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

Isabelle Lebars (1) , Bertrand Vileno (2) , Sarah Bourbigot (1) , Philippe Turek (2) , Philippe Wolff (3) , Bruno Kieffer (1)

1 IGBMC - Institut de génétique et biologie moléculaire et cellulaire
2 Institut de Chimie de Strasbourg
3 ARN - Architecture et Réactivité de l'ARN

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.

Domaines: Biophysique; Biologie structurale [q-bio.BM]

Isabelle Lebars, Bertrand Vileno, Sarah Bourbigot, Philippe Turek, Philippe Wolff, et al.. A fully enzymatic method for site-directed spin labeling of long RNA. Nucleic Acids Research, 2014, 42 (15), pp.e117-e117. ⟨10.1093/nar/gku553⟩. ⟨hal-03009308⟩