Design of a Fluorescent mRNA Biosensor

Currently, to measure the amount of mRNA produced during transcription, a post transcription assay is required, such as gel electrophoresis or reverse transcription- quantitative polymerase chain reaction (RT-qPCR) which all require RNA purification steps. Both methods are subject to high error and in addition, RT-qPCR is a long process which can take hours to run. This study describes a method in which post transcriptional mRNA can be qualitatively measured between reaction conditions or quantitatively measured after calibration. The post transcription assay utilises a fluorescent Escherichia coli single stranded binding protein (SSB). In this study, SSB showed similar binding properties to mRNA, to that of its native substrate, ssDNA. A mutant of SSB -SSB G26C- used in previous studies, was fluorescently labelled with 7-Diethylamino-3-((((2-Maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC). A fluorescence increase occurs when the SSB is bound to both ssDNA and ssRNA, and this increase is dependent on substrate concentration. This study shows the MDCC-SSB can be used as a comparative measurement of mRNA concentrations following in vitro transcription. After finding this method unsuitable for real time studies, we investigated potential causes for this behaviour and comment on improvements to the biosensor