Intracellular miRNA Imaging Research Articles

Live-Cell RNA Imaging with a DNA-Functionalized Metal-Organic Framework-Based Fluorescent Probe.

Live-cell fluorescence imaging of tumor-associated miRNAs is significant for understanding the cancer onset and progression and the diagnosis and prognosis of clinical diseases. In this protocol, we describe the construction of a DNA-functionalized metal-organic framework-based fluorescent probe and demonstrate that the probe can be used to detect miRNA in vitro and live-cell imaging. The formed DNA-MOF probe can specifically target miRNA with high sensitivity and specificity. The detection limit of the extracellular assay is as low as the picomolar level. In addition, we found that the probe could permeate cells and can be successfully applied in intracellular miRNA imaging, even achieving the distinction of cancer cells and normal cells, as well as the cancer cell lines with different miRNA expression levels.

A cancer-targeted glutathione-gated probe for self-sufficient ST/CDT combination therapy and FRET-based miRNA imaging.

Acancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. TheCGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin.After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

Endogenous Enzyme-Driven Amplified DNA Nanocage Probe for Selective and Sensitive Imaging of Mature MicroRNAs in Living Cancer Cells.

Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this goal remains challenging because of the interference of precursor miRNAs (pre-miRNAs) and the low abundance of mature miRNAs. Herein, we develop an endogenous enzyme-driven amplified DNA nanocage probe (Acage) for the selective and sensitive imaging of mature miRNAs in living cells. The Acage consists of a microRNA-responsive probe, an endogenous enzyme-driven fuel strand, and a DNA nanocage framework with an inner cavity. Benefiting from the size selectivity of DNA nanocage, smaller mature miRNAs rather than larger pre-miRNAs are allowed to enter the cavity of DNA nanocage for molecular recognition; thus, Acage can significantly reduce the signal interference of pre-miRNAs. Moreover, with the driving force of an endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) for efficient signal amplification, Acage enables sensitive intracellular miRNA imaging without an additional external intervention. With these features, Acage was successfully applied for intracellular imaging of mature miRNAs during drug treatment. We believe that this strategy provides a promising pathway for better understanding the functions of mature microRNAs in biological processes and medical diagnostics.

Toehold Strand Displacement-Mediated Exponential HCR for Highly Sensitive and Specific Analysis of miRNA in Living Cells.

As an important disease biomarker, the development of sensitive detection strategies for miRNA, especially intracellular miRNA imaging strategies, is helpful for early diagnosis of diseases, pathological research, and drug development. Hybridization chain reaction (HCR) is widely used for miRNA imaging analysis because of its high specificity and lack of biological enzymes. However, the classic HCR reaction exhibits linear amplification with low efficiency, limiting its use for the rapid analysis of trace miRNA in living cells. To address this problem, we proposed a toehold-mediated exponential HCR (TEHCR) to achieve highly sensitive and efficient imaging of miRNA in living cells using β-FeOOH nanoparticles as transfection vectors. The detection limit of TEHCR was as low as 92.7 fM, which was 8.8 × 103 times lower compared to traditional HCR, and it can effectively distinguish single-base mismatch with high specificity. The TEHCR can also effectively distinguish the different expression levels of miRNA in cancer cells and normal cells. Furthermore, TEHCR can be used to construct OR logic gates for dual miRNA analysis without the need for additional probes, demonstrating high flexibility. This method is expected to play an important role in clinical miRNA-related disease diagnosis and drug development as well as to promote the development of logic gates.

A DNA nanowire based-DNAzyme walker for amplified imaging of microRNA in tumor cells.

Sensitive imaging of microRNAs (miRNAs) in tumor cells holds great significance in the domains of pathology, drug development, and personalized diagnosis and treatment. DNA nanostructures possess excellent biostability and programmability and are suitable as carriers for intracellular imaging probes. With its highly controllable motion mechanism and remarkable target recognition specificity, the DNA walker is an ideal tool for living cell imaging. Here, we report a DNA nanowire based-DNAzyme Walker (D-Walker), which loads the DNAzyme based-molecular beacon (D-MB) onto DNA nanowires (NWs) functionalized with aptamers. The experimental results demonstrated that the intracellular target miRNA can specifically activate the pre-locked DNAzyme through a strand displacement reaction, thereby triggering the cleavage of its substrate molecular beacon (MB) and subsequent fluorescence emission. NWs decorated with aptamers can effectively prevent the degradation of the D-Walker by nuclease, and can enter target cells without any transfection reagents, which enhances the stability and reliability of cell imaging. Furthermore, the D-Walker exhibited a remarkable sensitivity with a limit of detection (LOD) of 61 pM and was capable of distinguishing miRNA-21 from other closely related family members. This study provides a novel strategy for intracellular miRNA imaging, offering a promising tool for cancer diagnosis and treatment.

Engineering of a Self-Regulatory Bidirectional DNA Assembly Circuit for Amplified MicroRNA Imaging.

The engineering of catalytic hybridization DNA circuits represents versatile ways to orchestrate a complex flux of molecular information at the nanoscale, with potential applications in DNA-encoded biosensing, drug discovery, and therapeutics. However, the diffusive escape of intermediates and unintentional binding interactions remain an unsolved challenge. Herein, we developed a compact, yet efficient, self-regulatory assembly circuit (SAC) for achieving robust microRNA (miRNA) imaging in live cells through DNA-templated guaranteed catalytic hybridization. By integrating the toehold strand with a preblocked palindromic fragment in the stem domain, the proposed miniature SAC system allows the reactant-to-template-controlled proximal hybridization, thus facilitating the bidirectional-sustained assembly and the localization-intensified signal amplification without undesired crosstalk. With condensed components and low reactant complexity, the SAC amplifier realized high-contrast intracellular miRNA imaging. We anticipate that this simple and template-controlled design can enrich the clinical diagnosis and prognosis toolbox.

Endogenous miRNA and K+ Co-Activated Dynamic Assembly of DNA Coacervates for Intracellular miRNA Imaging and Mitochondrial Intervention.

Intracellular dynamic assembly of DNA structures may be beneficial for the development of multifunctional nanoplatforms for the regulation of cell behaviors, providing new strategies for disease diagnosis and intervention. Herein, we propose the dynamic assembly of DNA coacervates in living cells triggered by miRNA-21 and K+, which can be used for both miRNA imaging and mitochondrial intervention. The rationale is that miRNA-21 can trigger the hybridization chain reaction to generate G-quadruplex precursors, and K+ can mediate the assembly of G-quadruplex-based coacervates, allowing the colorimetric detection of miRNA-21 ranging from 10 pM to 10 μM. Moreover, the as-formed DNA coacervates can specifically target mitochondria in MCF-7 breast cancer cells using the MCF-7 cell membrane as delivery carriers, which further act as an anionic shielding to inhibit communication between mitochondria and environments, with a significant inhibitory effect on ATP production and cellular migration behaviors. This work provides an ideal multifunctional nanoplatform for rationally interfering with cellular metabolism and migration behaviors through the dynamic assembly of DNA coacervates mediated by endogenous molecules, which has a large number of potential applications in the biomedical field, especially theranostics for cancer metastasis.

Trigger-activated autonomous DNA machine for amplified liver cancer biomarker microRNA21 imaging.

MicroRNA-21 (miRNA-21) is a kind of RNA that exists in biological fluids such as blood, urine and saliva. It has over expression in liver cancer and has different expression in different stages of cancer. However, due to the characteristics of small base number, short length, low abundance and easy degradation of miRNA-21, the detection of miRNA-21 is a challenging subject. Visualization, sensitive, specific and stable detection of tumor suppressor or oncogene microRNAs (miRNAs) remains challenging and is highly significant for clinical diagnostics. To solve this problem, we have developed a target-triggered hybridization assembly DNA machine for intracellular miRNA imaging based on strand displacement amplification (SDA) and branched hybridization chain reaction (B-HCR). In this approach, the target miRNA could hybridize with the template probe to trigger the SDA, resulting in the formation of nicked fragments (NFs) that hybridized with hairpin probe1 (HP1). The opened HP1 could hybridize with hairpin probe2 (HP2), leading to the self-assembly of hyperbranched DNA nanostructures through B-HCR. As expected, the newly developed method exhibits a detection limit down to 11.3pM miRNA-21 and achieves high selectivity toward miRNA-21 against other interfering miRNAs. Due to its superior sensitivity and selectivity, our method can be further used to detect miRNA-21 in human serum samples. By taking advantage of intelligent design, the proposed method was also used for image miRNA-21 expression levels in different cell lines. This method shows a broad application in clinical diagnosis.

Dual-Signal Cascaded Nucleic Acid Amplification Circuit-Loaded Metal-Organic Frameworks for Accurate and Robust Imaging of Intracellular MicroRNA.

Cascaded signal amplification technologies play an important role in the sensitive detection of lowly expressed biomarkers of interests yet are constrained by severe background interference and low cellular accessibility. Herein, we constructed a metal-organic framework-encapsulating dual-signal cascaded nucleic acid sensor for precise intracellular miRNA imaging. ZIF-8 nanoparticles load and deliver FAM-labeled upstream catalytic hairpin assembly (CHA) and Cy5-modified downstream hybridization chain reaction (HCR) hairpin reactants to tumor cells, enabling visualization of the target-initiated signal amplification process for double-insurance detection of analytes. The pH-responsive ZIF-8 nanoparticles effectively protect DNA hairpins from degradation and allow the release of them in the acid tumor microenvironment. Then, intracellular target miRNAs orderly trigger cascaded nucleic acid signal amplification reaction, of which the exact progress is investigated through the analysis of the fluorescence recovering process of FAM and Cy5. In addition, DNA@ZIF-8 nanoparticles improve measurement accuracy by dual-signal colocalization imaging, effectively avoiding nonspecific false-positive signals and enabling in situ imaging of miRNAs in living cells. A dual-signal colocalization strategy allows accurate target detection in living cells, and DNA@ZIF-8 provides a promising intracellular sensing platform for signal amplification and visual monitoring.

Simultaneous detection and imaging of two specific miRNAs using DNA tetrahedron-based catalytic hairpin assembly

Improving the accuracy, sensitivity and speed of intracellular miRNA imaging is essential for early diagnosis of cancer. To achieve this goal, we herein present a strategy for imaging two distinct miRNAs by DNA tetrahedron-based catalytic hairpin assembly (DCHA). Two nanoprobes, DTH-13 and DTH-24, were prepared by one-pot synthesis. The resultant structures were DNA tetrahedrons functionalized with two sets of CHA hairpins, which respectively responded to miR-21 and miR-155. Using these structured DNA nanoparticles as the carriers, the probes could easily enter living cells. The presence of miR-21 or miR-155 could trigger CHA between DTH-13 and DTH-24, leading to independent fluorescence signals of FAM and Cy3. In this system, the sensitivity and kinetics were significantly enhanced owing to the strategy of DCHA. The sensing performance of our method was thoroughly investigated in buffers, fetal bovine serum (FBS) solutions, living cells, and clinical tissue samples. The results validated the potential of DTH nanoprobes as a diagnostic tool for early stages of cancer.

An intelligent DNA nanomachine for amplified MicroRNA imaging and MicroRNA-Guided efficient gene silencing

The DNA nanomachines as excellent synthetic biological tools have been widely used for the sensitive detection of intracellular microRNA (miRNA) and DNAzyme-involved gene silencing. However, intelligent DNA nanomachines which have the ability to sense intracellular specific biomolecules and respond to external information in complex environments still remain challenging. Herein, we develop a miRNA-responsive DNAzyme cascaded catalytic (MDCC) nanomachine to perform multilayer cascade reactions, enabling the amplified intracellular miRNA imaging and miRNA-guided efficient gene silencing. The intelligent MDCC nanomachine is designed based on multiple DNAzyme subunit-encoded catalyzed hairpin assembly (CHA) reactants sustained by the pH-responsive Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles. After cellular uptake, the MDCC nanomachine degrades in acidic endosome and releases three hairpin DNA reactants and Zn2+, and the latter can act as an effective cofactor for DNAzyme. In the presence of miRNA-21, a catalytic hairpin assembly (CHA) reaction is triggered, which produces a large number of Y-shaped fluorescent DNA constructs containing three DNAzyme modules for gene silencing. The construction of Y-shaped DNA modified with multisite fluorescence and the circular reaction realizes ultrasensitive miRNA-21 imaging of cancer cells. Moreover, miRNA-guided gene silencing inhibits the cancer cell proliferation through the DNAzyme-specific recognition and cleavage of target EGR-1 (Early Growth Response-1) mRNA, which is one key tumor-involved mRNA. The strategy may provide a promising platform for highly sensitive determination of biomolecules and accurate gene therapy of cancer cells.

Logic Control of Directional Long-Range Resonance Energy Transfer On 2D DNA Nanosheet.

By arranging fluorophores in a directional way on a 2D DNA nanosheet that transfers energy from the initial donor to the acceptor through homogeneous Förster resonance energy transfer (homo-FRET), it is found that the photonic wires (PWs) based on cascade long-range resonance energy transfer (LrRET) up to 15.6nm can be effectively achieved through the rational selection of the fluorophores and the adjustment of their position with different distance. Then, logic control of directional energy transfer is achieved with the blocking of the energy transfer pathway, making two tumor-associated microRNA (miRNA) inputs produce an obvious output with the association of tumor diagnosis only when they present simultaneously. This research provides a new thought for development of PWs on 2D DNA nanosheets and a smart application of LrRET-based DNA AND logic control of intracellular miRNA imaging and tumor cells recognition.

A ZIF-8@DNAzyme-based DNA walker nanosystem for microRNAs sensing in living cells with high accuracy

It is a challenge to establish efficient, simplicity and precision miRNA imaging methods in living cells. Herein, we report a ZIF-8 @DNAzyme-based DNA walker strategy for intracellular miRNA imaging with high accuracy. Compared with traditional DNAzyme-based DNA walker, DNAzyme-based DNA walker can be effectively delivered into the cytoplasm by packaging in ZIF-8, thus ensuring the fidelity of intracellular miRNA imaging. In addition, ZIF-8 with acid-degrading properties can be degraded to generate cofactor Zn2+ in the acidic environment of tumors, achieving in the synchronous transport of DNAzyme-based DNA walker and metal ions. This strategy not only displays extremely low background signals in live- cell miRNA imaging, but also can distinguish the expression level of miRNA between tumor cells and normal cells, showing great potential in disease diagnosis. We believe this strategy can further promote the application of DNA-based motors in live-cell miRNA imaging, and provide new ideas for the joint application of MOFs and nucleic acid detection.

Chemiluminescence resonance energy transfer-based multistage nucleic acid amplification circuits for MiRNA detection with low background.

Chemiluminescence resonance energy transfer (CRET)-based assays have shown great potential in biosensing due to their negligible background autofluorescence, yet are still limited by their low sensitivity and short half-life luminescence. Herein, a multistage CRET-based DNA circuit was constructed with amplified luminescence signals for accurate miRNA detection and fixed reactive oxygen species (ROS) signals for cell imaging. The DNA circuit is designed through an ingenious programmable catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and the use of DNAzyme to realize target-triggered precise regulation of distance between the donor and acceptor for CRET-mediated excitation of photosensitizers. In detail, the analyte catalyzes the hybridization of CHA reactants, which leads to the assembly of multiple HCR-mediated DNAzyme nanowires. Subsequently, DNAzymes catalyze the oxidation of luminol by H2O2, and the adjacent photosensitizer chlorin e6 (Ce6) anchored on the DNA nanostructure is stimulated by the CRET process, resulting in the amplified long-wavelength luminescence and the generation of single oxygen signals through further energy transfer to oxygen. The biomarker miRNA can be detected with great sensitivity by integrating the recognition module into a universal platform. Furthermore, the DNA circuit enables CRET-mediated intracellular miRNA imaging, by detecting singlet oxygen signals through a ROS probe. The significant amplification effect is attributed to the robust multiple recognition of the target and the guaranteed transduction of the CRET signal through programmable engineering of DNA nanostructures. The CRET-based DNA circuit achieves amplified long-wavelength luminescence for accurate miRNA detection with low background and ROS-mediated signal fixation for cell imaging, making it a promising candidate for early diagnosis and theranostics.

Intracellular miRNA Imaging Based on a Self-Powered and Self-Feedback Entropy-Driven Catalyst-DNAzyme Circuit.

DNAzyme-based signal amplification circuits promote the advances in low-abundant miRNA imaging in living cells. However, due to the insufficient cofactor in living cells and unsustainable target utilization, self-powered and self-feedback DNAzyme amplification circuits have rarely been achieved. Here, a MnO2 nanosheet-mediated self-powered and self-feedback entropy-driven catalyst (EDC)-DNAzyme nanoprobe (MnPFEDz) was demonstrated for sensitive imaging of intracellular microRNA (miRNA). In this strategy, MnPFEDz was formed by adsorbing EDC modules and substrate probes on MnO2 nanosheets. The MnO2 nanosheets acted not only as glutathione (GSH)-responsive nanocarriers for efficient delivery of DNA probes but also as a DNAzyme cofactor supplier to power the DNAzyme biocatalysis and promote signal transduction in a feedback way. When entering the cells, GSH could decompose MnO2 nanosheets to generate numerous Mn2+ ion cofactors, leading to the release of DNA probes. Subsequently, the target miRNA initiated EDC cycles to generate amplified fluorescence signals and exposed the complete DNAzyme. Meanwhile, each of the exposed DNAzyme then cleaved the substrate probes with the help of Mn2+ ion cofactors and released a new trigger analogue for the next round of EDC cycles, initiating additional fluorescence signals in a feedback way. As a multiple signal amplification strategy, the MnPFEDz nanoprobe facilitated the effective detection of intracellular molecules with enhanced sensitivity and provided a versatile strategy for the construction of self-powered and self-feedback DNA circuits in living cells.

Trojan Horse Delivery of Spherical Nucleic Acid Probes into the Cytoplasm for High-Fidelity Imaging of MicroRNAs.

We report a Trojan horse strategy to efficiently deliver the spherical nucleic acid probes (namely, nanoflares) into the cytoplasm for microRNA (miRNA) imaging with high fidelity, breaking through the cytoplasmic transport dilemma of RNA probes in living cells. The nanoflare is encapsulated into a "Trojan horse" consisting of zwitterionic choline phosphates (CPs) and acid-degradable crosslinkers; the former effectively promotes cell uptake and the latter triggers instantaneous liberation of the nanoflare probes from the lysosome to the cytoplasm. The exposed nanoflares in the cytoplasm can be lightened up by the target miRNAs specifically. Compared with the conventional nanoflares as well as the improved ones in previous reports, the "Trojan horse" nanoflares avoid nuclease degradation and thiol displacement during the delivery process, providing unprecedentedly high accuracy for intracellular miRNA imaging.

A two-photon metal-organic framework nanoprobe with catalytic hairpin assembly for amplified MicroRNA imaging in living cells and tissues

Monitoring miRNAs can provide critical information for the generation and progress of various cancers. However, the existing DNA amplification strategies-based fluorescent nanoprobes for intracellular miRNAs imaging are often confronted with problems such as shallow light-penetration depth, enzymatic degradation, and low DNA loading efficiency. Herein, we developed a new two-photon nanoprobe (TP-CHA-MOFs) by using MOF as the nanocarrier and hairpins as a two-photon fluorescence signal amplifier for intracellular miRNA imaging. After cell penetration, intracellular phosphate could coordinate with Zr atoms in the MOFs, leading to the release of hairpins from the surfaces of TP-CHA-MOFs. The intracellular miRNA could trigger the formation of the H1-H2 duplex, which led to a turn-on TP fluorescence signal, realizing amplified detection of target miRNA. TP-CHA-MOFs nanoprobe showed high loading capacity of nucleic acid molecules, high biostability toward enzyme, and good biocompatibility. Because of the employment of TP fluorophore, TP-CHA-MOFs achieved sensitive imaging of intracellular miRNA and differentiated tumor cells with different miRNA levels. Moreover, sensitive imaging of miRNA in deep tumor tissues was realized with a penetration depth of 160 µm. The TP-CHA-MOFs nanoprobe is highly promising for future application in medical diagnostics.

Poly-adenine-mediated spherical nucleic acid probes for live cell fluorescence imaging of tumor-related microRNAs.

Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells. polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells. The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

Framework Nucleic Acid-Based Spatial-Confinement Amplifier for miRNA Imaging in Living Cells.

Real-time in situ monitoring of miRNAs in living cells is often appealed to signal amplifiers to tackle their low abundance challenges. However, the poor kinetics of amplifiers and potential interferences from the complex intracellular environment hamper its widespread applications in vivo. Herein, we report a framework nucleic acid (FNA)-based nonenzymatic spatial-confinement amplifier for rapid and reliable intracellular miRNA imaging. The amplifier consists of a localized catalytic hairpin assembly (L-CHA) reactor encapsulated in the inner cavity of an FNA (a 20 bp cube). The L-CHA reactor is certainly confined to the internal frame by integrating two probes (H1 and H2) of the L-CHA within a DNA strand and harnessing it to the opposite angles of the cube. We find that the stability of the amplifier is remarkably improved due to the protection of the FNA. More importantly, the spatial-confinement effect of the FNA can endow the confined L-CHA amplifier with enhanced local concentrations of reagents (5000-fold), thereby accelerating the reaction rate and improving the dynamic performance (up to 14.34-fold). With these advantages, the proposed amplifier can enable accurate and effective monitoring of miRNA expression levels in living cells and poses great potential in medical diagnostics and biomedical research.

Endogenous miRNA-Activated DNA Nanomachine for Intracellular miRNA Imaging and Gene Silencing.

The development of multifunctional nanoplatforms that integrate both diagnostic and therapeutic functions has always been extremely desirable and challenging in the cancer combat. Here, we report an endogenous miRNA-activated DNA nanomachine (EMDN) in living cells for concurrent sensitive miRNA imaging and activatable gene silencing. EMDN is constructed by interval hybridization of two functional DNA monomers (R/HP and F) to a DNA nanowire generated by hybridization chain reaction. After the target cell-specific transportation of EMDN, intracellular let-7a miRNA initiates the DNA nanomachine by DNA strand displacement cascades, resulting in an amplified fluorescence resonance energy-transfer signal and the release of many free HP sequences. The restoration of HP hairpin structures further activates the split-DNAzyme to identify and cleave the EGR-1 mRNA to realize gene silencing therapy. The proposed EMDN shows efficient cell internalization, good biological stability, rapid reaction kinetics, and the ability to avoid false-positive signals, thus ensuring reliable miRNA imaging in living cells. Meanwhile, the controlled activation of the split-DNAzyme activity regulated by the intracellular specific miRNA may be promising in the precise treatment of cancer. Collectively, this strategy provides a valuable nanoplatform for early clinical diagnosis and activatable gene therapy of tumors.