bioRxiv Biochemistry
bioRxiv Biochemistry
5 years ago

A Reagentless Biosensor For mRNA: A New Tool To Study Transcription

C. P., Y., Toseland, Cook, Hari-Gupta, A.
Gene expression, catalysed by RNA polymerases, is one of the most fundamental processes in living cells. Yet, the means to study their activity are currently limited. The majority of methods to quantify mRNA are based upon initial purification of the nucleic acid. This leads to experimental inaccuracies and loss of product. Here we describe the use of a reagentless mRNA fluorescent biosensor based upon the single stranded binding (SSB) protein. In this study, SSB showed similar binding properties to mRNA, to that of its native substrate, ssDNA. Furthermore, MDCC-SSB gave the same fluorescence response with both ssDNA and ssRNA, in a concentration dependent manner. When directly compared to RT-qPCR, we found the biosensor to be more reproducible with no product lost through purification. Therefore, the MDCC-SSB can be used as a comparative measurement of mRNA concentrations following in vitro transcription.

Publisher URL: http://biorxiv.org/cgi/content/short/142794v1

DOI: 10.1101/142794

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