Preparation of partially (2)H/(13)C-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 ((13)C, (15)N, (2)H) 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-(2)H-1,2,3,4,5,6-(13)C-d-glucose (d(7)-(13)C(6)-d-glucose) into [1,2,3,4,5,6(R)-(2)H-1,2,3,4,5,6-(13)C]-d-glucose (d(6)-(13)C(6)-d-glucose). [1′,3′,4′,5′′-(2)H-1′,2′,3′,4′,5′-(13)C]GTP (d(4)-(13)C(5)-GTP) was then produced from d(6)-(13)C(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 ∼60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d(4)-(13)C(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. ((13)C,(1)H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly (13)C/(15)N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with (13)C-labelled non-deuterated RNA.