Trausch Jeremiah J, Batey Robert T
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado, USA.
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado, USA.
Methods Enzymol. 2015;550:41-71. doi: 10.1016/bs.mie.2014.10.031. Epub 2014 Dec 19.
Genetically encodable RNA devices that directly detect small molecules in the cellular environment are of increasing interest for a variety of applications including live cell imaging and synthetic biology. Riboswitches are naturally occurring sensors of intracellular metabolites, primarily found in the bacterial mRNA leaders and regulating their expression. These regulatory elements are generally composed of two domains: an aptamer that binds a specific effector molecule and an expression platform that informs the transcriptional or translational machinery. While it was long established that riboswitch aptamers are modular and portable, capable of directing different output domains including ribozymes, switches, and fluorophore-binding modules, the same has not been demonstrated until recently for expression platforms. We have engineered and validated a set of expression platforms that regulate transcription through a secondary structural switch that can host a variety of different aptamers, including those derived through in vitro selection methods, to create novel chimeric riboswitches. These synthetic switches are capable of a highly specific regulatory response both in vitro and in vivo. Here we present the methodology for the design and engineering of chimeric switches using biological expression platforms.
能够在细胞环境中直接检测小分子的基因编码RNA装置，在包括活细胞成像和合成生物学在内的各种应用中越来越受到关注。核糖开关是细胞内代谢物的天然传感器，主要存在于细菌mRNA前导序列中并调节其表达。这些调控元件通常由两个结构域组成：一个结合特定效应分子的适体和一个向转录或翻译机制传递信息的表达平台。虽然早就确定核糖开关适体是模块化且可移植的，能够指导包括核酶、开关和荧光团结合模块在内的不同输出结构域，但直到最近，表达平台才被证明具有同样的特性。我们设计并验证了一组表达平台，这些平台通过一个二级结构开关来调节转录，该开关可以容纳各种不同的适体，包括那些通过体外筛选方法获得的适体，从而创建新型嵌合核糖开关。这些合成开关在体外和体内都能够产生高度特异性的调控反应。在这里，我们介绍使用生物表达平台设计和构建嵌合开关的方法。
