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 MnO₂ nanosheet-mediated and target-triggered assembly of hairpins. The MnO₂ nanosheets can adsorb and exhibit an excellent quenching effect to the dye labeled hairpin probes. Besides, due to their biodegradability, the MnO₂ 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 MnO₂ 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

Simultaneous and Sensitive Sensing of Intracellular MicroRNA and mRNA for the Detection of the PI3K/AKT Signaling Pathway in Live Cells

Simultaneous detection of the concentration variations of microRNA-221 (miRNA-221) and PTEN mRNA molecules in the PI3K/AKT signaling pathway is of significance to elucidate cancer cell migration and invasion, which is useful for cancer diagnosis and therapy. In this work, we show the biodegradable MnO2 nanosheet-assisted and target-triggered DNAzyme recycling signal amplification cascaded approach for the specific detection of the PI3K/AKT signaling pathway in live cells via simultaneous and sensitive monitoring of the variation of intracellular miRNA-221 and PTEN mRNA. Our nanoprobes enable highly sensitive and multiplexed sensing of miRNA-221 and PTEN mRNA with low detection limits of 23.6 and 0.59 pM in vitro, respectively, due to the signal amplification cascades. Importantly, the nanoprobes can be readily delivered into cancer cells and the MnO2 nanosheets can be degraded by intracellular glutathione to release the Mn2+ cofactors to trigger multiple DNAzyme recycling cycles to show highly enhanced fluorescence at different wavelengths to realize sensitive and multiplexed imaging of PTEN mRNA and miRNA-221 for detecting the PI3K/AKT signaling pathway. Moreover, the regulation of PTEN mRNA expression by miRNA-221 upon stimulation by various drugs can also be verified by our method, indicating its promising potentials for both disease diagnosis and drug screening

Multicolor-Encoded Reconfigurable DNA Nanostructures Enable Multiplexed Sensing of Intracellular MicroRNAs in Living Cells

Despite the widespread utilization of gold nanoparticles and graphene for in vivo applications, complex steps for the preparation and functionalization of these nanomaterials are commonly required. In addition, the cytotoxicity of such materials is currently still under debate. In this work, by taking the significant advantages of DNA in terms of biocompatibility, nontoxicity, and controllability as building blocks for DNA nanostructures, we describe the construction of a reconfigurable, multicolor-encoded DNA nanostructure for multiplexed monitoring of intracellular microRNAs (miRNAs) in living cells. The DNA nanostructure nanoprobes containing two fluorescently quenched hairpins can be obtained by simple thermal annealing of four ssDNA oligonucleotides. The presence of the target miRNAs can unfold the hairpin structures and recover fluorescent emissions at distinct wavelengths to achieve multiplexed detection of miRNAs. Importantly, the DNA nanostructure nanoprobes exhibit significantly improved stability over conventional DNA molecular beacon probes in cell lysates and can steadily enter cells to realize simultaneous detection of two types of intracellular miRNAs. The demonstration of the self-assembled DNA nanostructures for intracellular sensing thus offers great potential application of these nanoprobes for imaging, drug delivery and cancer therapy in vivo