A fully enzymatic method for site-directed spin labeling of long RNA.
A fully enzymatic method for site-directed spin labeling of long RNA.
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DOI：
10.1093/nar/gku553
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发表时间：
2014-09
影响因子：
14.9
通讯作者：
Kieffer B
中科院分区：
文献类型：
--
作者：
Lebars I;Vileno B;Bourbigot S;Turek P;Wolff P;Kieffer B
位点定向自旋标记正在成为研究RNA结构和动力学特征的重要工具。我们在这里提出了一种酶的方法，它允许在RNA分子的特定位置插入顺磁中心。该技术基于使用T4 RNA连接酶2的连接协议的分段方法。一个转录的受体RNA与一个供体RNA连接，其中一个硫代修饰的核苷酸通过T7 RNA聚合酶的体外转录在其5 '端引入。顺磁硫醇特异性试剂随后附着在RNA连接产物上。通过将顺磁探针引入枯草芽孢杆菌tyrS T-Box先导RNA中对应K-turn和Specifier Loop结构域的55个核苷酸长的RNA中，证明了这种新策略。采用质谱法、电子顺磁共振（EPR）和核磁共振（NMR）对偶联反应的效率和自旋标记RNA的质量进行了评价。这种方法可以实现同位素片段标记和自旋标记方案的各种组合，这是一种通过核磁共振和EPR光谱研究大型RNA复合物的结构和动力学特性的特别有趣的策略。
Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies.
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影响因子：
5.6
作者：
Gillespie, JR;Shortle, D
通讯作者：
Shortle, D
影响因子：
14.9
作者：
Lebars I;Martinez-Zapien D;Durand A;Coutant J;Kieffer B;Dock-Bregeon AC
通讯作者：
Dock-Bregeon AC
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
影响因子：
3.5
作者：
Buettner, Lea;Seikowski, Jan;Hoebartner, Claudia
通讯作者：
Hoebartner, Claudia
影响因子：
2.9
作者：
Cai, Sheng;Zhu, Lingyang;Chen, Yuan
通讯作者：
Chen, Yuan