Design criteria for synthetic riboswitches acting on transcription

Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

核糖开关（Riboswitches）是一类基于RNA的基因表达调控因子，由配体识别适配体结构域与重叠的表达平台构成。其调控可发生于转录水平（通过形成终止子或抗终止子结构）或翻译水平（通过暴露或遮蔽核糖体结合位点）。由于具备模块化组成特性，这类元件可通过组合不同适配体与表达平台进行改造，因此成为合成生物学中调控基因表达的实用工具。

本研究利用人工设计的依赖茶碱的核糖开关，证实终止子发夹稳定性与折叠陷阱这两个参数对设计构建体的功能具有关键性影响，需在设计阶段予以审慎考量。

此外，将多个独立核糖开关进行串联排布，可大幅提升诱导态与未诱导态基因活性间的激活比率，并使报告基因表达呈现线性剂量依赖性增长。这类合成核糖开关的串联排布与其天然同源物高度相似，有望成为用于细胞内特定靶分子检测的简易定量读出系统的基础。