In Vivo, Large-Scale Preparation of Uniformly (15)N- and Site-Specifically (13)C-Labeled Homogeneous, Recombinant RNA for NMR Studies.

Knowledge of how ribonucleic acid (RNA) structures fold to form intricate, three-dimensional structures has provided fundamental insights into understanding the biological functions of RNA. Nuclear magnetic resonance (NMR) spectroscopy is a particularly useful high-resolution technique to investigate the dynamic structure of RNA. Effective study of RNA by NMR requires enrichment with isotopes of C-13 or N-15 or both. Here, we present a method to produce milligram quantities of uniformly N-15- and site-specifically C-13-labeled RNAs using wild-type K12 and mutant tktA Escherichia coli in combination with a tRNA-scaffold approach. The method includes a double selection protocol to obtain an E. coli clone with consistently high expression of the recombinant tRNA-scaffold. We also present protocols for the purification of the tRNA-scaffold from a total cellular RNA extract and the excision of the RNA of interest from the tRNA-scaffold using DNAzymes. Finally, we showcase NMR applications to demonstrate the benefit of using in vivo site-specifically C-13-labeled RNA.