PREPARATION OF ISOTOPICALLY LABELED RIBONUCLEOTIDES FOR MULTIDIMENSIONAL NMR-SPECTROSCOPY OF RNA

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 N-15-labeling, E.coli is grown on N-15-ammonium sulfate, whereas for C-13-labeling, Methylophilus methylotrophus is grown on C-13-methanol, which is more economical than C-13-glucose. To demonstrate the feasibility and utility of this method, uniformly C-13-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.