Abstract
High-resolution investigations of the structure and dynamics of RNA molecules by nuclear magnetic resonance (NMR) methodologies require the production of 13C/15N-isotopically labeled samples. A common strategy entails the preparation of 13C/15N-enriched ribonucleoside 5′-triphosphates (rNTPs) to be incorporated into RNA oligomers by in vitro transcription. Here, we describe the methods to obtain isotopically labeled rNTP in a uniform or selective fashion from bacterial cultures, using common and versatile E. coli strains. This chapter also covers procedures for extraction and digestion of the total RNA from bacterial cells, purification of the ribonucleoside 5′-monophosphates and their enzymatic phosphorylation to rNTPs.
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Acknowledgements
MRC is indebted to the Wellcome Trust. LM acknowledges the European Molecular Biology Organization (EMBO) for a long-term fellowship.
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Martino, L., Conte, M.R. (2013). Biosynthetic Preparation of 13C/15N-Labeled rNTPs for High-Resolution NMR Studies of RNAs. In: Conn, G. (eds) Recombinant and In Vitro RNA Synthesis. Methods in Molecular Biology, vol 941. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-113-4_17
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DOI: https://doi.org/10.1007/978-1-62703-113-4_17
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