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 nanosheet-mediated and target-triggered assembly of hairpins. The MnO nanosheets can adsorb and exhibit an excellent quenching effect to the dye labeled hairpin probes. Besides, due to their biodegradability, the MnO 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 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.
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.
The detection of specific intracellular microRNAs (miRNAs) in living cells can potentially provide insight into the causal mechanism of cancer metastasis and invasion. However, because of the characteristic nature of miRNAs in terms of small sizes, low abundance, and similarity among family members, it is a great challenge to monitor miRNAs in living cells, especially those with much lower expression levels. In this work, we describe the establishment of a DNA-fueled and catalytic molecule machinery in cell signal amplification approach for monitoring trace and under-expressed miRNAs in living cells. The presence of the target miRNA releases the hairpin sequences from the dsDNA (containing the fluorescence resonance energy transfer (FRET) pair-labeled and unfolded hairpin sequences)-conjugated gold nanoparticles (dsDNA-AuNPs), and the DNA fuel strands assist the recycling of the target miRNA sequences via two cascaded strand displacement reactions, leading to the operation of the molecular machine in a catalytic fashion and the release of many hairpin sequences. As a result, the liberated hairpin sequences restore the folded hairpin structures and bring the FRET pair into close proximity to generate significantly amplified signals for detecting trace miRNA targets. Besides, the dsDNA-AuNP nanoprobes have good nuclease stability and show low cytotoxicity to cells, and the application of such a molecular system for monitoring trace and under-expressed miRNAs in living cells has also been demonstrated. With the advantages of in cell signal amplification and reduced background noise, the developed method thus offers new opportunities for detecting various trace intracellular miRNA species.
The monitoring and imaging of intracellular microRNAs (miRNAs) with specific sequences plays a vital role in cell biology as it can potentially elucidate many cellular processes and diseases related to miRNAs in living cells with accurate information. However, the detection of trace amounts of under-expressed intracellular miRNAs in living cells represents one of the current major challenges. In an effort to address this issue, we describe the establishment of an in cell catalytic hairpin assembly (CHA) signal amplification strategy for imaging under-expressed intracellular miRNAs in this work. Gold nanoparticles functionalized with FAM- and TAMRA-labeled hairpins with disulfide bonds in the stems are readily delivered into cells via endocytosis. Glutathione with evaluated concentrations in cancer cells cleaves the disulfide bonds in the hairpins by reduction to release the hairpins, and the target miRNAs further trigger CHA between the two hairpins to form many DNA duplexes, which bring the FAM and TAMRA labels into close proximity to generate apparently enhanced fluorescence resonance energy transfer (FRET) for the sensitive monitoring of low amounts of under-expressed miRNAs in live cancer cells. Using CHA to amplify the signal output and FRET to reduce the background noise, a significantly enhanced signal-to-noise ratio, thereby high sensitivity, over conventional fluorescence imaging can be realized, making our method particularly suitable for monitoring low levels of intracellular species.
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