Synthesis of Specifically Deuterated Nucleotides for NMR Studies on RNA

Abstract

We propose a strategy for NMR structure determination of RNA based on deuteration and use of specific labeling patterns. This strategy involves the use of NTPs that are deuterated in the ribose ring except for specific positions, e.g. H2′, and that are either unlabeled or uniformly labeled in ¹³C and ¹⁵N in either the ribose or the base or both. Incorporation of these NTPs into an RNA sequence reduces both resonance line-width and spectral overlap. A limited number of combinations of these differently labeled NTPs in an RNA sequence suffices to obtain all relevant proton resonance assignments and structure parameters necessary for structure determination of larger systems (≫ 50 nucleotides). We describe the in vitro synthesis of the deuterated and/or ¹³C/¹⁵N-labeled NTPs from glucose via separate enzymatic reactions. First, enzymes from the pentose-phosphate pathway efficiently convert glucose into ribose and enzymes from nucleotide biosynthesis and salvage pathways subsequently convert the ribose into nucleosides triphosphates (NTPs). The enzymes from the pentosephosphate pathway are all commercially available; the remaining enzymes have been purified from over-expressing strains. Separate enzymatic reactions were used to convert ²H₇- ¹³C₆-glucose into [1′,3′,4′,5′,5″-²H₅-1′,2′,3′,4′,5′,2,4,5,6-¹³C₉-1,3-¹⁵N₂]UTP and ²H₇-glucose into [1′,3′,4′,5′,5″-²H₅]ATP, [1′,3′,4′,5′,5″-²H₅]GTP, and [1′,3′,4′,5′,5″-²H₅] CTP. The synthesis yields up to 1 gram of NTPs from 1 gram of glucose, which is about 5 to 10 times as efficient extraction for E. Coli grown on glucose. The synthesis presented here, is a modification of the method described by Tolbert & Williamson (1,2). ¹D and ²D NMR spectra were acquired to demonstrate the utility of the new labeling patterns. The enzymatically synthesized NTPs were used in the synthesis of a 31-nucleotide RNA derived from the primer binding site of Hepatitis B virus genomic RNA to asses their efficiency in transcription.