutl-2-3-5 sequence indicating that the ensemble of K-ras WT G-quadruplex motifs includes a quadruplex incorporating a kinked out base (T7) and a large lateral or end loop having as many as 11 unstructured bases. The two mutant sequences (Mutl-3-4-5 and Mutl-2-4-5) exhibit CD and DMS footprinting spectra that indicate essentially no G-quadruplex formation. DSC experiments demonstrate that the WT K-ras sequence folds into a mixture of at least two thermodynamically unique conformers with the two overlapping melting transitions having 7m values of 58.5 °C and 71.2 °C. In comparison to simpler G-quadruplex forming sequences, e.g. c-MYC or even Bcl-2, formation of stable K-ras promoter sequence G-quadruplexes requires the incorporation of large lateral or end loops and/or a corner backbone kinked out base.