Combining computational design and molecular evolution to devise RNA aptamers directed against members of the TetR and GntR family of bacterial repressors

短和长的非编码RNA是所有生命王国中基因表达的重要调节因素。因此，RNA分子在合成生物学中变得重要起来，小的调节RNA、核糖开关和核酶正被用作合成遗传电路和网络设计中的调节装置。可以通过体外选择(SELEX，指数富集型配体的系统进化)来鉴定与其靶标具有极高亲和力和特异性的RNA分子。这些所谓的适配子可以采用定义的三维结构，如结合口袋或裂隙状相互作用表面，类似于在抗体中发现的结构。申请人Beatrix Suess的实验室之前已经选择并鉴定了一种RNA适配子，它可以与细菌转录抑制蛋白TetR结合，从而竞争其DNA结合。我们演示了该适配子如何作为一种合成装置来调节基因表达的条件控制，例如作为剪接装置。最近，申请人Suess和Muller的实验室联手解决了TetR-RNA适配子复合体的晶体结构，并使用定点突变、大小排除层析、电泳迁移率改变分析和滴定量热法综合表征了TetR-RNA适配子与TetR-操纵子DNA的相互作用(Grau等人，2020年，NAR，PMID：32052019)。在本提案中，申请人试图探索两种方法的协同潜力，即计算设计和分子进化，以设计不仅针对TetR而且针对GntR/HutC细菌前驱体家族成员的新型RNA适配子。申请者在过去对这些阻遏物进行了广泛的研究，同时在体外选择和计算设计领域展示了专业知识。该提案还旨在扩大现有计算机软件的能力，以包括设计新的RNA-蛋白质界面，从而也包括DNA-蛋白质界面的设计。功能结合表位和结构结合表位的性质将在这些新的RNA适体-细菌抑制子复合体中得到详细的表征，并与天然操纵子DNA-抑制子复合体的性质进行比较。初步实验将探索这些新型适体-抑制子对作为合成生物学工具的潜力。结合这些研究，这些研究将为设计新的逻辑门创造新的机会，这些逻辑门可以整合到合成遗传电路和调控网络中。同时，这种协同方法将为成功设计蛋白质-RNA甚至蛋白质-DNA复合体开辟新的场所。

Short and long non-coding RNAs are important regulators of gene expression in all kingdoms of life. Consequently, RNA molecules have become prominent in synthetic biology, and small regulatory RNAs, riboswitches and ribozymes are being applied as regulatory devices in the design of synthetic genetic circuits and networks. RNA molecules that bind their target with extraordinarily affinity and specificity can be identified de novo by in vitro selection (SELEX, Systematic Evolution of Ligands by Exponential enrichment). These so-called aptamers can adopt defined three-dimensional structures such as binding pockets or cleft-like interaction surfaces similar to those found in antibodies.The lab of the applicant Beatrix Suess has previously selected and characterized an RNA aptamer that can bind to the bacterial transcription repressor protein TetR and thus compete for its DNA binding. We demonstrated how this aptamer can be used as a synthetic device to regulate the conditional control of gene expression, for example as a splicing device. Recently, the labs of both applicants Suess and Muller joined forces to solve the crystal structure of the TetR-RNA aptamer complex and comprehensively characterize the TetR-RNA aptamer versus TetR-operator DNA interaction using site-directed mutagenesis, size exclusion chromatography, electrophoretic mobility shift assays and titration calorimetry (Grau et al., 2020, NAR, PMID: 32052019).In the present proposal, the applicants seek to explore the synergistic potential of two approaches, i.e. computational design and molecular evolution, for the design of novel RNA aptamers directed against members of not only the TetR but also the GntR/HutC family of bacterial repressors. The applicants have extensively studied these repressors in the past and, at the same time, demonstrated expertise in the field of in vitro selection and computational design. The proposal also aims to expand the abilities of currently available computer software to include the design of novel RNA-protein interfaces, and hence also DNA-protein interfaces. The properties of the functional versus structural binding epitopes will be characterized in detail in these novel RNA aptamer-bacterial repressor complexes and compared to those of the natural operator DNA-repressor complexes. Initial experiments will be conducted to explore the potential of these novel aptamer-repressor pairs as tools in synthetic biology.In combination, these studies will generate new opportunities for the design of novel logic gates that could be incorporated into synthetic genetic circuits and regulatory networks. At the same time, the synergistic approach will open up new venues for the successful design of protein-RNA or even protein-DNA complexes.