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.
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