A simple spectral-driven procedure for the refinement of DNA structures by NMR spectroscopy

Abstract

We have developed a simple and quantitative procedure (SPEDREF) for the refinement of DNA structures using experimental two-dimensional nuclear Overhauser effect (2D NOE) data. The procedure calculates the simulated 2D NOE spectrum using the full matrix relaxation method on the basis of a molecular model. The volume of all NOE peaks is measured and compared between the experimental and the calculated spectra. The difference of the experimental and simulated volumes is minimized by a conjugated gradient procedure to adjust the interproton distances in the model. An agreement factor (analogous to the crystallographic R-factor) is used to monitor the progress of the refinement. The procedure is an The agreement is considered to be complete when several parameters, including the R-factor, the energy associated with the molecule, the local conformation (as judged by the sugar pseudorotation), and the global conformation (as judged by the helical x-displacement), are refined to their respective convergence. With the B-DNA structure of d(CGATCG) as an example, we show that DNA structure may be refined to produce calculated NOE spectra that are in excellent agreement with the experimental 2D NOE spectra. This is judged to be effective by the low R-factor of approximately 15%. Moreover, we demonstrate that not only are NOE data very powerful in providing details of the local structure but, with appropriate weighting of the NOE constraints, the global structure of the DNA double helix can also be determined, even when starting with a grossly different model. The reliability and limitations of a DNA structure as determined by NMR spectroscopy are discussed.

Human chromosomal centromere (AATGG)n sequence forms stable structures with unusual base pairs

“…2A, there is no interruption of the connectivity for sequential assignment and every aromatic proton (H 6 and H s) has crosspeaks to the HZlH z' of its own sugar and the sugar on the 3' side, confirming the helical nature of the backbone. To have a more definitive view of the possible structure, several models (including blunt-ended 6-bp duplexes with type-I or type-II G-A mismatches, or a 5-bp duplex with a 3'-overhang G, all with an overall B or A conformation) were constructed and subjected to NOE-constrained refinement by the procedure SPEDREF [11] and a simulated annealing (SA) procedure using X-PLOR [12,15]. Our results indicated that the most plausibe model is a 6-bp B-DNA duplex with type-I G-A base pairs.…”

“…The deblocked oligonucleotides were purified by Sepharose G-50 column chromatography. Solutions of the DNA oligomers (-5 mM duplex) with 150 mM NaCI and 50 mM 'phosphate buffer (pH 7.0) were prepared in 0.5 mi of I)20 as described earlier [11,12]. NMR spectra were collected on either a GE GN500 or a Varian VXR500 500 MHz spectrometer and the data were processed with FELIX via [13].…”

“…This procedure allowed us to obtain 1088 NOE integrals involving all the non-exchangeable protons for the bexamer. Refinement of the starting model was carried out by the sequence of procedures comprising the SPEDREF package [11]. This included a complete relaxation matrix calculation of the NOEs for the model, with comparison of the experimental and simulated spectra to deconvolute overlapped areas of the spectra.…”

Monofunctional platinum amine complexes destabilize DNA significantly

“…Integrals from the two-dimensional NOESY data set were measured as described by Robinson and Wang [26]. Refinement of the starting model was carried out by the sequence of procedures comprising the SPEDREF package [26].…”

Sequence-specific local structural variations in solution structures of d(CGXX′CG)2 and d(CAXX′TG)2 self-complementary deoxyribonucleic acids

“…For DNA hexamer duplex, the overall molecular tumbling is effectively isotropic and is well-approximated by a single correlation time t c (Robinson & Wang, 1992). Assuming simple and rigid molecular motion, t c is related to T 1 and T 2 by equation (1) (Suzuki et al, 1986):…”