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Synthetic biochemical circuits (e.g., DNA circuits) remain at the forefront of intracellular biosensing tasks yet are hindered by the undesired off‐site activation and the accompanying signal leakage. Herein, the study attempts to overcome this limitation by developing a simple‐yet‐powerful endogenous glutathione (GSH)‐regulating tactic that permits robust and distinguishable on‐site microRNA (miRNA) imaging under disturbed redox homeostasis. Specifically, a hierarchically activated catalytic DNA (HAD) circuit is fabricated by grafting the disulfide linkage within entropy‐driven DNA circuitry (EDC) reactants. It is exemplified that this HAD system promises the spatiotemporally selective microRNA‐21 (miR‐21) imaging in living cells and the robust differentiation of tumor cells from normal cells. The correlationship between GSH and miRNA is extensively explored in live cells, and can substantially expand the toolbox of DNA circuits for profiling intracellular biochemical processes. [ABSTRACT FROM AUTHOR]

The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self‐assembly system. Herein, a catalytic self‐defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre‐sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self‐assembly via a microRNA (miRNA)‐powered self‐sorting catalytic hybridization reaction. With minimal strand complexity, this self‐sustainable CSC system streamlined the circuit component and achieved localization‐intensified cascaded signal amplification. Profiting from the self‐adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para‐carcinoma tissues. The CSC‐sustained self‐assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Laser‐free photodynamic therapy (PDT) is a promising noninvasive therapeutic modality for deep‐seated tumor, yet is constrained by low efficiency due to the limited stimulation strategies. Herein, a novel miRNA‐responsive laser‐free PDT was developed through metal‐organic frameworks (MOFs)‐mediated chemiluminescence resonance energy transfer (CRET) nanoplatform. The photosensitizer chlorin e6 (Ce6)‐loaded MOFs were functionalized with hairpin nucleic acids for sensitive responsiveness of tumor biomarker miRNA through catalytic hairpin assembly (CHA), which enabled the amplified assembly of horseradish peroxidase (HRP)‐mimicking hemin/G‐quadruplex DNAzyme on MOFs. Simultaneously, the on‐MOF assembled DNAzymes efficiently catalyzed chemiluminescence reaction to stimulate adjacent Ce6 in the presence of luminol and H2O2, thus allowing the CRET‐mediated Ce6 luminescence and reactive oxygen species (ROS) generation for self‐illuminating PDT. The CRET nanoplatform achieved significant malignant cell apoptosis and tumor inhibition effects without external laser irradiation. It is envisioned that the miRNA‐amplified CRET nanoplatform might be a selective and highly efficient antitumor nanomedicine for precise theranostic. [ABSTRACT FROM AUTHOR]

Herein, we developed a reliable and portable biosensor (TDR-PGM nanomachine) for the sensitive detection of microRNA by integrating an efficient toehold-mediated strand displacement reaction module (TDR) and a personal glucose meter (PGM). The system provides a versatile methodology for microRNA detection in real samples and holds broad prospects in point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lowly expressed analyte in complex cytoplasmic milieu necessitates the development of non‐enzymatic autocatalytic DNA circuits with high amplification and anti‐interference performance. Herein, we engineered a versatile and robust stimuli‐responsive autocatalytic hybridization assembly (AHA) circuit for high‐performance in vivo bioanalysis. Under a moderately confined condition, the initiator motivated the autonomous and cooperative cross‐activation of cascade hybridization reaction and catalytic DNA assembly for generating an exponentially amplified readout without the parasite steric hindrance and random diffusion side effects. The AHA circuit was systematically investigated by a series of experimental studies and theoretical simulations. The successively guaranteed target recognition and synergistically accelerated signal‐amplification enabled the sensitive and selective detection of analyte, and realized the robust miRNA imaging in living cells and mice. This autocatalytic DNA circuit could substantially expand the toolbox for accurate diagnosis and programmable therapeutics. [ABSTRACT FROM AUTHOR]

The systemic therapeutic utilisation of RNA interference (RNAi) is limited by the non-specific off-target effects, which can have severe adverse impacts in clinical applications. The accurate use of RNAi requires tumour-specific on-demand conditional activation to eliminate the off-target effects of RNAi, for which conventional RNAi systems cannot be used. Herein, a tumourous biomarker-activated RNAi platform is achieved through the careful design of RNAi prodrugs in extracellular vesicles (EVs) with cancer-specific recognition/activation features. These RNAi prodrugs are assembled by splitting and reconstituting the principal siRNAs into a hybridisation chain reaction (HCR) amplification machine. EVs facilitate the specific and efficient internalisation of RNAi prodrugs into target tumour cells, where endogenous microRNAs (miRNAs) promote immediate and autonomous HCR-amplified RNAi activation to simultaneously silence multiantenna hypoxia-related genes. With multiple guaranteed cancer recognition and synergistic therapy features, the miRNA-initiated HCR-promoted RNAi cascade holds great promise for personalised theranostics that enable reliable diagnosis and programmable on-demand therapy. The therapeutic application of small interfering RNA (siRNA) is challenging due to its non-specific targeting and delivery issues. Here, the authors report an endogenous micro-RNA guided and hybridisation chain reaction-promoted siRNA delivery system encapsulated in tumour-derived extracellular vesicles, with cancer-specific activation, and achieve silencing of hypoxia-related genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The autocatalytic HCR-DNAzyme platform was constructed as a versatile amplification platform for intracellular microRNA imaging by integrating hybridization chain reaction (HCR) circuit with DNAzyme biocatalysis. The HCR-assembled multifunctional DNAzyme nanowires produce new HCR triggers and numerous transducer DNAzyme amplifier, and thus shows great promise in earlier cancer diagnosis. [ABSTRACT FROM AUTHOR]

DNAzymes have been recognized as promising transducing agents for visualizing endogenous biomarkers, but their inefficient intracellular delivery and limited amplification capacity (including insufficient cofactor supply) preclude their extensive biological application. Herein, an autocatalytic DNAzyme (ACD) biocircuit is constructed for amplified microRNA imaging in vivo based on a hybridization chain reaction (HCR) and DNAzyme biocatalysis, sustained by a honeycomb MnO2 nanosponge (hMNS). The hMNS not only delivers DNA probes, but also supplies Mn2+ as a DNAzyme cofactor and magnetic resonance imaging (MRI) agent. Through the subsequent cross‐activation of HCR and DNAzyme amplicons, the ACD amplifies the limited signal resulting from miRNA recognition. The hMNS/ACD system was used to image microRNA in vivo, thus demonstrating its great promise in cancer diagnosis. [ABSTRACT FROM AUTHOR]

Benefiting from the simple DNAzyme and a duplex-specific nuclease, a series of microRNA-stimulated DNAzyme logic gates were rationally assembled for the highly accurate detection of multiple low-abundant microRNA biomarkers that correspond to the specific aberrant microRNA expression patterns of cancer tissues, thus showing a great potential in early diagnosis. [ABSTRACT FROM AUTHOR]

A facile cross-catalytic circuit is engineered for intracellular microRNA (miRNA) imaging by facilely integrating a catalytic hairpin assembly (CHA) amplifier and DNAzyme biocatalyst through an ingenious feedback loop. These two indispensable catalytic reactions play vital roles in executing high-performance signal amplification, as demonstrated experimentally and theoretically. [ABSTRACT FROM AUTHOR]

Abstract: The development of effective strategies for sensitive and specific detection of microRNAs will facilitate clinical diagnostics. Here, we utilize cascade hybridization chain reaction as an enzyme‐free isothermal amplified strategy, and construct label‐free electrochemical biosensors for microRNAassay. MicroRNA initiated autonomous assembly of DNA nanostructures through the toehold‐mediated strand displacement reaction. The assembly behavior was examined by gel electrophoresis and morphology of branched DNA nanostructure was verified by atomic force microscope. This cascade hybridization chain reaction leads to extended growth of DNA chains and higher amplification efficiency compared with the regular hybridization chain reaction. The performance of the sensors was characterized by electrochemical impedance spectroscopy, differential pulse voltammetry, and chronocoulometric analysis. The prepared biosensor exhibits high sensitivity and a lower detection limit of 11 pM for the determination of microRNA‐21. This electrochemical assay based on cascade hybridization chain reaction is specific and can distinguish between highly homologous targets such as fully complementary target microRNA‐21, single, or two‐base mismatched sequences and other family members. These findings open the possibility of using cascade hybridization chain reaction as possible amplified electrochemical sensing platforms. [ABSTRACT FROM AUTHOR]

DNA Circuit, Fluorescence, RNA, Signal Amplification, Tumor Imaging Der am Wirkort aktivierte CCD-Schaltkreis hat erhebliches Potenzial für die hochpräzise In-vivo-Bildgebung von microRNA. Keywords: DNA Circuit; Fluorescence; RNA; Signal Amplification; Tumor Imaging EN DNA Circuit Fluorescence RNA Signal Amplification Tumor Imaging 1 1 1 11/03/22 20221107 NES 221107 B Eine nicht-enzymatische b Aktivierungsstrategie mittels multipler molekularer Erkennungsereignisse und progressiv beschleunigter Signalverstärkung führte zu einem orthogonal gesteuerten katalytischen DNA-Schaltkreis (CCD), wie Fuan Wang et al. in ihrem Forschungsartikel berichten (e202206529). [Extracted from the article]

Back Cover: On-Site Non-enzymatic Orthogonal Activation of a Catalytic DNA Circuit for Self-Reinforced In Vivo MicroRNA Imaging (Angew. Keywords: DNA Circuit; Fluorescence; RNA; Signal Amplification; Tumor Imaging EN DNA Circuit Fluorescence RNA Signal Amplification Tumor Imaging 1 1 1 11/03/22 20221107 NES 221107 B A non-enzymatic circuitry b activation strategy can realize an orthogonally controlled catalytic DNA (CCD) circuit through multiple molecular recognition events and progressively accelerated signal amplification, as reported by Fuan Wang and co-workers in their Research Article (e202206529). DNA Circuit, Fluorescence, RNA, Signal Amplification, Tumor Imaging. [Extracted from the article]

Catalytic DNA circuit, a versatile synthetic molecular nanodevice, shows great potential for in vivo bioimaging application, yet is distorted by its off-site signal leakage. The endogenously activated DNA circuit could guarantee the specific and sensitive in vivo imaging utility yet was still unexplored. In this work, we engineered an endogenously and sequentially activated DNA circuit by using the specific enzymatic regulation strategy. This smart DNA circuit is consisting of two successive reaction modules, the initial site-specific circuitry exposure module and the subsequent circuitry activation module for amplified in vivo biosensing. Initially, the catalytic circuitry reactant was caged with a long elongated duplex, encoding with high energy barriers of circuitry crosstalk, to prevent the undesirable off-target signal leakage prior to its arrival in targeting cells. Subsequently, the as-integrated functional duplex could be specifically cleaved and removed by the endogenously overexpressed DNA excision repairing enzyme of cancer cells, thus liberating the DNA circuitry reactant for participating the catalyzed and amplified imaging of intracellular analyte, e.g. , microRNA. Meanwhile, the catalytic DNA circuit stayed inert in normal cells for lacking the indispensable cell-specific exposure of circuitry reactant. Through the sequential circuitry exposure (by endogenous enzyme of specific cells) and activation (by target of interest) procedure, our multiply guaranteed DNA circuit realized the robust in vivo imaging of tumor cells with high precision and reliability, thus supplementing a powerful toolbox for cancer diagnosis and treatment. [Display omitted] • An endogenously activated DNA circuit enables the intracellular imaging of miRNA through the enzyme-modulated methodology. • This system acquires the high-contrast imaging of miRNA in live mice by preventing the annoying off-site signal leakage. • The enzyme-regulation of DNA circuit facilitates the efficient and site-specific circuitry activation in specific live cells. • This work provided a simple yet powerful strategy to regulate various DNA circuits with biologically important functions. [ABSTRACT FROM AUTHOR]

Deoxyribozyme (DNAzyme) is single-stranded catalytic DNA that possesses various physical and chemical features, including small molecular weight, higher stability, excellent programmability, and cost-effectiveness. Considerable efforts are spent on the incorporation of RNA-cleaving DNAzyme (RCD) into biosensor and biomedical researches. However, their in vivo application is constrained by off-target activation (always turn-on) in complex biological environment. Therefore, there is a pursuit to develop spatiotemporally controlled DNAzyme in desired locations and time-points. Herein, we provide a timely and comprehensive overview of various chemical modification and structural reconfiguration methods to realize the specific stimulation/regulation of RCD activities, including metal ions, small molecules, nucleic acids, proteins, and external photo/thermal stimuli. The specific examples of using chemically and/or structurally caged RCD for stimuli-responsive biosensing and biomedical research have also been reviewed within the past five years. Lastly, the challenges and perspectives of stimuli-responsive RCD are suggested to further advance the RCD toolbox in clinical application. • Stimuli-responsive RNA-cleaving DNAzyme (RCD) represents a powerful toolbox for biological research. • The off-site signal leakage of RCD can be eliminated by chemical or/and structural modifications. • The spatiotemporally controlled RCD is summarized for bioimaging and biomedical applications. • The ongoing challenge and future development of stimuli-responsive RCD are properly summarized. [ABSTRACT FROM AUTHOR]