Biosynthetic preparation of 13C/15N-labeled rNTPs for high-resolution NMR studies of RNAs

TY - JOUR

T1 - Biosynthetic preparation of 13C/15N-labeled rNTPs for high-resolution NMR studies of RNAs

AU - Martino, Luigi

AU - Conte, Maria

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

U2 - 10.1007/978-1-62703-113-4_17.

DO - 10.1007/978-1-62703-113-4_17.

M3 - Article

SN - 1064-3745

VL - 941

SP - 227

EP - 245

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -