Direct observation of dipolar couplings between distant protons in weakly aligned nucleic acids

Abstract

Under conditions where macromolecules are aligned very weakly with respect to an external magnetic field, Brownian diffusion no longer averages internuclear dipole-dipole interactions to zero. The resulting residual dipolar coupling, although typically 3 orders of magnitude weaker than in a fully aligned sample, can readily be measured by solution NMR methods. To date, application of this idea has focused primarily on pairs of nuclei separated by one or two covalent bonds, where the internuclear separation is known and the measured dipolar coupling provides direct information on the orientation of the internuclear vector. A method is described that allows observation of dipolar interactions over much larger distances. By decoupling nearest-neighbor interactions, it is readily possible to observe direct dipolar interactions between protons separated by up to 12 A. The approach is demonstrated for the DNA dodecamer d(CGCGAATTCGCG)2, where direct interactions are observed between protons up to three base pairs apart.

Fig. 1.

Comparison of the H1′/H5 region of 1D spectra of d(CGCGAATTCGCG)₂, recorded without (A and B) or with (C) homonuclear decoupling. Spectrum A corresponds to the partially aligned state [12% (wt/vol) ether bicelle], and spectra B and C have been recorded on the corresponding isotropic sample of DNA. The duration of the sampled free induction decay equals 307 ms for spectra A and B. Apodization using a cosine-squared function, followed by zero-filling, was used before Fourier transformation. Spectrum C has been acquired and processed as described in the legend to Fig. 3. In C,H_1′ and H₅ protons are labeled by their nucleotide numbers, including * for H₅ resonances.

Fig. 2.

Pulse schemes for homonuclear decoupled spectroscopy. (A) Basic pulse element for band-selective ¹H homonuclear decoupling. (B) Selective constant-time COSY pulse scheme, in which band-selective decoupling is implemented to specifically detect internucleotide residual dipolar coupling. Narrow and wide pulses correspond to flip angles of 90° and 180°, respectively. Unless specified, pulses are applied along the x axis. Shaped ¹H pulses are of the 180° REBURP type (39) (8-ms duration at 600 MHz, for a 500-Hz inversion bandwidth, centered at 5.8 ppm). Phase cycling: ϕ = x,–x; φ₁ = 2(x),2(–x); φ₂ = 4(x),4(–x); and receiver = y,–y,–y,y,–y,y,y,–y. (A) Quadrature detection in the indirect ¹H dimension is achieved by applying an extra ¹H 180° pulse just before the acquisition for half of the scans, storing signals separately, and using the usual “echo-antiecho” Fourier transform processing method (40, 41). All gradients are sine bell-shaped, with durations of 200 μs each, and (x,y,z) peak amplitudes in G/cm of (15,–33,20) for G₁, (9,9,12) for G₂, and (24,–24,32) for G₃.(B) Quadrature detection in F₁ is achieved by incrementing ϕ in the usual States-TPPI manner, and in F₂ by insertion on alternate scans of the extra ¹H 180° pulse just before acquisition (as in A). All gradients are sine bell-shaped, with durations of 200 μs, and (x,y,z) peak amplitudes in G/cm of (7,7,24) for G₁, (5,5,–7) for G₂, (12,12,17) for G₃, (15,–33,20) for G₄, (9,9,12) for G₅, and (24,–24,32) for G₆.

Fig. 3.

Processing scheme for diagonal unfolding. (A) The original spectrum, processed in the standard manner, in which the diagonal signal (dashed line) is repeatedly folded in the F₁ dimension. Spectra have been recorded with the pulse scheme of Fig. 2 A. The data matrix consists of 6*(t₁) × 1,024*(t₂) data points, with acquisition times of 144 ms (t₁) and 307 ms (t₂) (2T = 164 ms). Linear prediction was used to double the number of time-domain points in the F₁ dimension, and both dimensions were apodized by cosine-squared functions and zero-filled by a factor of two before Fourier transformation. (B)An F₁-dependent frequency shift is applied to the F₂ dimension, such that the diagonal signals are now positioned vertically. (C) Vertical bands from the shifted spectrum are transposed and reassembled horizontally to construct an unfolded spectrum in the F₁ dimension. (D) A 20-Hz band, corresponding to the center of the F₂ dimension in C, is extracted, apodized by a sine-bell function, and then projected onto the F₁ dimension, to yield a homonuclear decoupled 1D spectrum. The spectral widths for the horizontal and vertical dimensions are, respectively, 600 and 34.9 Hz. For display purposes, the vertical axis is expanded 2-fold relative to the horizontal axis.

Fig. 4.

¹H-¹H dipolar correlation spectra of d(CGCGAATTCGCG)₂, homonuclear band-selectively decoupled, recorded at 600 MHz in 12% ether bicelle medium. (A) Projection of the 3D spectrum along the F₃ axis onto the 2D (F₁,F₂) plane, displaying the dipolar connectivities between H_1′ and H₅ protons. (B)F₁ cross section, taken at the frequency of A5-H_1′ (6.03 ppm, marked by a dashed line in A). Spectra have been recorded with the pulse scheme of Fig. 2B. Total measurement time is 36 h, with the data matrix consisting of 40*(t₁) × 20*(t₂) × 256*(t₃) data points, with acquisition times of 62.4 ms (t₁), 60.8 ms (t₂) and 51 ms (t₃)(2 T_A = 2 T_B = 81 ms). Linear prediction was used to double the number of time-domain points in both the t₁ and t₂ dimensions, and all three dimensions were apodized by cosine-squared functions and zero-filled before Fourier transformation. Each (F₂,F₃) plane has been reduced to its homonuclear decoupled 1D equivalent, using the protocol of Fig. 3 (using a 60-Hz width for the projected central band). Intrastrand connectivities (intranucleotide, sequential, and long range) are labeled by their corresponding residue numbers on the lower right half of the spectrum, whereas the interstrand cross peaks are labeled in the upper left half of the spectrum. Only positive contour levels are displayed.

Fig. 5.

Long-range ¹H-¹H connectivities observed for the d(CGCGAATTCGCG)₂ dodecamer, with only half of the palyndromic oligomer shown. H1′ and H5 are displayed as spheres. Nucleotides are labeled by their nucleotide numbers, with red and blue marking the opposing strands. Intrastrand and interstrand long-range connectivities are indicated by red and green arrows, respectively.