Determination of sugar conformations by NMR in larger DNA duplexes using both dipolar and scalar data: Application to d(CATGTGACGTCACATG)₂

Different methods for determining sugar conformations in large oligonucleotides have been evaluated using both J-coupling and NOE data. In order to simulate COSY spectra, reliable estimates of line widths are required. We have measured T_1ρ (=T₂) values for a large number of protons of the hexadecamer d(CATGTGACGTCACATG)₂ using a new two-dimensional NMR experiment (T1RHOSY) to provide baseline information for the simulations. Both DQF-COSY and P.E.COSY cross-peaks have been systematically simulated as a function of line width, digitisation and signal-to-noise ratio. We find that for longer correlation times (τ ≥ 5 ns), where line widths are comparable to or larger than active couplings, only Σ₁, (³J_1′2′+³J _1′2″) is reasonably accurately determined (within ± 1 Hz). Under these conditions, additional information is needed to determine the sugar conformation. We have used apparent distances H1′-H4′ and H2″-H4′, which provide a range of P_s over an interval of ca. 20°. Complete analysis of time courses for intraresidue NOEs, with and without coupling constants, has also been evaluated for determining nucleotide conformations. Whereas P_s is poorly determined in the absence of both intrasugar NOEs and coupling constants, the range of solutions is decreased when intrasugar NOEs and Σ₁, are also available. DQF-COSY, P.E.COSY and NOESY spectra at different mixing times of the hexadecamer d(CATGTGACGTCACATG)₂ were recorded at three temperatures. A detailed analysis of the NOEs and coupling constants provided estimates of the sugar conformations in the hexadecamer. At 50°C, the sugar conformations are well determined by the scalar and dipolar data, with pseudorotation phase angles of 126-162° and mole fractions of the S conformation (f_s) of 0.86 ± 0.05. There was no statistically significant difference between f_s for the purines and the pyrimidines, although there was a small tendency for P_s of the purines to be larger than those of the pyrimidines. At 25°C, the sugar conformations were much less well determined, although the estimates of f_s were the same within experimental error as at 50°C. The experimental and theoretical results provide guidelines for the limits of conformational analysis of nucleic acids based on homonuclear NMR methods.