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Studies of physicochemical properties of N-H•••N hydrogen bonds in DNA, using selective 15N-labeling and direct 15N 1D NMR

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Abstract

15N-15N scalar coupling constants across base pair hydrogen bonds (2hJNN) were studied using residue- and atom-specifically 15N labeled DNA oligomers. The N3 atom selectively 15N enriched 2′-deoxycytidine and thymidine, and the uniformly 15N enriched 2′-deoxyadenosine and 2′-deoxyguanosine, were chemically prepared and incorporated into two DNA oligomers, d(CGCGAATTCGCG)2 and d(CGCAAAAAGCG)•d(CGCTTTTTGCG). This isotope labeling enabled us to determine the 2hJNN value from the splitting of the 15N 1D spectrum. Additionally, it enabled the determination of 2hJNN in D2O quite easily and highly quantitatively. The temperature and DNA sequence dependence were examined for these oligomers. The sequence dependence was not clear; however, a significant decrease of 2hJNN was observed by elevating the temperature. This temperature dependence was not due to the hydrogen exchange, since the addition of 20 mM NH3 did not change the 2hJNN values. The 2hJNN values in D2O were somewhat smaller than those in H2O. As compared to our 15N 1D method, the quantitative HNN-COSY method gave systematically smaller 2hJNN values in our system, due to the lower 15N fraction of our sample (79 and 88% for dA and the other nucleotides, respectively) and the insufficient power of the 15N RF pulse (B1=6.6 kHz). These systematic differences were recovered by theoretical correction of the 15N isotope fraction contribution, by using the composite 15N 180° pulse in a quantitative HNN-COSY experiment.

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Authors and Affiliations

  1. CREST (Core Research Evolutional Science and Technology), Japan Science and Technology Corporation (JST), Kawaguchi, Saitama , 332-0012, Japan

    Chojiro Kojima & Masatsune Kainosho

  2. Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Hachioji, Tokyo, 192-0397, Japan

    Akira Ono

Authors
  1. Chojiro Kojima
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  2. Akira Ono
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  3. Masatsune Kainosho
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Kojima, C., Ono, A. & Kainosho, M. Studies of physicochemical properties of N-H•••N hydrogen bonds in DNA, using selective 15N-labeling and direct 15N 1D NMR. J Biomol NMR 18, 269–277 (2000). https://doi.org/10.1023/A:1026717101063

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