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Journal of Biomolecular NMR

, Volume 59, Issue 4, pp 231–240 | Cite as

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

  • Barbara Krähenbühl
  • Peter Lukavsky
  • Gerhard WiderEmail author
Article

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.

Keywords

Nucleic acids NMR Projection spectroscopy APSY Automated assignment FLYA Novel sampling methods 

Notes

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].

Supplementary material

10858_2014_9841_MOESM1_ESM.pdf (2.5 mb)
Supplementary material 1 (PDF 2536 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Barbara Krähenbühl
    • 1
  • Peter Lukavsky
    • 2
  • Gerhard Wider
    • 1
    Email author
  1. 1.Institute of Molecular Biology and BiophysicsETH ZurichZurichSwitzerland
  2. 2.CEITEC – Central European Institute of TechnologyMasaryk UniversityBrnoCzech Republic

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