RNA-Mediated Genetic Selections for Small Molecules

RNA 介导的小分子遗传选择

基本信息

批准号：
7765577
负责人：
JUSTIN P GALLIVAN
金额：
$ 22.65万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7765577
关键词：
Abbreviations Address Affinity Alkaloids Anabolism Bacteria Behavior Beverages Binding Binding Sites Biochemical Biological Biological Assay Biological Factors Caffeine Catabolism Cells Characteristics Chemicals Cloning Codeine Coffea arabica (Plant)Coffee Coptis Cytochrome P-450 CYP1A2 Detection Development Engineering Enzymes Escherichia coli Evolution Future Gene Expression Gene-Modified Genes Genetic Genetic Screening Goals In Vitro Libraries Life Mediating Medicine Messenger RNA Metabolic Metabolic Pathway Methods Methylation Monitor Morphine Pathway interactions Plants Problem Solving Production Proteins RNA RNA Sequences Research Personnel Rest Ribosomes Sensitivity and Specificity Solutions Structure Tea Theophylline Translations United States National Institutes of Health Untranslated Regions aptamer base cDNA Library demethylation design dimethyl sulfate directed evolution enzyme activity genetic selection improved in vivo insight methyl group programs reconstitution research study response small molecule tool

项目摘要

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.

遗传选择提供了最强大的方法，用于测定大量生物分子的库以进行功能，并利用遗传选择的能力来检测特定的非遗传小分子靶标将极大地促进生物合成基因的克隆，酶的定向进化以及代谢途径的工程。虽然在发展体内遗传方面取得了重大进展小分子靶标的选择，库的大小和类型都存在限制可以选择的分子和化学转化。该建议详细介绍了一项全面的努力，以开发一种能够执行遗传筛查和的方法细菌中非源性小分子靶标的选择。提出的方法解决了许多当前方法的局限性，并基于两个公认的原则：第一个是可能的使用体外选择来生成紧密而专门识别小的RNA序列（适体）分子，同时区分相关结构。第二个是小分子代谢物可以通过称为的机制与Messenger RNA相互作用，以调节体内基因表达 Riboswitch控制。我们实验室和其他实验室的结果表明，可以将这些想法结合起来产生识别非遗传小分子和控制基因的合成核糖开关大肠杆菌中的表达。本文中，我们提出了一种产生能够介导遗传选择的合成核糖开关的方法小分子。该方法将通过使用合成核糖开关介导的“基因选择”来验证克隆并指导酶在两个重要的生物碱生物合成途径中的进化。并发的机械研究将提供新的合成核糖开关的证据可能阐明了控制天然核糖开关在体内功能的原则。