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Observation of H-bond mediated 3hJH2H3coupling constants across Watson–Crick AU base pairs in RNA

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Abstract

3hJH2H3trans-hydrogen bond scalar coupling constants have been observed for the first time in Watson-Crick AU base pairs in uniformly 15N-labeled RNA oligonucleotides using a new 2hJNN-HNN-E. COSY experiment. The experiment utilizes adenosine H2 (AH2) for original polarization and detection, while employing 2hJNNcouplings for coherence transfer across the hydrogen bonds (H-bonds). The H3 protons of uracil bases are unperturbed throughout the experiment so that these protons appear as passive spins in E. COSY patterns. 3hJH2H3coupling constants can therefore be accurately measured in the acquisition dimension from the displacement of the E. COSY multiplet components, which are separated by the relatively large 1JH3N3coupling constants in the indirect dimension of the two-dimensional experiment. The 3hJH2H3scalar coupling constants determined for AU base pairs in the two RNA hairpins examined here have been found to be positive and range in magnitude up to 1.8 Hz. Using a molecular fragment representation of an AU base pair, density functional theory/finite field perturbation theory (DFT/FPT) methods have been applied to attempt to predict the relative contributions of H-bond length and angular geometry to the magnitude of 3hJH2H3coupling constants. Although the DFT/FPT calculations did not reproduce the full range of magnitude observed experimentally for the 3hJH2H3coupling constants, the calculations do predict the correct sign and general trends in variation in size of these coupling constants. The calculations suggest that the magnitude of the coupling constants depends largely on H-bond length, but can also vary with differences in base pair geometry. The dependency of the 3hJH2H3coupling constant on H-bond strength and geometry makes it a new probe for defining base pairs in NMR studies of nucleic acids.

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Author notes

  1. Burkhard Luy

    Present address: Institut für Organische Chemie und Biochemie II, TU München, Lichtenberg-Str. 4, D-85747, Garching, Germany

Authors and Affiliations

  1. Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, 9600 Gudelsky Dr., Rockville, MD, 20850, U.S.A

    Burkhard Luy, Uwe Richter, Eric S. DeJong & John P. Marino

  2. Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, D-13125, Berlin, Germany

    Uwe Richter

  3. Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK 2500, Valby, Denmark

    Ole W. Sørensen

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Luy, B., Richter, U., DeJong, E.S. et al. Observation of H-bond mediated 3hJH2H3coupling constants across Watson–Crick AU base pairs in RNA. J Biomol NMR 24, 133–142 (2002). https://doi.org/10.1023/A:1020919131801

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