Ponchon Luc, Dardel Frédéric
Cristallographie & RMN Biologiques, Université Paris Descartes, CNRS, 4 avenue de l'Observatoire, 75006, Paris, France.
Nat Methods. 2007 Jul;4(7):571-6. doi: 10.1038/nmeth1058. Epub 2007 Jun 10.
RNA has emerged as a major player in most cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. So far, this has been a bottleneck, as the only methods for producing such pure RNA have been in vitro syntheses. Here we describe a generic approach for expressing and purifying structured RNA in Escherichia coli, using tools that parallel those available for recombinant proteins. Our system is based on a camouflage strategy, the 'tRNA scaffold', in which the recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular RNases. This opens the way to large-scale structural and molecular investigations of RNA function.
RNA已成为大多数细胞过程中的主要参与者。要在分子水平上理解这些过程,需要用于结构、生化和药理研究的均一RNA样本。到目前为止,这一直是个瓶颈,因为生产这种纯RNA的唯一方法是体外合成。在此,我们描述了一种在大肠杆菌中表达和纯化结构化RNA的通用方法,该方法使用的工具与用于重组蛋白的工具类似。我们的系统基于一种伪装策略,即“tRNA支架”,其中重组RNA被伪装成天然RNA,从而利用宿主机制,避开细胞内的核糖核酸酶。这为RNA功能的大规模结构和分子研究开辟了道路。