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Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10

Krähenbühl, Barbara ; Lukavsky, Peter ; Wider, Gerhard

In: Journal of Biomolecular NMR, 2014, vol. 59, no. 4, p. 231-240

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    Titre
    • Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10
    Auteur
    • Krähenbühl, Barbara. Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland
    • Lukavsky, Peter. CEITEC - Central European Institute of Technology, Masaryk University, 62500, Brno, Czech Republic
    • Wider, Gerhard. Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Journal of Biomolecular NMR, 2014, vol. 59, no. 4, p. 231-240. Springer Netherlands
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:325377
    Summary
    A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [13C, 15N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80% of the resonances of the 13C-1H moieties at the 1′, 2′, 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY.