Kinetics of DNA hydration

The hydration of the d(CGCGAATTCGCG) B-DNA duplex in solution was studied by nuclear magnetic relaxation dispersion (NMRD) of the water nuclei ¹H, ²H, and ¹⁷O, and by nuclear Overhauser effects (NOEs) in high-resolution two-dimensional ¹H NMR spectra. By comparing results from the free duplex with those from its complex with netropsin, water molecules in the 'spine of hydration' in the AATT region of the minor groove could be distinguished from hydration water elsewhere in the duplex. The ²H and ¹⁷O relaxation dispersions yield a model-independent residence time of 0.9(±0.1) ns at 4°C for five highly ordered water molecules in the spine. When corrected for frequency offset effects, the NOE data yield the same residence time as the NMRD data, giving credence to both methods. At 27°C, the residence time is estimated to 0.2 ns, a factor of 40 shorter than the tumbling time of the duplex. The NMRD data show that all water molecules associated with the duplex, except the five molecules in the spine, have residence times significantly shorter than Ins at 4°C. There is thus no long-lived hydration structure associated with the phosphate backbone. In contrast to ²H and ¹⁷O, the ¹H relaxation dispersion is dominated by labile DNA protons and therefore provides little information about DNA hydration.