RNA-Mediated Genetic Selections for Small Molecules

RNA 介导的小分子遗传选择

• 批准号: 7031315
• 负责人: JUSTIN P GALLIVAN
• 金额: $ 25.19万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031315
• 关键词: Escherichia coli; alkaloids; bacterial genetics; biosynthesis; biotechnology; chemical binding; chemical genetics; chemical synthesis; directed evolution; gene expression; genetic screening; genetic techniques; genetic translation; intermolecular interaction; messenger RNA; metabolomics; method development; molecular cloning; nucleic acid sequence; oligonucleotides; plant proteins; small molecule; theophylline

Genetic selection provides the most powerful method to assay large libraries of biomolecules for function, and harnessing the power of genetic selection for the detection of specific, non-endogenous small molecule targets would greatly facilitate the cloning of biosynthesis genes, the directed evolution of enzymes, and the engineering of metabolic pathways. While significant progress has been made in developing in vivo genetic selections for small molecule targets, there remain limitations to both the sizes of libraries and the types of molecules and chemical transformations that may be selected for. This proposal details a comprehensive effort to develop a method capable of performing genetic screens and selections for non-endogenous small molecule targets in bacteria. The proposed method addresses many of the limitations of current methods and rests on two well-established principles: The first is that it is possible to use in vitro selection to generate RNA sequences (aptamers) that tightly and specifically recognize small molecules, while discriminating against related structures. The second is that small molecule metabolites can interact with messenger RNAs to modulate gene expression in vivo through a mechanism known as riboswitch control. Results from our lab and from others show that it is possible to combine these ideas to generate synthetic riboswitches that recognize non-endogenous small molecules and control gene expression in E. coli. Herein we propose a method for producing synthetic riboswitches capable of mediating genetic selections for small molecules. This approach will be validated by using synthetic riboswitch-mediated genetic selections to clone and direct the evolution of enzymes within two important alkaloid biosynthesis pathways. Concurrent mechanistic studies will provide evidence of how the new synthetic riboswitches function and will likely illuminate principles that govern the function of natural riboswitches in vivo.

遗传选择提供了一种最有力的方法，可以分析用于功能的大型生物分子的库，并利用遗传选择的能力来检测特定的，非遗传的小分子靶标，这极大地将极大地促进生物合成基因的克隆，酶的定向演化，以及代理途径的工程。尽管在为小分子靶标开发体内遗传选择方面取得了重大进展，但文库的大小以及可以选择的分子和化学转化的类型仍然存在局限性。该建议详细介绍了一种全面的努力，以开发一种能够执行细菌中非遗传小分子靶标的遗传筛选和选择的方法。所提出的方法解决了当前方法的许多局限性，并基于两个公认的原则：第一个是可能 使用体外选择来生成紧密而专门识别小分子的RNA序列（适体），同时歧视相关结构。第二个是小分子代谢产物可以通过称为核糖开关控制的机制与信使RNA相互作用，以调节体内基因表达。我们实验室和其他实验室的结果表明，可以将这些思想结合起来，以产生识别非遗传小分子并控制大肠杆菌中的基因表达的合成核糖开关。 本文中，我们提出了一种产生能够介导小分子遗传选择的合成核糖开关的方法。该方法将通过使用合成核糖开关介导的遗传选择来克隆并将酶的演化定向在两个重要的生物碱生物合成途径中。并发的机械研究将提供证据证明新的合成核糖开关如何发挥作用，并可能阐明控制天然核糖开关在体内功能的原理。