Biodegradable MnO₂ Nanosheet-Mediated Signal Amplification in Living Cells Enables Sensitive Detection of Down-Regulated Intracellular MicroRNA

The monitoring of intracellular microRNAs plays important roles in elucidating the biological function and biogenesis of miRNAs in living cells. However, because of their sequence similarity, low abundance, and small size, it is a great challenge to detect intracellular miRNAs, especially for those with much lower expression levels. To address this issue, we have developed an in cell signal amplification approach for monitoring down-regulated miRNAs in living cells based on biodegradable MnO 2 nanosheet-mediated and target-triggered assembly of hairpins. The MnO 2 nanosheets can adsorb and exhibit an excellent quenching effect to the dye labeled hairpin probes. Besides, due to their biodegradability, the MnO 2 nanosheets feature highly reduced cytotoxicity to the target cells. Upon entering cells, the surface-adsorbed FAM- and Tamra (TMR)-conjugated hairpins can be released due to the displacement reactions by other proteins or nucleic acids and the degradation of the MnO 2 nanosheets by cellular GSH. Subsequently, the down-regulated target miRNA-21 triggers cascaded assembly of the two hairpins into long dsDNA polymers, which brings the fluorescence resonance energy transfer (FRET) pair, FAM (donor), and TMR (acceptor) into close proximity to generate significantly enhanced FRET signals for detecting trace miRNA-21 in living cells. By carefully tailoring the sequences of the hairpins, the developed method can offer new opportunities for monitoring various trace intracellular miRNA targets with low expression levels in living cells.