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表达的未翻译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。核糖开关在细菌中广泛分布，并对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，利用这一自然系统作为报告感兴趣代谢物水平的新技术。

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.