PREPARATION OF C-13 AND N-15 LABELED RNAS FOR HETERONUCLEAR MULTIDIMENSIONAL NMR-STUDIES

A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence. Uniformly labelled nucleotide 5' triphosphates (NTPs) were prepared from E.coli grown on C-13 and/or N-15 isotopically enriched media. These procedures routinely yield 180-mu-moles of labelled NTPs per gram of C-13 enriched glucose. The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription. Several C-13 and/or N-15 labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC). Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively. These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues. The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs. Examples of several such heteronuclear NMR experiments are shown.