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A novel cascade assembly reaction is applied to sensitively distinguish between low abundance miRNAs and pre-miRNAs in living cells., Mature microRNAs (miRNAs) produced from precursor microRNAs (pre-miRNAs) by the RNase Dicer have showed significant potential for cancer diagnosis and prognosis due to their key regulatory roles in various pathological processes. However, discriminatory detection of low-abundance miRNAs and pre-miRNAs remains a key challenge since the mature sequence is also present in the pre-miRNA forms. Herein, we report a novel cascade reaction to sensitively distinguish miRNAs versus pre-miRNAs in living cells based on two pairs of programmable hairpin oligonucleotide probes with a simple sequence design. The programmable hairpin probes can metastably coexist until the introduction of miRNAs or pre-miRNAs, which can trigger a specific hybridization chain reaction (HCR), respectively, leading to the self-assembly of nicked DNA duplex structures and a remarkable specific fluorescence intensity increase. The system can readily and sensitively assess the miRNA or pre-miRNA abundance in a homogeneous solution. The intracellular miRNA and pre-miRNA expression level assessment in different living cells is realized. Thus, we provide a novel investigation tool for discriminatorily and accurately assessing miRNA and pre-miRNA abundance, which could be useful for the biomedical application of miRNAs.

We designed a porous 3D Au–DNA hydrogel network for simultaneously imaging multiplex miRNAs in living cells through dual-signal amplification., Non-invasively imaging multiplex microRNAs (miRNAs) in living cells is pivotal to understanding their physiological functions and pathological development due to the key regulatory roles of miRNAs in gene expression. However, developing smart delivery systems with large gene loading capacity, biocompatibility and responsiveness remains a significant challenge. Herein, we successfully incorporated DNA-capped Au nanoparticles (NPs) and their complementary fluorescent DNA sequences into a porous 3D hydrogel network (AuDH), in which hairpin-locked DNAzyme strands and active metal ions were loaded (AuDH/Mn+/H) for simultaneously imaging multiplex miRNAs in living cells. After transfection into cells, the specific miRNAs trigger the strand-displacement reaction and sequentially activate the DNAzyme-assisted target recycling, leading to a strong increase in the corresponding fluorescence intensity for imaging. This enables simultaneous assessment of the abundance of multiplex cancer-related miRNAs, even if at a very low expression level, in different cells through the different fluorescence intensities due to the dual signal amplification, and the change in abundance of miRNAs induced by siRNA or miRNA mimics in living cells can also be efficiently monitored. The versatile and responsive DNA hydrogel system holds great potential for miRNA biomedical applications.

Herein, we design a smart autonomous ATP self-powered strand displacement cascade amplification (SDCA) system for highly sensitive multiple intracellular miRNA detection., Herein, we design a smart autonomous ATP self-powered strand-displacement cascade amplification (SDCA) system for highly sensitive multiple intracellular miRNA detection. Rationally engineered Y-motif DNA structures are functionalized on mesoporous silica-coated copper sulfide nanoparticles loaded with numerous ATPs (CuS@mSiO2-Y/ATP) through pH stimulus-responsive disulfide bonds. The SDCA system is implemented by endogenous specific miRNA as a trigger and ATP as fuel released from the nanocarrier at acidic pH and photothermal stimuli-responsive CuS. The ATP self-powered SDCA process presents higher sensitivity compared to that without amplification for intracellular miRNA imaging. Two-color simultaneous and sensitive imaging of multiple cancer-related miRNAs in living cells is also confirmed. This enables facile and accurate differentiation between normal cells and different types of cancer cell using intracellular miRNA imaging, which improves the veracity and timeliness for early cancer diagnosis.