A Fully Enzymatic Method for Site-directed Spin Labeling of Long RNA

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2014 Jul 2

PMID 24981512

Citations 10

Authors

Isabelle Lebars

Bertrand Vileno

Sarah Bourbigot

Philippe Turek

Philippe Wolff

Bruno Kieffer

Affiliations

Soon will be listed here.

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.

Citing Articles

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Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR.

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