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  • Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies.

Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies.

Chembiochem : a European journal of chemical biology (2014-06-24)
Luigi J Alvarado, Regan M LeBlanc, Andrew P Longhini, Sarah C Keane, Niyati Jain, Zehra F Yildiz, Blanton S Tolbert, Victoria M D'Souza, Michael F Summers, Christoph Kreutz, T Kwaku Dayie
要旨

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).

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Sigma-Aldrich
D-(-)-リボース, ≥99% (GC)
Sigma-Aldrich
D-(-)-リボース, 98%
Sigma-Aldrich
D-(-)-リボース, suitable for cell culture, BioReagent
Sigma-Aldrich
D-(-)-リボース, ≥98%