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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biochemistry / methods
  • Electron Spin Resonance Spectroscopy
  • Isotope Labeling
  • Magnetic Resonance Spectroscopy
  • RNA / biosynthesis
  • RNA / chemistry*
  • RNA Ligase (ATP)
  • Spin Labels*
  • Thionucleotides / biosynthesis
  • Thionucleotides / chemistry
  • Viral Proteins

Substances

  • Spin Labels
  • Thionucleotides
  • Viral Proteins
  • RNA
  • RNA Ligase (ATP)
  • bacteriophage T4 RNA ligase 2