The Transition from Unfolded to Folded G-Quadruplex DNA Analysed and Interpreted by Two-Dimensional Infrared Spectroscopy

A class of DNA folds/structures known collectively as G-quadruplexes (G4) commonly form in guanine-rich areas of genomes. G4 DNA is thought to have a functional role in the regulation of gene transcription and telomerase-mediated telomere maintenance and is therefore a target for drugs. The details of the molecular interactions that cause stacking of the guanine tetrads are not well-understood, which limits a rational approach to the drugability of G4 sequences. To explore these interactions further, we employed Electron-Vibration-Vibration two-dimensional Infra-red (EVV 2DIR) spectroscopy to measure extended vibrational coupling spectra for a parallel-stranded G-quadruplex DNA formed by Myc2345 nucleotide sequence. We also tracked the structural changes associated with G4 folding as a function of K+ ion concentration to generate further insight. In order to classify the structural elements that the folding process generates in terms of vibrational coupling characteristics, we used quantum-chemical calculations utilising density functional theory. This resulted in predictions of the coupling spectra associated with a given structure, which are compared against the experimental coupling data obtained from the EVV 2DIR spectroscopy. Overall, 102 coupling peaks are experimentally identified and followed during the folding process. A number of phenomena are noted and associated with the formation of the folded form. This includes frequency shifting, changes in cross-peak intensity and the appearance of new coupling peaks. The new peaks can be assigned to the coupling between specific chemical groups within the complex, and we use the 2DIR data to propose a folding sequence for this particular type of G4 under our experimental conditions. Overall, the combination of experimental 2DIR data and DFT calculations suggests that guanine quartets may already be present in the initial DNA before the addition of potassium ions, but that these quartets are unstacked until potassium ions are added, at which point the full G4 structure is formed.