Biophysical studies of human telomeric DNA

The major focus of this thesis was to examine the thermodynamic properties of human tiny telomere DNA system under different monocation (Na+ and K+) conditions. Three-dimensional UV and CD melting methods as well as differential scanning calorimetry (DSC) were applied to obtain thermodynamic parameters of this system. The quadruplex structures demonstrate multiple transition states during melting process. Adding dT nucleotides sequence to 5' end of quadruplexes can destabilize their structures. All quadruplex forms in K+ solution are observed to be more stable than those in Na+ solution.; We report the results of biophysical and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K+ solutions. Sedimentation coefficients were determined experimentally in both Na+ and K+ solutions, and were compared to values calculated from the reported NMR and crystal structures using bead models. While the NMR structure accurately predicted the observed S value in Na+ solution, the crystal structure predicted an S value that differed dramatically from that experimentally observed in K+ solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic substitution of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K+ yielded results at odds with quantitative predictions from the reported crystal structure. Fluorescence studies on 2-aminopurine substituted quadruplexes reveal that the Na+ forms ("basket") demonstrate very similar quenching pattern to those in K+ solution, which is radically different from the "propeller" structure observed by X-ray crystallography.; Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K+ solutions, but not in Na+ solutions, suggesting that the crystal environment may have selected for a particular conformational form. Therefore, molecular dynamics simulations were performed to yield model structures for the K+ quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K+ solution is not the one seen in crystals, but rather is a chair conformation with a compact loop structure.