Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
J Am Chem Soc. 2012 May 2;134(17):7558-69. doi: 10.1021/ja302148g. Epub 2012 Apr 19.
We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed.
我们提出了一种基于 (13)C 的 RNA 同位素标记方案。通过使用 (6-(13)C)嘧啶核苷亚磷酰胺构建块，可以通过化学寡核苷酸固相合成将特异性位点标记物掺入靶 RNA 中。这种标记方案特别适用于通过 NMR 光谱研究毫微秒到微秒的动力学，因为孤立的自旋体系是应用 Carr-Purcell-Meiboom-Gill (CPMG) 弛豫弥散型实验的关键前提。我们通过将 (6-(13)C)尿嘧啶和胞嘧啶标记物掺入具有生物学相关性的 RNA 中，证明了该方案在各种时间尺度上用于表征和检测功能动力学的适用性。我们研究了参与由 preQ(1) 核糖体开关 I 类控制的基因调控过程的双稳态终止子抗终止子片段的重折叠动力学。使用 (13)C CPMG 弛豫弥散 NMR 光谱，研究了 HIV-1 反式激活反应元件 RNA 和 Varkud 卫星茎环 V 基序的毫微秒到微秒动力学。
