Identification and functional validation of miR-190b-5p and miR-296-3p as novel therapeutic attenuators of liver fibrosis.

BACKGROUND & AIMS: Liver fibrosis and its end-stage form known as cirrhosis contributes to millions of deaths annually. The lack of robust anti-fibrotic molecules is in part attributed to absence of any functional screens to identify molecular regulators using patient-derived primary human hepatic myofibroblasts, which are key drivers of fibrosis. METHODS: Here, to identify robust regulators of fibrosis, we performed functional microRNA screenings in primary human hepatic myofibroblasts followed by in vivo validation in three independent mouse models of fibrosis (toxin, cholestasis and MASH). RESULTS: We identified miR-190b-5p and miR-296-3p as robust anti-fibrotic miRNAs that suppress liver fibrosis. Notably, the expression of miR-190b-5p and miR-296-3p was found significantly reduced in human livers with fibrosis. Mechanistically, we discovered hyaluronan synthase 2 (HAS2) and integrin alpha-6 (ITGA6) as novel targets of miR-190b-5p and miR-296-3p, respectively. Furthermore, we demonstrated that the anti-fibrotic properties of miR-190b-5p and miR-296-3p are, at least in part, dependent on HAS2 and ITGA6. Finally, we showed the anti-fibrotic function of both miRNAs in a human liver bud model, which mimics multiple features of human liver. CONCLUSIONS: Collectively, in our study we discovered miR-190b-5p and miR-296-3p as two novel anti-fibrotic miRNAs, and that HAS2 and ITGA6 contribute to miR-190b-5p- and miR-296-3p-mediated inhibition of liver fibrosis. These results provide a foundation for future research to explore the clinical utility of miR-190b-5p and miR-296-3p in liver injuries with fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis and cirrhosis contribute to millions of deaths world-wide and, till date, remain as unmet medical needs. In this study, we discovered two microRNAs, miR-190b-5p and miR-296-3p, which suppress liver fibrosis in preclinical mouse models and a human liver bud model. Our promising results encourage further studies that aim to develop both miRNAs for the treatment of liver fibrosis in patients.

Endogenous Glutathione-Activated Nucleic Acid Molecular Circuitry for Cell-Specific MicroRNA Imaging.

Sensitive and reliable microRNA imaging in living cells has significant implications for clinical diagnosis and monitoring. Catalytic DNA circuits have emerged as potent tools for tracking these intracellular biomarkers and probing the corresponding biochemical processes. However, their utility is hindered by the low resistance to external interference, leading to undesired off-site activation and consequent signal leakage. Therefore, achieving the endogenous control of the DNA circuit's activation is preferable to the reliable target analysis in living cells. In this study, we attempted to address this challenge by engineering a simple yet effective endogenous glutathione (GSH)-regulated hybridization chain reaction (HCR) circuit for acquiring high-contrast miRNA imaging. Initially, the HCR hairpin reactants were blocked by the engineered disulfide-integrated DNA duplex, thus effectively passivating their sensing function. And the precaged HCR hairpin was liberated by the cell-specific GSH molecule, thus initiating the HCR system for selectively amplified detection of microRNA-21 (miR-21). This approach prevented unwanted signal leakage before exposure into target cells, thus ensuring robust miR-21 imaging with high accuracy and reliability in specific tumor cells. Moreover, the endogenously responsive HCR circuit established a link between the small regulatory factors and miRNA, thereby enhancing the signal gain. In summary, the endogenously activatable DNA circuit represents a versatile toolbox for robust bioanalysis and exploration of potential signaling pathways in living cells.

A Methylation-Gated DNAzyme Circuit for Spatially Controlled Imaging of MicroRNA in Cells and Animals.

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

Direct reduction of NO into N2 catalyzed by fullerene-supported rhodium clusters.

Catalytic conversion of NO has long been a focus of atmospheric pollution control and diesel vehicle exhaust treatment. Rhodium is one of the most effective metals for catalyzing NO reduction, and understanding the nature of the active sites and underlying mechanisms can help improve the design of Rh-based catalysts towards NO reduction. In this work, we investigated the detailed catalytic mechanisms for the direct reduction of NO to N2 by fullerene-supported rhodium clusters, C60Rh4+, with density functional theory calculations. We found that the presence of C60 facilitates the smooth reduction of NO into N2 and O2, as well as their subsequent desorption, recovering the catalyst C60Rh4+. Such a process fails to be completed by free Rh4+, emphasizing the critical importance of C60 support. We attribute the novel performance of C60Rh4+ to the electron sponge effect of C60, providing useful guidance for designing efficient catalysts for the direct reduction of NO.

Bioorthogonal Disassembly of Hierarchical DNAzyme Nanogel for High-Performance Intracellular microRNA Imaging.

Rolling circle amplification (RCA) enables the facile construction of compact and versatile DNA nanoassemblies which are yet rarely explored for intracellular analysis. This is might be ascribed to the uncontrollable and inefficient probe integration/activation. Herein, by encoding with tandem allosteric deoxyribozyme (DNA-cleaving DNAzyme), a multifunctional RCA nanogel was established for realizing the efficient intracellular microRNA imaging via the successive activation of the RCA-disassembly module and signal amplification module. The endogenous microRNA stimulates the precise degradation of DNA nanocarriers, thus leading to the efficient exposure of RCA-entrapped DNAzyme biocatalyst for an amplified readout signal. Our bioorthogonal DNAzyme disassembly strategy achieved the robust analysis of intracellular biomolecules, thus showing more prospects in clinical diagnosis.

Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly.

Isothermal autocatalytic DNA circuits have been proven to be versatile and powerful biocomputing platforms by virtue of their self-sustainable and self-accelerating reaction profiles, yet they are currently constrained by their complicated designs, severe signal leakages, and unclear reaction mechanisms. Herein, we developed a simpler-yet-efficient autocatalytic assembly circuit (AAC) for highly robust bioimaging in live cells and mice. The scalable and sustainable AAC system was composed of a mere catalytic DNA assembly reaction with minimal strand complexity and, upon specific stimulation, could reproduce numerous new triggers to expedite the whole reaction. Through in-depth theoretical simulations and systematic experimental demonstrations, the catalytic efficiency of these reproduced triggers was found to play a vital role in the autocatalytic profile and thus could be facilely improved to achieve more efficient and characteristic autocatalytic signal amplification. Due to its exponentially high signal amplification and minimal reaction components, our self-stacking AAC facilitated the efficient detection of trace biomolecules with low signal leakage, thus providing great clinical diagnosis and therapeutic assessment potential.

Miniature Hierarchical DNA Hybridization Circuit for Amplified Multiplexed MicroRNA Imaging.

Accurate diagnosis requires the development of multiple-guaranteed DNA circuits. Nevertheless, for reliable multiplexed molecular imaging, existing DNA circuits are limited by poor cell-delivering homogeneity due to their cumbersome and dispersive reactants. Herein, we developed a compact-yet-efficient hierarchical DNA hybridization (HDH) circuit for in situ simultaneous analysis of multiple miRNAs, which could be further exploited for specifically discriminating cancer cells from normal ones. By integrating the traditional hybridization chain reaction and catalytic hairpin assembly reactants into two highly organized hairpins, the HDH circuit is fitted with condensed components and multiple response domains, thus permitting the programmable multiple microRNA-guaranteed sequential activations and the localized cascaded signal amplification. The synergistic multi-recognition and amplification features of the HDH circuit facilitate the magnified detection of multiplex endogenous miRNAs in living cells. The in vitro and cellular imaging experimental results revealed that the HDH circuit displayed a reliable sensing performance with high selective cell-identification capacity. We anticipate that this compact design can provide a powerful toolkit for accurate diagnostics and pathological evolution.

Efficient Intracellular MicroRNA Imaging Based on the Localized and Self-Sustainable Catalytic DNA Assembly Circuit.

Building dynamic biological networks, especially DNA circuits, has provided a powerful prospect for exploring the intrinsic regulation processes of live cells. Nevertheless, for efficient intracellular microRNA analysis, the available multi-component circuits are constrained by their limited operating speed and efficiency due to the free diffusion of reactants. Herein, we developed an accelerated Y-shaped DNA catalytic (YDC) circuit for high-efficiency intracellular imaging of microRNA. By grafting the catalytic hairpin assembly (CHA) reactants into an integrated Y-shaped scaffold, the CHA probes were concentrated in a compact space, thus achieving high signal amplification. Profiting from the spatially confined reaction and the self-sustainably assembled DNA products, the YDC system facilitated reliable and in situ microRNA imaging in live cells. Compared with the homogeneously dispersed CHA reactants, the integrated YDC system could efficiently promote the reaction kinetics as well as the uniform delivery of CHA probes, thus providing a robust and reliable analytical tool for disease diagnosis and monitoring.

Early Predictors of Infected Pancreatic Necrosis in Severe Acute Pancreatitis: Implications of Neutrophil to Lymphocyte Ratio, Blood Procalcitonin Concentration, and Modified CT Severity Index.

BACKGROUND: Infected pancreatic necrosis (IPN) accounts for 30% mortality in severe acute pancreatitis (SAP). Early prediction of IPN occurrence is critical for prophylactic measures to be taken. This study aimed to evaluate the predicting value for IPN of combined markers at early stages of SAP. METHODS: The clinical records of 324 SAP patients admitted within 48 h after disease onset were retrospectively analyzed. As potential predictors, the neutrophil to lymphocyte ratio (NLR), blood procalcitonin (PCT) concentration on the 1st, 4th, and 7th day post admission, as well as modified computerized tomography severity index (MCTSI) on the 5-7th day post admission, were extracted. Correlations between these features with IPN were analyzed using logistic regression, and predictive values were estimated using the receiver operating characteristic curve analyses. RESULTS: NLR, PCT, body mass index, and MCTSI were significantly higher in the IPN group (p < 0.001) compared to the control, among which NLR, PCT, and MCTSI were identified as independent predictors for IPN in logistic regression model. Combination of these parameters yielded significant predicting values with an area under curve of 0.92, sensitivity of 97.2%, and specificity of 77.2% in receiver operating characteristic curve analysis. CONCLUSION: Combination of NLR, PCT, MCTSI might facilitate the prediction of IPN occurrence in SAP patients.

Ablation of Death-Associated Protein Kinase 1 Changes the Transcriptomic Profile and Alters Neural-Related Pathways in the Brain.

Death-associated protein kinase 1 (DAPK1), a Ca2+/calmodulin-dependent serine/threonine kinase, mediates various neuronal functions, including cell death. Abnormal upregulation of DAPK1 is observed in human patients with neurological diseases, such as Alzheimer's disease (AD) and epilepsy. Ablation of DAPK1 expression and suppression of DAPK1 activity attenuates neuropathology and behavior impairments. However, whether DAPK1 regulates gene expression in the brain, and whether its gene profile is implicated in neuronal disorders, remains elusive. To reveal the function and pathogenic role of DAPK1 in neurological diseases in the brain, differential transcriptional profiling was performed in the brains of DAPK1 knockout (DAPK1-KO) mice compared with those of wild-type (WT) mice by RNA sequencing. We showed significantly altered genes in the cerebral cortex, hippocampus, brain stem, and cerebellum of both male and female DAPK1-KO mice compared to those in WT mice, respectively. The genes are implicated in multiple neural-related pathways, including: AD, Parkinson's disease (PD), Huntington's disease (HD), neurodegeneration, glutamatergic synapse, and GABAergic synapse pathways. Moreover, our findings imply that the potassium voltage-gated channel subfamily A member 1 (Kcna1) may be involved in the modulation of DAPK1 in epilepsy. Our study provides insight into the pathological role of DAPK1 in the regulatory networks in the brain and new therapeutic strategies for the treatment of neurological diseases.

A Smart Deoxyribozyme-Programmable Catalytic DNA Circuit for High-Contrast MicroRNA Imaging.

Synthetic catalytic DNA circuits have been recognized as a promising signal amplification toolbox for sensitive intracellular imaging, yet their selectivity and efficiency are always constrained by uncontrolled off-site signal leakage and inefficient on-site circuitry activation. Thus, the endogenously controllable on-site exposure/activation of DNA circuits is highly desirable for achieving the selective imaging of live cells. Herein, an endogenously activated DNAzyme strategy was facilely integrated with a catalytic DNA circuit for guiding the selective and efficient microRNA imaging in vivo. To prevent the off-site activation, the circuitry constitute was initially caged without sensing functions, which could be selectively liberated by DNAzyme amplifier to guarantee the high-contrast microRNA imaging in target cells. This intelligent on-site modulation strategy can tremendously expand these molecularly engineered circuits in biological systems.

Sensitive Autocatalytic Hybridization Circuit for Reliable In Situ Intracellular Polynucleotide Kinase Imaging.

Exploring the characteristic functions of polynucleotide kinase (PNK) could substantially promote the elucidation of PNK-related mechanistic pathways. Yet, the sensitive and reliable detection of intracellular PNK still presents a challenging goal. Herein, we propose a simple autocatalytic hybridization circuit (AHC) for in situ intracellular imaging of PNK with high reliability. The AHC amplifier consists of two mutually activated hybridization chain reaction (HCR) modules for magnified signal transduction. The PNK is transduced into initiator I by phosphorylation and cleavage of mediator Hp. Initiator I activates the initial HCR-1 module, leading to the formation of long dsDNA nanowires that carry numerous initiator T. Then, T-initiated feedback HCR-2 module generates branched products that contain plentiful initiator I, thus realizing an autocatalytic HCR amplification reaction. Simultaneously, the HCR-2 module is also assembled as a versatile signal transduction unit for generating the amplified readout. Based on the mutually sustained accumulation of two initiators for the reciprocal activation of two reaction modules, continuous signal amplification and assembly of high-molecular-weight copolymers endow the AHC system with high sensitivity and robustness for the PNK assay. Moreover, the PNK-sensing AHC system achieves reliable imaging of intracellular PNK, thus showing great potential to decipher the correlation between PNK and related diseases.

Multiply Guaranteed and Successively Amplified Activation of a Catalytic DNA Machine for Highly Efficient Intracellular Imaging of MicroRNA.

DNA amplification machines show great promise for intracellular imaging, yet are always constrained by off-site machinery activation or signal leakage, originating from the inherent thermodynamically driven hybridization between machinery substrates. Herein, an entropy-driven catalytic DNA amplification machine is integrated with the on-site amplified substrate exposure procedure to realize the high-contrast in vivo imaging of microRNA (miRNA). The key machinery substrate (fuel strands) is initially split into substrate subunits that are respectively grafted into an auxiliary DNA polymerization amplification accessory for eliminating the undesired signal leakage. Meanwhile, in target cells, the auxiliary polymerization accessory can be motivated by cell-specific mRNA for successively restoring their intact machine-propelling functions for guaranteeing the on-site amplified imaging of miRNA with high specificity. This intelligent on-site multiply guaranteed machinery can improve the specificity of catalytic DNA machines for discriminating different cell types and, thus, can provide a remarkable prospect in biomedical diagnosis.

Functional Zeolitic Imidazolate Framework for Robust l-Deoxyribozyme-Based Therapy.

Programmable chiral biocatalysis represents a promising therapeutic strategy for its high stereospecific control over various biotransformations (e.g., chiral Aß isomerization) of living entities yet is rarely explored. With an extraordinary resistance to nuclease digestion, the non-natural left-handed deoxyribozyme (l-DNAzyme) therapy is constrained by inefficient delivery/release and insufficient cofactors supply. Herein, an efficient adenosine triphosphate (ATP)-stimulated disassembly of l-histidine (l-His)-integrated ZIF-8 (l-His-ZIF-8) is reported for sustaining the l-DNAzyme-amplified photodynamic therapy. This self-sufficient l-therapeutic platform can intelligently release the l-DNAzyme probe and simultaneously supply l-His DNAzyme cofactors via endogenous ATP. Then, the intrinsic microRNA-21 catalyzes the generation of robust l-DNAzyme via the catalytic hybridization reaction for activating the photosensitizer with multiplied guaranteed therapeutic operation. This l-therapeutic strategy opens up great prospects for more precise diagnosis and customized gene silencing-based therapy.

Correlation Between Severity of Illness and Levels of Free Triiodothyronine, Interleukin-6, and Interleukin-10 in Patients with Acute Pancreatitis.

BACKGROUND Acute pancreatitis (AP) is a common acute abdominal disease. Rapid evaluation of the severity is important for AP prognosis and treatment. Free triiodothyronine (fT3) level is associated with the prognosis of AP patients. This study aimed to investigate the fT3 level in patients with acute pancreatitis; early warning signs of inflammation, including interleukin-6 (IL-6) and interleukin-10 (IL-10); and the correlation of fT3 level with illness severity. MATERIAL AND METHODS Enrolled AP patients (N=312) were divided into an SAP group (N=92) and a non-SAP group (N=220) according to the Revision of Atlanta classification. Blood or tissue samples and baseline clinical characteristics were recorded. The t test and chi-square test were used to evaluate differences between the 2 groups. Multivariate logistic regression analysis and receiver operating characteristic (ROC) curves were used to investigate protective factors. One-way repeated measures analysis of variance was used to evaluate the prognosis of SAP patients. RESULTS In our study, compared with APACHII score (AUC 0.829 [95% CIs 0.769-0.889]) and Ranson score (AUC 0.629 [95% CIs 0.542-0.715]), our predictive model (AUC 0.918 [95% CIs 0.875-0.961]) showed better prognostic performance in predicting poor patient outcomes. In the SAP group, changes in fT3 level were significantly associated with prognosis (P<0.05). CONCLUSIONS The predictive model can improve the diagnostic accuracy and prediction of the severity of disease. FT3 level could be used as an independent risk factor to predict the mortality of SAP patients.

miR-143-3p Inhibits Aberrant Tau Phosphorylation and Amyloidogenic Processing of APP by Directly Targeting DAPK1 in Alzheimer's Disease.

The neuropathology of Alzheimer's disease (AD) is characterized by intracellular aggregation of hyperphosphorylated tau and extracellular accumulation of beta-amyloid (Aß). Death-associated protein kinase 1 (DAPK1), as a novel therapeutic target, shows promise for the treatment of human AD, but the regulatory mechanisms of DAPK1 expression in AD remain unclear. In this study, we identified miR-143-3p as a promising candidate for targeting DAPK1. miR-143-3p directly bound to the 3' untranslated region of human DAPK1 mRNA and inhibited its translation. miR-143-3p decreased tau phosphorylation and promoted neurite outgrowth and microtubule assembly. Moreover, miR-143-3p attenuated amyloid precursor protein (APP) phosphorylation and reduced the generation of Aß40 and Aß42. Furthermore, restoring DAPK1 expression with miR-143-3p antagonized the effects of miR-143-3p in attenuating tau hyperphosphorylation and Aß production. In addition, the miR-143-3p levels were downregulated and correlated inversely with the expression of DAPK1 in the hippocampus of AD patients. Our results suggest that miR-143-3p might play critical roles in regulating both aberrant tau phosphorylation and amyloidogenic processing of APP by targeting DAPK1 and thus offer a potential novel therapeutic strategy for AD.

Blocking ERK-DAPK1 Axis Attenuates Glutamate Excitotoxicity in Epilepsy.

Glutamate excitotoxicity induces neuronal cell death during epileptic seizures. Death-associated protein kinase 1 (DAPK1) expression is highly increased in the brains of epilepsy patients; however, the underlying mechanisms by which DAPK1 influences neuronal injury and its therapeutic effect on glutamate excitotoxicity have not been determined. We assessed multiple electroencephalograms and seizure grades and performed biochemical and cell death analyses with cellular and animal models. We applied small molecules and peptides and knocked out and mutated genes to evaluate the therapeutic efficacy of kainic acid (KA), an analog of glutamate-induced neuronal damage. KA administration increased DAPK1 activity by promoting its phosphorylation by activated extracellular signal-regulated kinase (ERK). DAPK1 activation increased seizure severity and neuronal cell death in mice. Selective ERK antagonist treatment, DAPK1 gene ablation, and uncoupling of DAPK1 and ERK peptides led to potent anti-seizure and anti-apoptotic effects in vitro and in vivo. Moreover, a DAPK1 phosphorylation-deficient mutant alleviated glutamate-induced neuronal apoptosis. These results provide novel insight into the pathogenesis of epilepsy and indicate that targeting DAPK1 may be a potential therapeutic strategy for treating epilepsy.

On-Site Non-enzymatic Orthogonal Activation of a Catalytic DNA Circuit for Self-Reinforced In Vivo MicroRNA Imaging.

The wide extracellular-intracellular distribution of microRNA requires the on-site, robust and efficient activation of catalytic DNA circuits inside live cells. Herein, we develop an efficient non-enzymatic circuitry activation strategy to realize the orthogonally controlled catalytic DNA (CCD) circuit for achieving high-fidelity in vivo microRNA imaging through multiply guaranteed molecular recognition and progressively accelerated signal amplification. For predictable on-site activation and useful catalytic efficiency, the dominating circuitry fuel strand was initially split into inactive fuel subunits that were grafted into an auxiliary catalytic circuit. There, the in-cell-specific mRNA triggered the orthogonal amplification of the active fuel strands for sensitive target detection through the chief entropy-driven catalytic DNA circuit. We believe that the on-site orthogonal circuitry activation method can contribute to clinical diagnosis and prognosis.

Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.

BACKGROUND & AIMS: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. METHODS: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. RESULTS: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. CONCLUSION: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. LAY SUMMARY: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

A Smart Theranostic Nanocapsule for Spatiotemporally Programmable Photo-Gene Therapy.

The therapeutic performance of DNAzyme-involved gene silencing is significantly constrained by inefficient conditional activation and insufficient cofactor supply. Herein, a self-sufficient therapeutic nanosystem was realized through the delicate design of DNAzyme prodrugs and MnO2 into a biocompatible nanocapsule with tumor-specific recognition/activation features. The indocyanine green (ICG)-modified DNA prodrugs are designed by splitting the DNAzyme and then reconstituted into the exquisite catalyzed hairpin assembly (CHA) amplification circuit. Based on the photothermal activation of ICG, the nanocapsule was disassembled to expose the MnO2 ingredient which was immediately decomposed into Mn2+ ions to supplement an indispensable DNAzyme cofactor on-demand with a concomitant O2 generation for enhancing the auxiliary phototherapy. The endogenous microRNA catalyzes the amplified assembly of DNA prodrugs via an exquisite CHA principle, leading to the DNAzyme-mediated simultaneous silencing of two key tumor-involved mRNAs. This self-activated theranostic nanocapsule could substantially expand the toolbox for accurate diagnosis and programmable therapeutics.