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

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Oxford University Press

RESUMO

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 ∼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-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.

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