title: Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies.
abstract: Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically (13) C-labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile (13) C and (15) N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).
authors: Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, 1115 Biomolecular Sciences Building, College Park, MD 20782 (USA).
authors: Luigi J
authors: LJ
authors: Alvarado
authors: Regan M
authors: RM
authors: LeBlanc
authors: Andrew P
authors: AP
authors: Longhini
authors: Sarah C
authors: SC
authors: Keane
authors: Niyati
authors: N
authors: Jain
authors: Zehra F
authors: ZF
authors: Yildiz
authors: Blanton S
authors: BS
authors: Tolbert
authors: Victoria M
authors: VM
authors: D'Souza
authors: Michael F
authors: MF
authors: Summers
authors: Christoph
authors: C
authors: Kreutz
authors: T Kwaku
authors: TK
authors: Dayie
mesh_terms: D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011742:Pyrimidine Nucleotides / Q000096:biosynthesis* / Q000737:chemistry; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D011742:Pyrimidine Nucleotides; D012313:RNA
keywords: NMR spectroscopy; RNA; dynamics; labeling; structure