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Nucleic Acids Res. 2014 Sep;42(15):e117. doi: 10.1093/nar/gku553. Epub 2014 Jun 30.

A fully enzymatic method for site-directed spin labeling of long RNA.

Author information

1
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Structurale, Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé et de la Recherche Médicale (INSERM) U964/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, 67404 Illkirch cedex, France lebars@igbmc.fr.
2
Institut de Chimie, Laboratoire Propriétés Optiques & Magnétiques des Architectures Moléculaires, Université de Strasbourg, UMR 7177 CNRS, 4 rue Blaise Pascal, CS 90032, 67081 Strasbourg Cedex, France.
3
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Structurale, Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé et de la Recherche Médicale (INSERM) U964/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, 67404 Illkirch cedex, France.
4
Institut de Biologie Moléculaire et Cellulaire, Plateforme Protéomique Strasbourg Esplanade, FRC 1589 CNRS, 15 rue René Descartes, 67084 Strasbourg Cedex, France Institut de Biologie Moléculaire et Cellulaire, Architecture et Réactivité des ARN, Université de Strasbourg, UPR 9002 CNRS, 15 rue René Descartes, 67084 Strasbourg Cedex, France.

Abstract

Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5'-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies.

PMID:
24981512
PMCID:
PMC4150755
DOI:
10.1093/nar/gku553
[Indexed for MEDLINE]
Free PMC Article

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