title: Synthesis of (6-(13)C)pyrimidine nucleotides as spin-labels for RNA dynamics.
abstract: We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed.
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Wunderlich
authors.forename: Romana
authors.initials: R
authors.lastname: Spitzer
authors.forename: Tobias
authors.initials: T
authors.lastname: Santner
authors.forename: Katja
authors.initials: K
authors.lastname: Fauster
authors.forename: Martin
authors.initials: M
authors.lastname: Tollinger
authors.forename: Christoph
authors.initials: C
authors.lastname: Kreutz
mesh_terms: D002247:Carbon Isotopes / Q000138:chemical synthesis / Q000737:chemistry; D003562:Cytidine / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000737:chemistry; D011742:Pyrimidine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques / Q000379:methods; D013113:Spin Labels / Q000138:chemical synthesis*; D014529:Uridine / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D009943:Organophosphorus Compounds; D011742:Pyrimidine Nucleotides; D012367:RNA, Viral; D013113:Spin Labels; C434331:phosphoramidite; D003562:Cytidine; D012313:RNA; D014529:Uridine