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

结合计算设计和分子进化来设计针对细菌抑制因子 TetR 和 GntR 家族成员的 RNA 适体

• 批准号: 491295859
• 负责人: Professor Dr. Yves André Muller
• 金额: --
• 依托单位: Fachgebiet Molecular Genetics
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/491295859?language=en
• 关键词: Combining; computational; design; molecular; evolution

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.

短链和长链非编码rna是所有生命体中重要的基因表达调控因子。因此，RNA分子在合成生物学中占有重要地位，小的调控RNA、核开关和核酶正被用作设计合成遗传电路和网络的调控装置。通过体外选择（SELEX, Systematic Evolution of Ligands by Exponential enrichment），可以重新鉴定具有非凡亲和力和特异性结合靶标的RNA分子。这些所谓的适体可以采用明确的三维结构，如结合口袋或类似于抗体中发现的裂隙状相互作用表面。申请人Beatrix Suess的实验室之前已经选择并鉴定了一种RNA适体，该适体可以与细菌转录抑制蛋白TetR结合，从而竞争其DNA结合。我们演示了该适体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tetrr - rna适体复合物的晶体结构，并利用定点诱变、尺寸排除色谱、电泳迁移量转移测定和滴定量热法全面表征了tetrr - rna适体与tetrr -算子DNA的相互作用（Grau等，2020，NAR, PMID: 32052019）。在目前的提案中，申请人寻求探索两种方法的协同潜力，即计算设计和分子进化，用于设计针对细菌抑制因子的新型RNA适体，不仅针对TetR成员，还针对GntR/HutC家族成员。申请人过去曾广泛研究过这些抑制因子，同时在体外选择和计算设计领域表现出专业知识。该提案还旨在扩展目前可用的计算机软件的能力，包括设计新的rna -蛋白质界面，从而也包括dna -蛋白质界面。功能与结构结合表位的特性将在这些新的RNA适体-细菌阻遏物复合物中详细表征，并与天然操作符dna -阻遏物复合物进行比较。将进行初步实验，以探索这些新型适配体-抑制因子对作为合成生物学工具的潜力。综上所述，这些研究将为新型逻辑门的设计创造新的机会，这些逻辑门可以被纳入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复合物开辟新的途径。