MnO2 nanosheet-mediated target-binding-induced FRET strategy for multiplexed microRNAs detection and imaging in living cells

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 miRN-Aassociated biological research and disease diagnosis and treatment. Herein, a MnO2 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 MnO2 nanosheets by physisorption to form DNA/MnO2 nanocomposite probes. When the DNA/MnO2 nanocomposite probes were taken up by cells, the MnO2 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-MB231 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.

Automated summary

In this study, a MnO2 nanosheet-mediated target-binding-induced fluorescence resonance energy transfer (FRET) strategy was developed for detection and imaging of multiplexed miRNAs in living cells. The strategy successfully enabled simultaneous imaging of miRNA-373 and miRNA-96 in MDA-MB-231 and L02 cells, revealing higher expression levels of target miRNAs in MDA-MB-231 cells compared to L02 cells. This approach provides an alternative method for simultaneous accurate detection and imaging of multiplexed miRNAs with potential biomedical applications.