NMR scalar couplings across Watson-Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy

Abstract

This paper describes the NMR observation of 15N---15N and 1H---15N scalar couplings across the hydrogen bonds in Watson-Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6-7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2-3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.

Figure 1

NMR observation of scalar ¹⁵N—¹⁵N couplings across hydrogen bonds in DNA, ^hJ_NN. (A Top and Middle) Contour plots of the region containing signals of A⩵T base pairs from [¹⁵N,¹H]-TROSY spectra (3, 5) of the partially and uniformly ¹³C,¹⁵N-labeled DNA duplex, respectively. (A Bottom) Contour plot showing the relayed cross-peaks obtained with 2D ^hJ_NN-correlation-[¹⁵N,¹H]-TROSY, where the broken contours indicate negative spectral intensities. The direct [¹⁵N₃(T),¹H₃(T)]-correlations (Top and Middle) and the relayed [¹⁵N₁(A),¹H₃(T)] cross-peaks (Bottom) of the A⩵T base pairs are shown. Chemical shifts are relative to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS). The DNA sequence is shown in the Top, where the ¹⁵N- and ¹³C-containing nucleotides in the partially ¹³C,¹⁵N-labeled DNA (see text) are underlined. (Insert) Definition of the scalar couplings ^hJ_NN and ^hJ_HN in Watson–Crick base pairs. (B) Same presentation as A for the GC base pairs. (C) Cross sections along ω₁(¹⁵N) through the individual cross peaks in the spectra (A). (C Lower) Direct correlation cross-peaks in the uniformly ¹³C,¹⁵N-labeled DNA duplex. (C Upper) Direct correlation cross peaks in the partially labeled duplex. (D) Same presentation as C for cross sections along ω₁(¹⁵N) taken through the individual cross peaks in the spectra (B).

Figure 2

Experimental scheme for the 2D ^hJ_NN-correlation-[¹⁵N,¹H]-TROSY experiment used to correlate the chemical shifts of pairs of hydrogen bond-related ¹⁵N spins and to measure ^hJ_HN. On the lines marked ¹H, ¹⁵N, and ¹³C, narrow and wide bars stand for nonselective 90° and 180° radio frequency pulses, respectively, with the carrier frequencies at 12, 190, and 145 ppm, respectively. The delays are τ₁ = 2.7 ms, τ₂ = 18 ms, and τ₃ = τ₂ − τ₁. The line marked PFG indicates the pulsed magnetic field gradients applied along the z axis: G₁, amplitude 30 G/cm, duration 1 ms; G₂, 42 G/cm, 1 ms; G₃, 45 G/cm, 1 ms; G₄, 40 G/cm, 1 ms; and G₅, 48 G/cm, 1 ms. Two data sets, I and II, are measured in the interleaved manner with the following phase cycling schemes: (I), φ₁ = {−x, x}; φ₂ = {2(−x),2x}; ψ₁ = {4y,4(−y)}; ψ₂(receiver) = {y,−y,−y, y,−y, y,y,−y}; (II), φ₁ = {−y, y}; φ₂ = {2(−y),2y}; ψ₁ = {4(−x),4x}; ψ₂(receiver) = {−x, x,x,−x, x,−x,−x, x}; x on all other pulses. For both data sets, quadrature detection in the t₁ dimension is obtained by using States-TPPI (38) by simultaneously incrementing each of the phases φ₂ and ψ₂ by 90°. The transfer of ¹⁵N magnetization to ¹H for detection of single transition-to-single transition polarization transfer (5) was used. Fourier transformation of the sum and difference of the two data sets results in two 2D ^hJ_NN- correlation-[¹⁵N,¹H]-TROSY spectra in which the relative cross-peak positions are determined in E.COSY manner by the chemical shifts and the scalar-coupling constants (–30). For example, both the direct and the relayed correlation cross-peaks that appear in the two spectra are shifted by ¹J_HN along ω₂(¹H). Along ω₁(¹⁵N), the direct peaks also are shifted by ¹J_HN, but the separation of the relayed cross-peaks is determined exclusively by ^hJ_HN. To enhance the signals of rapidly exchanging imino protons, water saturation is minimized by keeping the water magnetization along +z during the entire experiment, using the water-selective 90° pulses indicated by curved shapes on the line ¹H.

Figure 3

NMR observation of scalar ¹H—¹⁵N couplings across hydrogen bonds in DNA, ^hJ_HN. The plots show comparisons of the sum and difference 2D ^hJ_NN-correlation-[¹⁵N,¹H]-TROSY spectra recorded with the uniformly ¹³C,¹⁵N-labeled DNA duplex (Fig. 1A) on a Bruker DRX-750 spectrometer, using the experimental scheme of Fig. 2. (A and B) Contour plots with signals from GC base pairs and A⩵T base pairs, respectively. To facilitate comparison, one spectrum was shifted relative to the other by ≈90 Hz along ω₂(¹H), so that corresponding peaks overlap along this dimension. The relayed cross-peaks from each of the two spectra are represented by two contour lines. For selected base pairs, superpositions of cross sections along ω₁(¹⁵N) taken through the original, unshifted peaks are shown above and below the two panels, where the two peaks to be compared have been normalized to the same intensity. The shift between the two peaks provides an E.COSY-type representation of ^hJ_HN (see text).