title: Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10.
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.
authors.affiliation: Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Krähenbühl
authors.forename: Peter
authors.initials: P
authors.lastname: Lukavsky
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wider
mesh_terms: D000465:Algorithms; D000818:Animals; D001331:Automation / Q000379:methods*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000295:instrumentation / Q000379:methods*; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry*; D014157:Transcription Factors
chemical_list: D029721:Drosophila Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; C088225:fs(1)K10 protein, Drosophila