title: Stable isotope-labeled RNA phosphoramidites to facilitate dynamics by NMR.
abstract: Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes, methods to synthesize these RNAs for biophysical studies are much needed. Here, we showcase the applicability of 6-(13)C-pyrimidine phosphoramidites to introduce isolated (13)C-(1)H spin pairs into RNAs up to 40 nucleotides long. The method allows the incorporation of 6-(13)C-uridine and -cytidine residues at any desired position within a target RNA. By site-specific positioning of the (13)C-label using RNA solid phase synthesis, these stable isotope-labeling patterns are especially well suited to resolve resonance assignment ambiguities. Of even greater importance, the labeling pattern affords accurate quantification of important functional transitions of biologically relevant RNAs (e.g., riboswitch aptamer domains, viral RNAs, or ribozymes) in the μs- to ms time regime and beyond without complications of one bond carbon scalar couplings. We outline the chemical synthesis of the 6-(13)C-pyrimidine building blocks and their use in RNA solid phase synthesis and demonstrate their utility in Carr Purcell Meiboom Gill relaxation dispersion, ZZ exchange, and chemical exchange saturation transfer NMR experiments.
authors.affiliation: Institute of Organic Chemistry and Center for Biomolecular Sciences Innsbruck, University of Innsbruck, Innsbruck, Austria.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Wunderlich
authors.affiliation: Institute of Organic Chemistry and Center for Biomolecular Sciences Innsbruck, University of Innsbruck, Innsbruck, Austria.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Juen
authors.affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA.
authors.forename: Regan M
authors.initials: RM
authors.lastname: LeBlanc
authors.affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Longhini
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
authors.affiliation: Institute of Organic Chemistry and Center for Biomolecular Sciences Innsbruck, University of Innsbruck, Innsbruck, Austria. Electronic address: christoph.kreutz@uibk.ac.at.
authors.forename: Christoph
authors.initials: C
authors.lastname: Kreutz
mesh_terms: D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*
chemical_list: D009943:Organophosphorus Compounds; C434331:phosphoramidite; D012313:RNA
keywords: Chemical RNA synthesis; NMR dynamics; RNA phosphoramidites; Relaxation dispersion; Stable isotope site-specific labeling