DOI：10.1038/nature14352 中国科研用户发表 作者：Yun-Xing Wang
摘要 : 一种能够按比例将修饰过的或标记过的核苷酸送入RNA分子中特定位置的方法的缺失，妨碍了RNA结构和动态研究。
一种能够按比例将修饰过的或标记过的核苷酸送入RNA分子中特定位置的方法的缺失，妨碍了RNA结构和动态研究。Yun-Xing Wang及同事现在报告了用于位置特定性、荧光或同位素标记的RNA合成的一个自动化固-液混合相转录过程。一个核糖开关(riboswitch)的标记显示对折叠没有影响，但的确显示了若干个构形状态。除了能够促进生物化学和生物物理研究外，被称为 “RNA的正选择标记”(PLOR)的这一标记方法也许可用来开发生物传感器和诊断方法。
Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid–liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch1 for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection2, and disease diagnostics.
来源： Nature 浏览次数：0