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Chembiochem. 2014 Jul 21;15(11):1573-7. doi: 10.1002/cbic.201402130. Epub 2014 Jun 20.

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

Author information

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

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).

KEYWORDS:

NMR spectroscopy; RNA; dynamics; labeling; structure

PMID:
24954297
PMCID:
PMC4127085
DOI:
10.1002/cbic.201402130
[Indexed for MEDLINE]
Free PMC Article
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