Genetically Encoded Dual-Color Light-Up RNA Sensor Enabled Ratiometric Imaging of MicroRNA
Genetically Encoded Dual-Color Light-Up RNA Sensor Enabled Ratiometric Imaging of MicroRNA
复制标题
基因编码双色发光 RNA 传感器可实现 MicroRNA 比例成像
DOI：
10.1021/acs.analchem.0c04588
复制
发表时间：
2021-02-02
影响因子：
7.4
通讯作者：
Jiang, Jian-Hui
中科院分区：
文献类型：
--
作者：
Dou, Cai-Xia;Liu, Chaoyang;Jiang, Jian-Hui
MicroRNAs(MiRNAs)在调控基因表达和细胞命运方面发挥着重要作用。然而，在活细胞中高精度地成像miRNAs仍然是一个巨大的挑战。在这里，我们报道了一种新型的基因编码的双色点亮RNA传感器，用于以芒果为内部参照物，以SRB2为传感器模块的miRNAs比率成像。这种基因编码的传感器是通过在单个启动子下表达内部参考和传感器模块的可拆分融合而设计的。这种设计策略允许两个模块在几乎不受干扰的情况下同步表达。活细胞成像研究表明，基因编码的比率RNA传感器对mir-224具有特异性反应。此外，传感器与芒果的荧光比率与mir-224的浓度呈线性相关，证实了它们测定活细胞中mir-224浓度的能力。我们的基因编码点亮RNA传感器还可以在不同的细胞系中对mir-224进行比率成像。这一策略可以为细胞内RNA的比率成像提供一种通用的方法，为询问活细胞中RNA的功能和丰度提供强大的工具。
MicroRNAs (miRNAs) play essential roles in regulating gene expression and cell fate. However, it remains a great challenge to image miRNAs with high accuracy in living cells. Here, we report a novel genetically encoded dual-color light-up RNA sensor for ratiometric imaging of miRNAs using Mango as an internal reference and SRB2 as the sensor module. This genetically encoded sensor is designed by expressing a splittable fusion of the internal reference and sensor module under a single promoter. This design strategy allows synchronous expression of the two modules with negligible interference. Live cell imaging studies reveal that the genetically encoded ratiometric RNA sensor responds specifically to mir-224. Moreover, the sensor-to-Mango fluorescence ratios are linearly correlated with the concentrations of mir-224, confirming their capability of determining mir-224 concentrations in living cells. Our genetically encoded light-up RNA sensor also enables ratiometric imaging of mir-224 in different cell lines. This strategy could provide a versatile approach for ratiometric imaging of intracellular RNAs, affording powerful tools for interrogating RNA functions and abundance in living cells.