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，由配体感应型适体结构域（aptamer domain）与紧随其后的重叠式表达平台构成。其调控可发生于转录或翻译层面：转录调控通过形成终止子或抗终止子结构实现，翻译调控则通过暴露或遮蔽核糖体结合位点完成。鉴于其模块化的结构组成，可通过组合不同的适体结构域与表达平台对这类元件进行工程化改造，因此成为合成生物学领域中用于调控基因表达的可靠工具。本研究利用计算机设计的茶碱（theophylline）依赖性核糖开关，证实终止子发夹稳定性与折叠陷阱（folding traps）这两个参数对所设计构建体的功能具有显著影响，因此在设计阶段需对其予以充分审慎的考量。此外，将单个核糖开关进行多拷贝串联排布，可显著提升诱导与未诱导状态下的基因活性比值，并使报告基因的表达呈现线性剂量依赖型递增。这类合成核糖开关的串联排布与其天然同源结构高度相似，有望为开发用于细胞内特定靶分子检测的简易定量读出系统奠定基础。