Wang Shuai, Zhang Liyan, Kan Ailing, Xu Xiaowen, Zhang Nan, Jiang Wei
School of Chemistry and Chemical Engineering, Shandong University, 250100, Jinan, Shandong, PR China.
Department of Oncology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, 250012, Jinan, Shandong, PR China.
Talanta. 2021 May 1;226:122202. doi: 10.1016/j.talanta.2021.122202. Epub 2021 Feb 6.
In the regulatory network, miRNAs play a regulatory role in a cooperative or antagonistic manner. Simultaneous accurate detection and imaging of multiplexed miRNAs in living cells are of great significance for miRNA-associated biological research and disease diagnosis and treatment. Herein, a MnO nanosheet-mediated target-binding-induced fluorescence resonance energy transfer (FRET) strategy was developed for detection and imaging of multiplexed miRNAs in living cells. Two pairs of DNA probes (P1-AF 488/P1'-Cy3 and P2-AF 488/P2'-AF 594) contained the complementary sequence to target miRNAs (miRNA-373 and miRNA-96) and labelled with different fluorescence dyes were designed. They were adsorbed onto MnO nanosheets by physisorption to form DNA/MnO nanocomposite probes. When the DNA/MnO nanocomposite probes were taken up by cells, the MnO nanosheets were reduced by intracellular glutathione, accompanying the release of DNA probe pairs. Then the DNA probe pairs specifically recognized and combined with miRNA-373 and miRNA-96 to form stable duplexes, respectively, bringing labelled fluorophores into close proximity to occur FRET. Based on this, the simultaneous imaging of miRNA-373 and miRNA-96 in MDA-MB-231 and L02 cells was successfully implemented. The results displayed a higher expression level of target miRNAs in MDA-MB-231 cells compared to L02 cells. The changes in expression levels of miRNA-96 induced by anti-miRNA-96 or mimics in MDA-MB-231 cells could also be monitored. In addition, the ratiometric detections of multiplexed miRNAs were achieved by utilizing the DNA probe pairs. The proposed strategy provides an alternative method for simultaneous accurate detection and imaging of multiplexed miRNAs and has potential application in biomedical applications.
在调控网络中，微小RNA（miRNA）以协同或拮抗的方式发挥调控作用。在活细胞中同时准确检测和成像多重miRNA对于与miRNA相关的生物学研究以及疾病的诊断和治疗具有重要意义。在此，开发了一种MnO纳米片介导的靶标结合诱导荧光共振能量转移（FRET）策略，用于活细胞中多重miRNA的检测和成像。设计了两对DNA探针（P1-AF 488/P1'-Cy3和P2-AF 488/P2'-AF 594），它们包含与靶标miRNA（miRNA-373和miRNA-96）互补的序列，并标记有不同的荧光染料。它们通过物理吸附吸附到MnO纳米片上，形成DNA/MnO纳米复合探针。当DNA/MnO纳米复合探针被细胞摄取时，MnO纳米片被细胞内的谷胱甘肽还原，同时释放出DNA探针对。然后，DNA探针对分别特异性识别并与miRNA-373和miRNA-96结合，形成稳定的双链体，使标记的荧光团紧密靠近以发生FRET。基于此，成功实现了MDA-MB-231和L02细胞中miRNA-373和miRNA-96的同时成像。结果显示，与L02细胞相比，MDA-MB-231细胞中靶标miRNA的表达水平更高。还可以监测抗miRNA-96或模拟物在MDA-MB-231细胞中诱导的miRNA-96表达水平的变化。此外，利用DNA探针对实现了多重miRNA的比率检测。所提出的策略为同时准确检测和成像多重miRNA提供了一种替代方法，在生物医学应用中具有潜在的应用价值。
