DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides.
Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C¿1H¿ NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.
Kojima
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Department of Pharmaceutical Chemistry, University of California, San Francisco, California, 94143-0446, USA.
Ono
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Kainosho
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James
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D000241:Adenosine / Q000032:analysis; D001412:Bacillus subtilis / Q000235:genetics; D002247:Carbon Isotopes; D004269:DNA, Bacterial / Q000032:analysis*; D003855:Deoxyribose / Q000032:analysis; D019791:Guanidine / Q000032:analysis; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D015394:Molecular Structure; D009038:Motion; D009587:Nitrogen Isotopes; D009841:Oligonucleotides / Q000032:analysis; D011685:Purine Nucleotides / Q000032:analysis*; D012399:Rotation
D002247:Carbon Isotopes; D004269:DNA, Bacterial; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D011685:Purine Nucleotides; D003855:Deoxyribose; D006859:Hydrogen; D019791:Guanidine; D000241:Adenosine