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

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Abstract

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 0.3 mM 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.

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Acknowledgments

We would like to gratefully acknowledge Prof. Peter Güntert and Elena Schmidt (both University Frankfurt) for support with the use of FLYA. We thank Prof. Frédéric Allain (ETH Zurich) for his manifold support. This work was supported by the Swiss National Science Foundation [Grant Numbers 120048, 140559].

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Authors and Affiliations

  1. Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland

    Barbara Krähenbühl & Gerhard Wider

  2. CEITEC – Central European Institute of Technology, Masaryk University, 62500, Brno, Czech Republic

    Peter Lukavsky

Authors
  1. Barbara Krähenbühl
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  2. Peter Lukavsky
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  3. Gerhard Wider
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Correspondence to Gerhard Wider.

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Krähenbühl, B., Lukavsky, P. & Wider, G. Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10 . J Biomol NMR 59, 231–240 (2014). https://doi.org/10.1007/s10858-014-9841-3

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