Quantitative and Systematic NMR Measurements of Sequence-Dependent A–T Hoogsteen Dynamics in the DNA Double Helix

NMR experiments, we quantitatively measured Watson-Crick to Hoogsteen dynamics for an A-T base pair in 13 trinucleotide sequence contexts. The Hoogsteen population and exchange rate varied 4-fold and 16-fold, respectively, and were dependent on both the 3'- and 5'-neighbors but only weakly dependent on monovalent ion concentration (25 versus 100 mM NaCl) and pH (6.8 versus 8.0). Flexible TA and CA dinucleotide steps exhibited the highest Hoogsteen populations, and their kinetics rates strongly depended on the 3'-neighbor. In contrast, the stiffer AA and GA steps had the lowest Hoogsteen population, and their kinetics were weakly dependent on the 3'-neighbor. The Hoogsteen lifetime was especially short when G-C neighbors flanked the A-T base pair. Our results uncover a unique conformational basis for sequence-specificity in the DNA double helix and establish the utility of NMR to quantitatively and comprehensively measure sequence-dependent DNA dynamics.