Measuring Metabolites using Riboswitch reports(RMI)

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。核糖开关是未翻译的信使核糖核酸区域，它结合代谢产物并调节信使核糖核酸的表达。结合在没有蛋白质或其他大分子参与的情况下发生。核糖开关广泛存在于细菌中，对氨基葡萄糖-6-磷酸(GlcN6P)和硫胺素焦磷酸(TPP)等中心分子起作用。我们建议应用我们在操纵核糖开关和创造功能RNA方面的专业知识来开发这一自然系统，作为一种报告感兴趣代谢物水平的新技术。 具体目标是： 1)通过体外进化改变天然glcN6P核糖开关的特异性。 2)克隆、测序并鉴定glcN6P核糖开关变异体。 3)测量具有变异克隆的细菌中glcN6P类似物的报告。 4)建立获得所需代谢物报告的自动化方法。 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代谢物的特异性的努力可以细分为定制设计核糖开关以报告或隔离任何所需代谢物的大规模计划。细菌的单个核糖开关可以作为真核生物中相匹配的代谢物的报告者进行研究。此外，生物信息学搜索可以扩大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.