Lincoln Scott - Enzymatic de novo pyrimidine nucleotide synthesis.

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  J Am Chem   Soc. 2011 Jan 19;133(2):297-304. doi:   10.1021/ja1059685. Epub 2010 Dec 17.


  The use of stable isotope labeling has revolutionized NMR studies   of nucleic acids, and there is a need for methods of   incorporation of specific isotope labels to facilitate specific   NMR experiments and applications. Enzymatic synthesis offers an   efficient and flexible means to synthesize nucleoside   triphosphates from a variety of commercially available   specifically labeled precursors, permitting isotope labeling of   RNAs prepared by in vitro transcription. Here, we recapitulate de   novo pyrimidine biosynthesis in vitro, using recombinantly   expressed enzymes to perform efficient single-pot syntheses of   UTP and CTP that bear a variety of stable isotope labeling   patterns. Filtered NMR experiments on (13)C, (15)N, (2)H-labeled   HIV-2 TAR RNA demonstrate the utility and value of this approach.   This flexible enzymatic synthesis will make implementing detailed   and informative RNA stable isotope labeling schemes substantially   more cost-effective and efficient, providing advanced tools for   the study of structure and dynamics of RNA molecules.

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