生物分子相互作用介导调控功能的重塑与实现

We will use the σ factor of E. coli and artificial riboswitches as specific targets for engineering, to reengineer or realize in vivo regulatory functions of biomolecules through directed change of the specificity of intermolecular interactions or through introducing connections between molecular interactions and regularoty functions. More specifically, our proposed research includes: (1) To reenengineer the transcription starting function of the σ factor of E. Coli using continuous directed evolution and known structure-function relations, and to obtain σ factors with host-orthogonal as well as mutually orthogonal promoter specificities. Such factors are suitable for modular control of downstream gene groups, and for synthetic gene circuits with host-orthogonal transcriptions. (2) To realize cellular responses to diverse chemical signals using RNA riboswitches through a new in vitro selection approach that selects directly for switch functions. Subsequently, in vivo RNA switches responding to metabolites and light-induced compounds can be engineered, to overcome the current limitation that artificial in vivo RNA riboswitches have been implemented for only a few particular compounds. Through the above, we try to address the following key scientific questions: (1) How to engineer very large or fundamental changes in the regulatory properties of biomolecules, such as inter-molecular interactions with specificities that are orthogonal to starting points? (2) How to make core cellular processes to accept regulations by more diverse chemical species

本项目以大肠杆菌σ因子和人工RNA开关为对象，分别将定向改变生物分子相互作用特异性、引入相互作用与功能效应之间的联系作为重塑与实现生物分子调控功能的切入点。研究内容与目标包括（1）重塑大肠杆菌σ因子的转录启动功能，基于细胞内连续定向进化以及结构-功能关系，获得特异性识别人工启动子的宿主正交、相互正交σ因子，用于模块化控制下游基因群的转录，实现宿主正交的基因转录调控网络；（2）实现人工RNA开关响应化学信号的调控功能，建立RNA开关的体外筛选方法，克服目前只有针对少数特定化合物的胞内人工RNA开关的局限，进一步研究响应细胞内代谢中间物和响应人工光控合成化合物的RNA开关。拟解决的关键科学问题包括：（1）怎样大幅度定向改变生物重要调控属性,如与出发点正交的相互作用特异性；（2）怎样让细胞内核心过程能够接受更多不同的化学小分子调控。

本项目对蛋白质、核酸等生物分子与大分子和小分子特异性性相互作用的定向改造和调控功能重塑开展研究。在大分子-大分子相互作用方面，在大分子-大分子相互作用方面，用噬菌体辅助蛋白质连续定向进化（PACE）重复实现了大肠杆菌sigma70转录因子基于胞内自突变的定向进化，获得了该因子DNA识别区改变且有显著预期表型效应的变体；利用光调控的蛋白-蛋白相互作用构建了光响应的T7-RNA聚合酶，可用作原核生物光激活转录开关。在大分子-小分子相互作用方面，完成了别构RNA筛选的实验流程，分析了各环节的作用及其对总体效果的影响；发展了对特定蛋白家族类型的蛋白质-小分子作用进行生物信息学分析的方法，用数据驱动设计策略提出蛋白质改造方案，与合作者的实验验证结合，构建了细胞内重要代谢分子NADPH的首个荧光蛋白探针。陪养博士3人，硕士2人。博士后2人。发表研究SCI收录的研究论文8篇；英文专著章节1章