Synthetic riboswitches: design of multi-ligand switches and novel regulatory principle

在合成生物学中，人工调控网络发挥着核心作用。尤其是基于RNA的基因表达调控是一种很有前途的应用，它允许对细胞行为进行多种控制。由于它们的模块组成和易于操作，调节RNA元件，如核糖开关，是特别感兴趣的。在最近的过去--部分由申请者--已经证明，在翻译和转录水平上作用的核糖开关可以在计算机上成功地设计出来。到目前为止，这种设计仅限于对单一配体的传感。在这里，我们建议研究如何设计对多个配体的特定浓度模式起反应的核糖开关。多个人工适配子结构域将以这样一种方式整合，即它们响应配基结合的单独结构变化与RNA结构的整体变化相结合，该变化对调节致动器元件施加明确的力，从而导致所需的整体生物效应。计算机预测将进行实验验证、定性和定量分析。基于计算机的预测和湿法实验室分析的迭代过程将导致算法的预测能力和产生的核糖开关的调节能力的不断改进。利用一组不同的适体元件，我们将开发一种普遍适用的方法来设计具有非平凡传感器逻辑的人造核糖开关，并将其扩展到进一步的调控水平，如tRNA加工反应。同时，我们将开发用户友好的软件，允许解决这类设计问题。

In synthetic biology, artificial regulatory networks play a central role. Especially RNA-based regulation of gene expression is a promising application that allows a versatile control of cell behavior. Due to their modular composition and easy manipulation, regulatory RNA elements like riboswitches are of particular interest. It has been demonstrated in the recent past - in part by the applicants - that riboswitches acting both at the translational and the transcriptional level can be designed successfully on the computer. To date, such designs have been limited to the sensing of a single ligand. Here, we propose to investigate how riboswitches that react to specific concentration patterns of multiple ligands can be designed. Multiple artificial aptamer domains will be integrated in such a way that their individual structural changes in response to ligand binding are combined to an overall change in RNA structure that exerts a well-defined force on the regulatory actuator elements leading to the desired overall biological effect. The in silico predictions will be experimentally tested and qualitatively as well as quantitatively analyzed. The iterative process of computer-based predictions and wet-lab analyses will lead to a continuous improvement of the predictive power of the algorithm and also of the regulatory power of the generated riboswitches. Making use of a collection of distinct aptamer elements, we will develop a universally applicable method to design artificial riboswitches with non-trivial sensor logic and expand this also to further regulatory levels like tRNA processing reactions. In parallel, we will develop user-friendly software that can allows to solve this kind of design problems.