Two-dimensional proton nuclear magnetic resonance investigation of the synthetic deoxyribonucleic acid decamer d(ATATCGATAT)

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Abstract
Two-dimensional NMR techniques (COSY and NOESY) were used in conjunction with 1-dimensional (1D) NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements were combined to systematically assign many of the resonances from the H1'' and H2'',2'''' sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6 and CH6) in right-handed helices can interact simultaneously with their own H2'',2'''' sugar protons and those of the adjacent (5''-3'') nucleotide attached to its 5'' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can be immediately identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5''-3'')adenine residue. These interactions are used to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., and Kearns, D.R. (1982) J. Am. Chem. Soc. 104, 5540]. Interactions between the base protons (PuH8 and PyH6) and the H1'' sugar protons are quite weak and probably arise from second-order NOEs (via the 2'',2'''' protons). The strong interactions between the base protons and the H2'',H2'''' sugar protons indicate that the base protons are in close proximity to the 2'' sugar proton on the same nucleotide unit and only slightly further away from the 2'''' sugar proton of the adjacent (5''-3'') nucleotide unit. This establishes that the nucleotides have an anti conformation of the bases relative to the sugar. The collection of inter- and intranucleotide interactions observed in the NOESY experiments places restrictions on the possible conformational state of this decamer. These results in conjunction with the results of a set of 1D-NMR experiments, provide the foundation for a more rigorous determination of the structure of this molecule in solution.