Quantitative Detection and Real-Time Monitoring of Endogenous mRNA at the Single Live Cell Level Using a Ratiometric Molecular Beacon
Quantitative Detection and Real-Time Monitoring of Endogenous mRNA at the Single Live Cell Level Using a Ratiometric Molecular Beacon
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使用比率分子信标在单个活细胞水平上定量检测和实时监测内源 mRNA
DOI：
10.1021/acsami.9b12394
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发表时间：
2019-08-14
影响因子：
9.5
通讯作者：
Liu，Fei
中科院分区：
文献类型：
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作者：
Feng，Xufei;Kang，Wenjie;Liu，Fei
信使核糖核酸在多种细胞过程中发挥着重要作用。然而，传统的技术依赖于mRNA的提纯或细胞固定，无法实现活细胞中的mRNA检测。为了在单个活细胞水平上实现实时和定量的mRNA检测，提出了一种由2‘-O-甲基RNA骨架上的硫代修饰的环结构域与报告染料、猝灭剂和参考染料组成的单链茎环结构比率分子信标，用于检测Hsp27mRNA作为模拟的内源mRNA。当人民币与靶杂交时，茎环结构打开，导致报告染料和猝灭剂分离，恢复报告荧光信号，从而可以根据报告荧光信号与参考荧光信号的比例来定量检测Hsp27 mRNA。硫代和2‘-O-甲基RNA修饰均明显减少了非特异性开放，添加的参考染料通过共定位分析保证了检测精度。这不仅消除了核酸酶降解产生的人民币片段引起的假阳性信号，而且还纠正了由于探针传递中的不均匀而在单个细胞水平上直接测量报告荧光强度所引起的差异。经体外实验和活细胞实验证明，该方法能检测到信噪比高、灵敏度高、特异性强、抗降解能力强的Hsp27基因。此外，它还被成功地应用于活细胞中Hsp27的亚细胞定位、定量拷贝数测量，甚至实时监测，表明所提出的人民币可以成为一种潜在的内源性mRNA定量检测工具，特别是在单个活细胞水平上。
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.