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NMR spectroscopy is an experimental method capable of providing high-resolution structural and dynamic information for RNA molecules in solution. The power of NMR as a structuralanalytical tool is greatly enhanced when heteronuclear methods can be applied to molecules enriched with C. These heteronuclear NMR methods have revolutionized the field of protein structure determination in the past several years. However, heteronuclear NMR has not yet been as widely exploited in the analysis of RNA, in large part due to the difficulty, inefficiency and expense of current methods of preparing isotopically enriched ribonucleotides. This currently presents a significant barrier to the application of NMR to the analysis of RNA. Here we describe an alternative method for the preparation of C enriched ribonucleotides, the essential precursors for either chemical or enzymatic in vitro synthesis of C enriched RNA. This method combines the advantage of using an inexpensive isotope source (sodium acetate) with the advantages of using Escherichia coli as the source of crude nucleotides. In addition, this method permits the distribution of C within the ribonucleotide products to be controlled by mixing various combinations of enriched and unenriched sodium acetate in the cell growth medium. This work is of particular significance to investigators using NMR methods to characterize the structure and dynamics of RNAs. Protocols have been previously reported for the preparation of C enriched ribonucleotides (1-4) and deoxyribonucleotides (5). These protocols call for using either glucose or methanol as the source of isotope. Procedures based on glucose involve growing E.coli on a defined medium containing C enriched glucose and isolating the cellular RNA, which is then digested to nucleotide monophosphates (NMPs). The NMPs are then converted to nucleotide triphosphates, the precursors for in vitro RNA synthesis. The main disadvantage of this procedure is that it requires an isotope source (glucose, C enriched at all six positions) that can be prohibitively expensive for large-scale preparations. Procedures based on methanol produce labeled ribonucleotides using raw material from the organism Methylophilus methylotrophus (1) or Methylobacterium extorquens (4). Although methanol is a relatively inexpensive source of C, the use of methylophiles have several significant disadvantages when compared to E.coli: they have a significantly lower ribonucleotide content than E.coli per gram of cells (1); the growth of E.coli is easier and more familiar to most researchers; and since E.coli is used by many investigators in preparing C-labeled proteins for NMR analysis (6), ribonucleotides can be isolated from E.coli as useful side products of protein preparations. In the work presented here, C enriched ribonucleotides were prepared from material isolated from E.coli grown using C sodium acetate as the source of carbon. The media contained the following components (per liter): 7 g Na2HPO4,3 g KH2PO4,1 g NH4CI, 0.5 g NaCl, 240 mg MgSO4,10 mg CaCl2,0.2 mg FeCl3, 0.15 mg ZnCl2,25 pig CoCl2,25 |ig CuSO4,25 u.g MnCl2,0.5 mg thiamine and 1.5 g of sodium acetate. The media was inoculated using cells from a culture growing in the same media but with ̂ -glucose rather than sodium acetate as the carbon source. Cells grown using acetate as the carbon source had significantly slower growth rates than cells grown on an equal mass of glucose, with typically 3 h doubling time versus 45 min. However, the cells ultimately reached the same density and total mass. Cells were harvested when the culture reached an absorbance of between 1.0 and 1.4 at 600 nm. The C-enriched ribonucleotides were isolated from the E.coli as described in excellent detail by Batey etal.(l). The harvested cells were resuspended in STE buffer (0.1 M NaCl, 10 mM Tris, 1 mM EDTA, pH 8) and frozen. After thawing, SDS was added up to a concentration of 1% to the cell lysate, which was then vigorously mixed. Cellular proteins were removed by phenol extraction. After centrifugation the RNAcontaining aqueous layer was removed from above the phenol and white protein-containing interface. Additonal STE buffer was added to the phenol-protein mixture, which was again mixed and centrifuged to extract additional RNA. The ribonucleotidecontaining aqueous layers were combined and extracted with 24:1 chloroform-isoamyl alcohol. The cellular RNA was precipitated with ethanol, and dissolved in acetate buffer at pH 5.2. The cellular RNA was digested to mononucleotides by adding 0.1 mM ZnCl2 and 30 U RNAse PI and incubating at 37°C overnight. The crude cell lysate contained 4100 A260 U/l cells. After phenol and chloroform extraction and ethanol precipitation, typically 1400 A26o U of nucleotides were obtained/1 of cells. In our hands, there was no significant difference in nucleotide yield from cells grown on glucose or either doubly or singly labeled acetate as an isotope source. With 99% C-enriched sodium acetate as the only significant source of carbon in the cell growth media, isotope enrichment of the ribonucleotides appeared to be nearly complete as judged by carbon-carbon coupling in C NMR spectra. This media was also used for preparing doubly

Preparation of carbon-13 labeled ribonucleotides using acetate as an isotope source