Alvarado Luigi J, LeBlanc Regan M, Longhini Andrew P, Keane Sarah C, Jain Niyati, Yildiz Zehra F, Tolbert Blanton S, D'Souza Victoria M, Summers Michael F, Kreutz Christoph, Dayie T Kwaku
Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, 1115 Biomolecular Sciences Building, College Park, MD 20782 (USA).
Chembiochem. 2014 Jul 21;15(11):1573-7. doi: 10.1002/cbic.201402130. Epub 2014 Jun 20.
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).
同位素标记彻底改变了小分子核酸的核磁共振（NMR）研究，但要将这项技术扩展到更大的RNA，就需要位点特异性标记工具来加快NMR结构和动力学研究。利用磷酸戊糖途径中的酶，我们将化学合成的尿嘧啶核苷碱基与特异性（13）C标记的核糖偶联，以近乎定量的产率合成了UTP和CTP。这种化学酶法提供了一种经济高效的标记制备方法，这是目前方法无法实现的。该方法可生成通用的（13）C和（15）N标记模式，与弛豫优化的NMR光谱法一起使用时，可有效缓解导致低分辨率和低灵敏度的快速弛豫问题。该方法已在各种大小、复杂性和功能的RNA上得到验证：外显子剪接沉默子3（27个核苷酸）、铁反应元件（29个核苷酸）、原tRNA（76个核苷酸）和HIV-1核心包装信号（155个核苷酸）。
