Measuring Metabolites using Riboswitch reports

使用 Riboswitch 报告测量代谢物

• 批准号: 7032348
• 负责人: RONALD R BREAKER
• 金额: $ 63.33万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2008-03-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7032348
• 关键词: NIH Roadmap Initiative tag; biotechnology; chemical binding; directed evolution; glucosamine; high throughput technology; messenger RNA; metabolomics; molecular cloning; reporter genes; ribosomes; small molecule; sugar phosphates; synthetic nucleic acid; technology /technique development

DESCRIPTION: A method is proposed to report the concentrations of cellular metabolites based on modified natural riboswitches. Riboswitches are untranslated mRNA regions that bind metabolites and regulate expression of the mRNA. Binding occurs without participation from proteins or other macromolecules. Riboswitches are widely distributed in bacteria and respond to such central molecules as glucosamine-6-phosphate (glcN6P) and thiamine pyrophosphate (TPP). We propose to apply our expertise in manipulating riboswitches and in creating functional RNAs to exploit this natural system as a new technology for reporting levels of metabolites of interest. The specific aims are to: 1) Alter the specificity of the natural glcN6P riboswitch using in vitro evolution. 2) Clone, sequence, and characterize glcN6P riboswitch variants. 3) Measure reporting of glcN6P analogs in bacteria with variant clones. 4) Establish an automated method for obtaining any desired metabolite reporter. This work can be expanded in numerous fruitful directions. For example, efforts to modify the specificity of natural riboswitches to metabolites of interest can segue into large-scale plans to custom design riboswitches to report or sequester any desired metabolite. Individual riboswitches from bacteria can be investigated as reporters of matched metabolites in eukaryotes. Moreover, bioinformatics searches can be expanded to uncover examples of known or new riboswitches in humans and other eukaryotes.

描述：提出了一种基于修饰的天然核糖开关来报告细胞代谢物浓度的方法。核糖开关是结合代谢物并调节 mRNA 表达的非翻译 mRNA 区域。结合的发生无需蛋白质或其他大分子的参与。核糖开关广泛分布于细菌中，对 6-磷酸葡萄糖胺 (glcN6P) 和焦磷酸硫胺素 (TPP) 等中心分子有反应。我们建议运用我们在操纵核糖开关和创建功能性 RNA 方面的专业知识，以利用这一自然系统作为报告感兴趣的代谢物水平的新技术。 具体目标是： 1) 利用体外进化改变天然glcN6P核糖开关的特异性。 2) 克隆、测序和表征 glcN6P 核糖开关变体。 3) 测量具有变异克隆的细菌中glcN6P类似物的报告。 4) 建立一种自动化方法来获取任何所需的代谢物报告基因。 这项工作可以在许多富有成效的方向上扩展。例如，修改天然核糖开关对感兴趣的代谢物的特异性的努力可以演变为大规模计划，以定制设计核糖开关以报告或隔离任何所需的代谢物。细菌中的单个核糖开关可以作为真核生物中匹配代谢物的报告基因进行研究。此外，生物信息学搜索可以扩展到揭示人类和其他真核生物中已知或新核糖开关的例子。