Two-dimensional nuclear Overhauser enhancement investigation of the solution structure and dynamics of the DNA octamer [d(GGTATACC)]2

Abstract

The resonances of nearly all 70 of the non-exchangeable protons of the duplex [d(GGTATACC)I2 in aqueous solution are assigned by proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra obtained in pure absorption phase at 500 MHz. Experimental and theoretical 2D NOE spectra are compared at each mixing time (100, 175,250 and 400 ms) using two B-DNA structures: a standard B-form and an energy-minimized form. The G G and CC ends of the octamer duplex are well represented by the regular B-DNA structure. But large discrepancies from these models are observed for the 'TATA' box. All 2D NOE data are consistent with nanosecond correlation times, as indicated by non-selective proton spin-lattice relaxation times, but small variations in the correlation time are observed, suggesting that there are some local differences in mobility within the octamer duplex structure in solution Since the discovery that sequence-dependent structural variations are present in DNA [l-31, studies of oligonucleotides have received more attention as these structural variations may play an important role in specific protein-DNA interactions. These investigations are now possible due to recent progress in oligodeoxynucleotide synthesis techniques. Detailed X-ray crystal structures of short DNA fragments are already available [l, 2,4-81. Studies in solution have recently begun which will allow comparisons with crystal data. Nuclear magnetic resonance is the most powerful spectroscopic method for probing details of the structures and dynamics of these oligonucleotide duplexes in aqueous solution. Using two-dimensional nuclear Overhauser effect (2D NOE) experiments, assignments of all or nearly all resonances in the proton spectra of oligonucleotides can be made [9 -131. Following assignment of resonances, detailed analysis of conformations in solution may be undertaken. We have already started a study on the dynamics and structure of the [d(GGAATTCC)I2 duplex in solution [13].In the present study, we report the first results of a detailed conformational investigation of the duplex [d(GGTATACC)], in solution, whose crystal and solution structures are of A-type [ 5 , 61 and B-type [14], respectively. Before examining conformational features, almost all 70 nonexchangeable proton resonances were assigned. Conformational details were explored by comparing experimental 2D NOE spectra (pure absorption phase) obtained at four mixing times with theoretical spectra calculated using either a standard B-form structure or an energy-minimized structure for DNA. The method of calculating theoretical spectra has previously been described MATERIALS AND METHODS Sample preparutionsThe self-complementary octamer [d(5'GGTATACC3')I2 was synthesized by the solid phase phosphoramidite method [12, 16, 171 and its purity was checked by HPLC, 31P NMR, ' H NMR and base composition analysis. An amount of 5.7mg of the oligonucleotide was dissolved in 0.4ml of a phosphate buffer (200 mM phosphate, 180 mM NaCI, 0.2 mM EGTA, pH = 7.0). This sample was lyophilized...

Resonance assignments of non-exchangeable protons in B type DNA oligomers, an overview

“…It is not so that these substantial differences between chemical shifts of very similar protons could not be rationalized: they are either due to differences in external conditions or caused by long-range effects involving more remote sites in the DNA duplex. Consider, for example, T(7) in 5'CGCGTA-TACGCG (20) and T(5) in 5'GGTATACC (25). All nonexchangeable protons of T (7) in the first sequence were reported to resonate at least 0.2 ppm downfield from the corresponding protons of T(5) in the second sequence, even though these two sequences have six nucleotides in common centered around T(7) and T(5) respectively.…”

Sequence-specific assignments and their use in NMR studies of DNA structure

“…Neither of these theoretical relaxation matrices exactly matched the NOESY data but the energy minimized structure generated theoretical NOESY spectra which mimic many of the experimental cross peak growth and decay rates. Similar comparisons between the timedependent NOESY data and the complete relaxation matrix for B-DNA were carried out on d(GGTATACC) 8 by Jamin et al (1985). Separate analysis of the two GC pairs at each end of the duplex revealed that this region could be simulated reasonably well by the relaxation matrix for standard B-DNA but the internal region of AT pairs could not, thus suggesting a different conformation for this region.…”

DNA and RNA: NMR studies of conformations and dynamics in solution

“…There has been recent progress in calculating proton NOESY spectra for a given DNA oligomer conformation (Broido et al 1985;Jamin et al 1985;Suzuki et al 1986) based on a relaxation matrix analysis which includes all proton-proton dipolar interactions and spin diffusion (Keepers & James, 1984;Olejniczak et al 1986). Thus, theoretical NOESY spectra were computed as a function of mixing time for standard B-DNA and an energy minimized analog and were compared with experimental NOESY spectra (mixing times 100, 175, 250 and 400 ms) for the d(GGAATTCC) duplex (Broido et al 1985), the d(GGTATACC) duplex (Jamin et al 1985) and the d(ATATATAT) duplex (Suzuki et al 1986). The comparison supports an effective isotropic correlation time in the nanosecond range for the octanucleotide duplex with some local fluctuations in mobility along the helix.…”