Characterization of synthetic riboswitch in cell-free protein expression systems

Riboswitches are RNA-based regulatory elements that utilize ligand-induced structural changes in the 5ʹ-untranslated region of mRNA to regulate the expression of associated genes. The majority of synthetic riboswitches have been selected and tested in cell-based systems. Cell-free protein expression systems (CFPS) have several advantages for the development and testing of synthetic riboswitches, including eliminating interactions with complex cellular networks, and the decoupling of transcription and translation processes. To gain a better understanding of the riboswitch regulatory mechanism, to allow for more efficient riboswitch optimization and use for biosensing applications, we studied the performance of a theophylline-responsive synthetic riboswitch coupled with the superfolder green fluorescent protein (sfGFP) reporter gene in E. coli cellular extract and PURE cell-free systems. To monitor the mRNA dynamics, a malachite green aptamer sequence was added to the 3ʹ-untranslated region of sfGFP mRNA. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of the riboswitch mRNA was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both E. coli extract and PURE CFPS. However, sfGFP expression in the riboswitch construct was one order of magnitude lower, even at the highest concentration of theophylline. A mathematical model of riboswitch activation governed by the kinetic trapping mechanism was developed. Two factors – a reduced fraction of mRNA in the ‘ON’ state and a considerably lower translation initiation rate in the riboswitch – contribute to the much lower level of protein expression in the theophylline riboswitch compared to the control construct.

核糖开关（riboswitches）是一类基于RNA的调控元件，通过配体诱导的构象变化作用于信使RNA（mRNA）的5'非翻译区（5'-UTR），以此调控相关基因的表达。当前绝大多数合成核糖开关均在细胞体系中完成筛选与验证。无细胞蛋白表达系统（cell-free protein expression systems, CFPS）在合成核糖开关的开发与验证中具备多项优势：可规避与复杂细胞网络的相互作用，且能实现转录与翻译过程的解偶联。
为更深入解析核糖开关的调控机制，实现更高效的核糖开关优化并将其应用于生物传感领域，本研究针对响应茶碱的合成核糖开关，将其与超折叠绿色荧光蛋白（superfolder green fluorescent protein, sfGFP）报告基因偶联，分别在大肠杆菌（E. coli）细胞提取物系统与PURE无细胞系统中检测其性能。为监测mRNA动态变化，我们在sfGFP mRNA的3'非翻译区（3'-UTR）引入了孔雀石绿适配体（malachite green aptamer）序列。同时将该茶碱响应核糖开关的性能与组成型表达sfGFP的对照组进行对比。
在大肠杆菌提取物与PURE CFPS两种体系中，对不同浓度茶碱进行测试的结果显示，核糖开关mRNA的转录动态与对照组mRNA的转录过程高度相似。然而，即便在最高浓度茶碱处理下，核糖开关构建体的sfGFP表达量仍较对照组低一个数量级。本研究还建立了基于动力学捕获机制的核糖开关激活数学模型。研究表明，相较于对照组构建体，茶碱响应核糖开关的蛋白表达水平显著偏低主要源于两大因素：一是处于‘ON’状态的mRNA占比降低，二是核糖开关的翻译起始速率显著更低。