Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

Nucleic Acids Research
Jenny CromsigtS S Wijmenga

Abstract

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-l...Continue Reading

References

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Jan 1, 2000·Journal of Biomolecular Structure & Dynamics·J A CromsigtS S Wijmenga

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Citations

Aug 19, 2007·Journal of Biomolecular NMR·Radovan Fiala, Vladimír Sklenár
Aug 19, 2008·ACS Chemical Biology·Heather L SchultheiszJames R Williamson
Jul 1, 2010·Nucleosides, Nucleotides & Nucleic Acids·Mark Lukin, Carlos de los Santos
Jun 28, 2008·Nucleic Acids Research·Frank H T NelissenSybren S Wijmenga
Jan 19, 2011·Progress in Nuclear Magnetic Resonance Spectroscopy·Cyril DominguezFrédéric H-T Allain
Aug 3, 2005·Chembiochem : a European Journal of Chemical Biology·Cameron D MackerethMichael Sattler
May 19, 2012·Biopolymers·Qin ZhaoGeorge E Fox
Feb 6, 2010·Chembiochem : a European Journal of Chemical Biology·Falk Wachowius, Claudia Höbartner
Apr 20, 2016·Methods : a Companion to Methods in Enzymology·Andrew P LonghiniT Kwaku Dayie
Aug 31, 2016·Progress in Nuclear Magnetic Resonance Spectroscopy·Frank H T NelissenHans A Heus

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