Measuring Metabolites using Riboswitch reports

描述：提出了一种基于修饰的天然核糖开关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。结合在没有蛋白质或其他大分子参与的情况下发生。核糖开关广泛分布于细菌中，并对诸如葡糖胺-6-磷酸（glcN 6P）和焦磷酸硫胺素（TPP）的中心分子作出反应。我们建议将我们的专业知识应用于操纵核糖开关和创建功能性RNA，以利用这种天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)使用体外进化改变天然glcN 6P核糖开关的特异性。 2)克隆、测序和表征glcN 6 P核糖开关变体。 3)测量具有变体克隆的细菌中glcN 6P类似物的报告。 4)建立一种自动化方法，以获得任何所需的代谢物报告。 这项工作可以在许多富有成效的方向上加以扩展。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发展成大规模的计划，以定制设计核糖开关来报告或隔离任何所需的代谢物。来自细菌的单个核糖开关可以作为真核生物中匹配代谢物的报告者进行研究。此外，生物信息学搜索可以扩展到发现人类和其他真核生物中已知或新的核糖开关的例子。

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.