Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA

Abstract

A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For ¹⁵ N-labeling, E.coli is grown on ¹⁵ N-ammonium sulfate, whereas for ¹³ C-labeling, Methylophilus methylotrophus is grown on ¹³ C-methanol, which is more economical than ¹³ C-glucose. To demonstrate the feasibility and utility of this method, uniformly ¹³ C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR.