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

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 (C-13, N-15, H-2) 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-H-2-1,2,3,4,5,6-C-13-d-glucose (d(7)-C-13(6)-d-glucose) into [1,2,3,4,5,6(R)-H-2-1,2,3,4,5,6-C-13]-d-glucose (d(6)-C-13(6)-d-glucose). [1',3',4',5''-H-2-1',2',3',4',5'-C-13]GTP (d(4)-C-13(5)-GTP) was then produced from d(6)-C-13(6)-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 similar to60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d(4)-C-13(5)-GTP, together with in vitro synthesised d(5)-UTP, d(5)-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. (C-13,H-1) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly C-13/N-15-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with C-13-labelled non-deuterated RNA.