Abstract
Gene expression, catalysed by RNA polymerases (RNAP), is one of the most fundamental processes in living cells. The majority of methods to quantify mRNA are based upon purification of the nucleic acid which leads to experimental inaccuracies and loss of product, or use of high cost dyes and sensitive spectrophotometers. Here, we describe the use of a fluorescent biosensor based upon the single stranded binding (SSB) protein. In this study, the SSB biosensor showed similar binding properties to mRNA, to that of its native substrate, single-stranded DNA (ssDNA). We found the biosensor to be reproducible with no associated loss of product through purification, or the requirement for expensive dyes. Therefore, we propose that the SSB biosensor is a useful tool for comparative measurement of mRNA yield following in vitro transcription.
Highlights
The information to develop and sustain life is encoded within DNA
This study has reinforced the idea that single stranded binding (SSB) is able to bind to multiple single stranded nucleic acid substrates
The fluorescence increase of the MDCC-SSB biosensor occurs in a substrate concentration dependent manner for both the ssRNA and single-stranded DNA (ssDNA)
Summary
The information to develop and sustain life is encoded within DNA. RNAPs facilitate the distribution of this information through the transcription of DNA into mRNA. The sample will need to be purified to remove both protein and DNA contamination, which leads to both, a loss of total RNA yield and increased experimental error. Both approaches require a high yield of mRNA and are not typically suitable for eukaryotic transcription assays. The latter typically use radioactively tagged nucleotides [6,7] or Reverse Transcription quantitative PCR (RT-qPCR) is a very sensitive method to quantify transcripts [8]. A need for a low cost, sensitive, easy to use reagent which can be added to the sample to directly compare in vitro transcription reactions without additional purification steps could be advantageous
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Biochemical and Biophysical Research Communications
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
