A METHOD FOR PRODUCTION OF C-13 N-15 DOUBLE-LABELED RNA IN ESCHERICHIA-COLI, AND SUBSEQUENT IN-VITRO SYNTHESIS OF RIBONUCLEOTIDE 5'-TRIPHOSPHATES/

In this paper we describe an enhanced method for the large scale produ ction of high quality C-13/N-15 labelled NTPs. High amounts of labelle d RNA was obtained from E. coli cells grown in C-13/N-15 enriched medi um and treated with chloramphenicol. Total RNA was extracted from sphe roplasted cells in the presence of SDS and proteinase K and subsequent ly degraded to Nh?Ps by nuclease P1 and high concentrations of nucleas e S1 in a low salt buffer. To avoid non-specific degradation of the RN A, nuclease digestion was performed in a short term reaction on native , not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographical ly separated and converted to the corresponding NTPs by a mixture of k inases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the C-13/N-15 labelled NTPs was tes ted by in vitro transcription.