Live cell imaging of single RNA molecules with fluorogenic Mango II arrays
Live cell imaging of single RNA molecules with fluorogenic Mango II arrays
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DOI：
10.1038/s41467-020-14932-7
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发表时间：
2020-03-09
影响因子：
16.6
通讯作者：
Rueda, David S.
中科院分区：
文献类型：
--
作者：
Cawte, Adam D.;Unrau, Peter J.;Rueda, David S.
RNA分子在许多细胞过程中起着至关重要的作用。以单分子分辨率可视化它们在活细胞中的动力学对于阐明它们在RNA代谢中的作用至关重要。RNA适体，如菠菜和芒果，最近出现作为一个强大的背景自由活细胞RNA成像技术，由于其荧光性质的配体结合。在这里，我们报告了一种新型的芒果II适体阵列，用于活细胞和固定细胞中的RNA成像，具有高对比度和单分子灵敏度。Mango II和MS 2-tdMCP-mCherry双标记mRNA的直接比较显示使用荧光Mango适体的信噪比显著改善。使用编码（β-肌动蛋白mRNA）和长非编码（NEAT 1）RNA，我们表明芒果阵列不影响细胞定位。此外，我们可以跟踪单个mRNA延长的时间段，可能是由于漂白的荧光团替换。这种特性使得阵列很容易与结构照明超分辨率显微镜兼容。
RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (beta-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.