Triple resonance experiments for the simultaneous correlation of H6/H5 and exchangeable protons of pyrimidine nucleotides in 13C,15N-labeled RNA applicable to larger RNA molecules |
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Authors: | Wöhnert Jens Görlach Matthias Schwalbe Harald |
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Affiliation: | (1) Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-Universität Frankfurt, Marie-Curie-Str. 11, 60439 Frankfurt/M, Germany;(2) Institut für Molekulare Biotechnologie, Beutenbergstr. 11, 07745 Jena, Germany |
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Abstract: | Triple-resonance two-dimensional H6/H5(C4N)H and C6/C5(C4N)H experiments are described that provide through-bond H6/H5 or C6/C5 to imino/amino correlations in pyrimidine bases in 13C,15N-labeled RNA. The experiments simultaneously transfer H6/H5 magnetization by an INEPT step to the C6/C5 nuclei and by homonuclear CC- and heteronuclear CN-TOCSY steps via the intervening C4 nucleus to the N3/N4 nuclei and then by a reverse INEPT step to the imino/amino hydrogens. The sensitivity of these experiments is high as demonstrated using a 30-nucleotide pyrimidine rich RNA at a concentration of 0.9 mM at temperatures of 10°C and 25°C. This indicates the general applicability of the experiments and the possibility to obtain correlations for imino resonances in non-canonical regions of the target RNA. |
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Keywords: | assignment 13C,15N-labeling NMR pyrimidine bases RNA TOCSY |
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