Proton nuclear magnetic resonance investigation of the conformation and dynamics in the synthetic deoxyribonucleic acid decamers d(ATATCGATAT) and d(ATATGCATAT)

Abstract

A variety of 1-dimensional proton NMR methods were used to investigate the properties of 2 synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of 2-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential melting of the A .cntdot. T base pairs and by comparison of the spectra of the 2 decamers. Below 20.degree. C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A .cntdot. T and G .cntdot. C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52.degree. C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8 and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5''ApT3''). The base protons (AH8, GH8 and TH6) appear to be located close (1.9-2.3 .ANG.) to sugar H2'',2'''' protons. A cross-strand AH2-AH2 interaction (interproton distance of 2.8 .+-. 0.2 .ANG.) is manifested in terms of a larger R2 value for bases A3 and A9 compared to A1 and A7. The spin-spin couplings (J1''2'' + J1''2'''') for the H1'' sugar resonances indicate that most sugar groups in the molecule are in S conformation, consistent with a B-family DNA conformation.