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

J Biomol NMR. 2014 Aug;59(4):231-40. doi: 10.1007/s10858-014-9841-3. Epub 2014 Jun 5.

Abstract

A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [(13)C, (15)N]-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 (13)C-(1)H 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Animals
  • Automation / methods*
  • Drosophila Proteins
  • Drosophila melanogaster
  • Nuclear Magnetic Resonance, Biomolecular / instrumentation
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Nuclear Proteins
  • Nucleic Acid Conformation*
  • RNA, Messenger / chemistry*
  • Transcription Factors

Substances

  • Drosophila Proteins
  • Nuclear Proteins
  • RNA, Messenger
  • Transcription Factors
  • fs(1)K10 protein, Drosophila