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    Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons

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        Abstract

        We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D, 4′-D, or 5′,5″-D 2). Our synthetic approach is inspired by the pioneering work of Tolbert and Williamson, who developed a method for the convenient one-pot enzymatic synthesis of nucleotides (Tolbert, T. J. and Williamson, J. R. (1996) J. Am. Chem. Soc. 118, 7929–7940). Our protocol consists of a comprehensive list of required chemical and enzymatic reagents and equipment, detailed procedures for enzymatic assays and nucleotide synthesis, and chromatographic procedures for purification of deuterated nucleotides. As an example of the utility of specifically deuterated nucleotides, we used them to synthesize specifically deuterated sarcin/ricin loop (SRL) RNA and measured the deuterium kinetic isotope effect on hydroxyl radical cleavage of the SRL.

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        Most cited references 21

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        SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments.

        Footprinting is a powerful and widely used tool for characterizing the structure, thermodynamics, and kinetics of nucleic acid folding and ligand binding reactions. However, quantitative analysis of the gel images produced by footprinting experiments is tedious and time-consuming, due to the absence of informatics tools specifically designed for footprinting analysis. We have developed SAFA, a semi-automated footprinting analysis software package that achieves accurate gel quantification while reducing the time to analyze a gel from several hours to 15 min or less. The increase in analysis speed is achieved through a graphical user interface that implements a novel methodology for lane and band assignment, called "gel rectification," and an optimized band deconvolution algorithm. The SAFA software yields results that are consistent with published methodologies and reduces the investigator-dependent variability compared to less automated methods. These software developments simplify the analysis procedure for a footprinting gel and can therefore facilitate the use of quantitative footprinting techniques in nucleic acid laboratories that otherwise might not have considered their use. Further, the increased throughput provided by SAFA may allow a more comprehensive understanding of molecular interactions. The software and documentation are freely available for download at http://safa.stanford.edu.
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          Isotope labeling strategies for NMR studies of RNA

          The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of long-range 1H–1H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, may provide the best opportunities for obtaining structural information by NMR. Here we review methods that have been developed for preparing and purifying isotopically labeled RNAs, as well as NMR strategies that have been employed for signal assignment and structure determination.
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            DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone

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              Author and article information

              Affiliations
              [1 ]Department of Chemistry, Boston University, Boston, MA 02215, USA
              [2 ]Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
              [3 ]Program in Bioinformatics, Boston University, Boston, MA 02215, USA
              Author notes
              [* ]Corresponding author's e-mail address: tullius@ 123456bu.edu
              Contributors
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              Journal
              SOR-LIFE
              ScienceOpen Research
              ScienceOpen
              2199-1006
              29 May 2015
              : 0 (ID: eb44f1b0-c408-4336-a2c0-aed203250898 )
              : 0
              : 1-20
              2751:XE
              10.14293/S2199-1006.1.SOR-LIFE.ALCJCN.v1
              © 2015 Azad et al.

              This work has been published open access under Creative Commons Attribution License CC BY 4.0 , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at www.scienceopen.com .

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              Figures: 17, Tables: 11, References: 20, Pages: 20
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