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Regio-Selective Chemical-Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

Authors

  • Dr. Luigi J. Alvarado,

    1. Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, 1115 Biomolecular Sciences Building, College Park, MD 20782 (USA)
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  • Regan M. LeBlanc,

    1. Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, 1115 Biomolecular Sciences Building, College Park, MD 20782 (USA)
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  • Andrew P. Longhini,

    1. Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, 1115 Biomolecular Sciences Building, College Park, MD 20782 (USA)
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  • Dr. Sarah C. Keane,

    1. Howard Hughes Medical Institute and Department of Chemistry & Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250 (USA)
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  • Dr. Niyati Jain,

    1. Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (USA)
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  • Dr. Zehra F. Yildiz,

    1. Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138 (USA)
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  • Prof. Blanton S. Tolbert,

    1. Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (USA)
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  • Prof. Victoria M. D'Souza,

    1. Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138 (USA)
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  • Prof. Michael F. Summers,

    1. Howard Hughes Medical Institute and Department of Chemistry & Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250 (USA)
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  • Prof. Christoph Kreutz,

    1. Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck (Austria)
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  • Prof. T. Kwaku Dayie

    Corresponding author
    1. Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, 1115 Biomolecular Sciences Building, College Park, MD 20782 (USA)
    • Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, 1115 Biomolecular Sciences Building, College Park, MD 20782 (USA)===

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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 13C-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 13C and 15N 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).

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