Transcription, catalysed by RNA polymerases, is one of the most fundamental processes in living cells (1). Characterizing the enzymatic process or RNA generation for use in CRISPR requires quantifying RNA transcripts. This typically requires puriﬁcation of the nucleic acids leading to experimental inaccuracies and loss of product. Synthetic ﬂuorescent dyes are available but are relatively expensive. Here, we describe the use of a ﬂuorescent biosensor based upon the single-stranded binding (SSB) protein (2), which has previously been established as an ssDNA biosensor (2). The protein is easily expressed in E. coli and can be used without prior puriﬁcation of the RNA, thereby providing a low cost, easy to use alternative for measuring mRNA.
SSB binds to 65-70 base lengths of ssDNA as a tetrameric protein. The binding site length represents the limiting resolution of the biosensor.
Materials & Methods
- Black 96-well microplate from Nunc, USA. #7605
- CLARIOstar®, BMG LABTECH, Germany.
- MDCC from Invitrogen, UK. # D10253
- E.Coli SSB G26C, Addgene, USA. Plasmid #78203. ssRNA70, Euroﬁns, UK
- HiScribe T7 RNA Synthesis Kit (NEB)
All other reagents supplied by Sigma Aldrich, UK
- Puriﬁcation and Labelling of SSB with MDCC
SSB protein expression, puriﬁcation and labelling with MDCC was performed as described by Dillingham et al (1) and Cook et al (2).
- Titration of Oligonucleotides to MDCC-SSB (calibration)
All reactions were performed at 25 °C in a buffer containing 50 mM Tris.HCl pH 7.5, 3 mM MgCl2 and 100 mM NaCl with 200 nM MDCC-SSB in a ﬁnal volume of 100 µL. SSB is a tetrameric protein therefore biosensor concentration is 50 nM. For the measurements, dispense 100 µl MDCC-SSB to the 10-20 wells. Serial dilute 100 µl of 2 µM ssRNA70. Fluorescence change is then calculated relative to an MDCC-SSB only control. Measurements were done on a CLARIOstar microplate reader with the settings indicated below.
- In vitro Transcription and mRNA quantiﬁcation
In vitro transcription was performed with the HiScribe T7 RNA Synthesis kit. The control template generates a 2225 base run-off transcript. Reactions were performed at 30 °C for 20 120 min. mRNA was puriﬁed using RNeasy® kit. Half of the sample was used for RT-qPCR quantiﬁcation with the QuantiFast SYBR Green qPCR kit. The remaining sample was transferred to a microplate and mixed with MDCC-SSB with the assay conditions outlined above.
|Optic settings||Fluorescence spectra, Top optic|
|Monochromator settings||Ex. 400-10->440-10/470-16
||Adjusted prior to measurement|
|General settings||Number of flashes||40|
|Settling time||0.1 s|
Results & Discussion
The MDCC-SSB ﬂuorescence emission increases 2.1 fold when binding to ssRNA70 (Fig. 2).
Fluorescence of the MDCC-SSB depends on the concentration of ssRNA70 and becomes saturated once all biosensor is bound to substrate (Fig. 3). The MDCC-SSB biosensor (200 nM) detects mRNA in a linear range over two orders of magnitude, 5-500 nM. Within this linear range the biosensor can be calibrated in terms of concentration of SSB binding sites.
In vitro transcription by T7 polymerase driven from a T7 promoter resulted in a 2225 nucleotide run-off transcript. Transcription assays of different reaction duration (20-120 min) yielded different amounts of mRNA products. After product puriﬁcation, MDCC-SSB was added to each mRNA sample and MDCC-SB binding to the transcripts was recorded (Fig. 4). The linear response in ﬂuorescence intensity versus amount of mRNA indicates that the biosensor detects differences in transcript yield.
To increase versatility of the biosensor, we tested if mRNA puriﬁcation is required. We found that MDCC-SSB binds to both puriﬁed and non-puriﬁed mRNA (Fig. 5). The latter prevents loss of product through the isolation steps. Performing the titration in Fig. 3 enables the ﬂuorescence signal to be calibrated in terms of SSB binding sites therefore the biosensor can generate quantitative data. Calibrations should be performed under the same experimental conditions as the transcription reactions.
The MDCC-SSB biosensor is a low cost, fast, easy to use and reliable tool for measuring mRNA from in vitro transcription assays. The tool does not require RNA puriﬁcation. Moreover, the biosensor can be used in both qualitative and quantitative assays. With this tool faster identiﬁcation of transcription components will lead to a better understanding of the complex nature of transcription. The CLARIOstar assisted in assay development and reliably quantiﬁ es mRNA with MDCC-SSB.
- Hahn, S. (2004) Nature Structural & Molecular Biology, 11, 394-403. DOI: 10.1038/nsmb763
- Cook, A. et al. (2018) Biochemical and biophysical research communications. DOI: 10.1016/j. bbrc.2018.01.147
- Dillingham, M. et al. (2008). Biophysical Journal, 95, 3330-3339. DOI: 10.1529/biophysj.108.133512