title: In vivo, large-scale preparation of uniformly (15)N- and site-specifically (13)C-labeled homogeneous, recombinant RNA for NMR studies.
abstract: 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 (13)C or (15)N or both. Here, we present a method to produce milligram quantities of uniformly (15)N- and site-specifically (13)C-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 (13)C-labeled RNA.
authors.affiliation: Department of Chemistry and Biochemistry,Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA.
authors.forename: My T
authors.initials: MT
authors.lastname: Le
authors.affiliation: Department of Chemistry and Biochemistry, Department of Cellular Biology and Molecular Genetics, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Brown
authors.affiliation: Department of Chemistry and Biochemistry, Department of Cellular Biology and Molecular Genetics, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Simon
authors.affiliation: Department of Chemistry and Biochemistry,Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA. Electronic address: dayie@umd.edu.
authors.forename: T Kwaku
authors.initials: TK
authors.lastname: Dayie
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D011995:Recombination, Genetic
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012313:RNA
keywords: (15)N, (13)C isotopes; Escherichia coli; HNCO Experiment; Nuclear magnetic resonance spectroscopy; Ribonucleic acid; Selective biomass method; Site-specific labeling; tRNA-scaffold