Quantitative Detection and Real-Time Monitoring of Endogenous mRNA at the Single Live Cell Level Using a Ratiometric Molecular Beacon.

Abstract

Messenger ribonucleic acid (mRNA) plays an important role in various cellular processes. however, traditional techniques cannot realize mRNA detections in live cells as they rely on mRNA purification or cell fixation. To achieve real-time and quantitative mRNA detections at a single live cell level, a single-strand stem-loop-structured ratiometric molecular beacon (RMB) composed of the phosphorothioate-modified loop domain on the 2'-O-methyl RNA backbone with a reporter dye, quencher, and reference dye is proposed to detect the Hsp27 mRNA as a modeled endogenous mRNA. When the RMB hybridizes with the target, the stem-loop structure opens, causing separation of the reporter dye and the quencher and restores the reporter fluorescent signals; therefore, the Hsp27 mRNA can be quantitatively detected according to the ratio of the reporter fluorescent signal to the reference fluorescent signal. Both the phosphorothioate and 2'-O-methyl RNA modifications obviously reduce the nonspecific opening, and the additional reference dye ensures the detection precision using co-localization analysis. Not only does this remove the false-positive signal caused by the nuclease degradation-generated RMB fragment, but it also corrects variations caused by direct measurement of reporter fluorescence intensities at a single cell level owing to inhomogeneity in probe delivery. The designed RMB could detect the Hsp27 mRNA with high signal-to-noise ratio and sensitivity as well as excellent specificity and antidegradation capability proved in vitro and in live cells. Furthermore, it was successfully adopted in subcellular localization, quantitative copy number measurements, and even real-time monitoring of Hsp27 mRNA in live cells, demonstrating that the proposed RMB can be a potential quantitative endogenous mRNA detection tool, especially at a single live cell level.