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。特别是基于RNA的基因表达调控是一个有前途的应用，它允许对细胞行为进行多方面的控制。由于它们的模块化组成和易于操作，RNA调控元件如核糖开关特别令人感兴趣。在最近的过去已经证明-部分地由申请人证明-在翻译和转录水平上起作用的核糖开关可以在计算机上成功地设计。迄今为止，这种设计仅限于检测单个配体。在这里，我们建议研究如何反应的多个配体的特定浓度模式的核糖开关可以设计。多个人工适体结构域将以这样的方式整合，使得它们响应于配体结合的个体结构变化组合成RNA结构的总体变化，该总体变化在调节致动元件上施加明确的力，导致所需的总体生物学效应。将对计算机模拟预测进行实验测试，并进行定性和定量分析。基于计算机的预测和湿实验室分析的迭代过程将导致算法的预测能力以及所生成的核糖开关的调节能力的持续改进。利用一系列不同的适体元件，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工核糖开关，并将其扩展到进一步的监管水平，如tRNA加工反应。同时，我们将开发用户友好的软件，可以允许解决这种设计问题。

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.