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Comprehensive Summary: A label‐free deoxyribozyme (DNAzyme) biosensor for m6A demethylase activity detection is developed. When demethylase FTO (Fat mass and obesity‐associated protein), an important m6A demethylase of ALKBH demethylase family is present, the m6A group is specifically demethylated, and the cleavage activity of DNAzyme is restored. This allows the cleavage product that contains G‐quadruplex sequence to bind to thioflavin T (ThT) and generate fluorescence signals. The biosensor shows high specificity and sensitivity, and fast reaction speed. Our study demonstrates a new design of allosteric DNAzyme for sensing. This method represents the first label‐free nucleic acid biosensor for FTO assay, providing a feasible route towards inhibitors screening. [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]

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]

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]

Polymerase/nicking enzymes and nucleic-acid scaffolds are implemented as DNA machines for the development of amplified DNA-detection schemes, and for the design of logic gates. The analyte nucleic acid target acts, also, as input for the logic gates. In the presence of two DNA targets, acting as inputs, and appropriate DNA scaffolds, the polymerase-induced replication of the scaffolds, followed by the nicking of the replication products, are activated, leading to the autonomous synthesis of the Mg2+-dependent DNAzyme or the Mg2+-dependent DNAzyme subunits. These biocatalysts cleave a fluorophore/quencher-functionalized nucleic-acid substrate, thus providing fluorescence signals for the sensing events or outputs for the logic gates. The systems are used to develop OR, AND, and Controlled-AND gates, and the DNA-analyte targets represent two nucleic acid sequences of the smallpox viral genome. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: A label‐free deoxyribozyme (DNAzyme) biosensor for m6A demethylase activity detection is developed. When demethylase FTO (Fat mass and obesity‐associated protein), an important m6A demethylase of ALKBH demethylase family is present, the m6A group is specifically demethylated, and the cleavage activity of DNAzyme is restored. This allows the cleavage product that contains G‐quadruplex sequence to bind to thioflavin T (ThT) and generate fluorescence signals. The biosensor shows high specificity and sensitivity, and fast reaction speed. Our study demonstrates a new design of allosteric DNAzyme for sensing. This method represents the first label‐free nucleic acid biosensor for FTO assay, providing a feasible route towards inhibitors screening. [ABSTRACT FROM AUTHOR]

Highlights • Fluorescent method for biosensing of Zn2+, pyrophosphate, and alkaline phosphatase activity. • Real-time detection based on assembly-induced fluorescence enhancement by gold nanoclusters or quantum dots. • Capable of screening enzyme inhibitor and analyzing biological samples. • Sensitive assay of alkaline phosphatase with a detection limit of 3.4 pM. Abstract Fluorescent nanoparticles exhibit unique optical properties for applications in imaging and sensing. Herein, the enhancement of luminescence emission from self-assembled nanoparticles including gold nanoclusters, cadmium sulphide and near-infrared silver selenide quantum dots are reported. Addition of zinc and pyrophosphate ions induces assembly and disassembly of the particle aggregates and the corresponding intensified and recovery of fluorescence intensity, respectively. The enhancement is due to assembly-enhanced emission of metal nanoclusters and passivation of the surface trap states of quantum dots. With the hydrolysis of pyrophosphate catalyzed by alkaline phosphatase, the released zinc ions from pyrophosphate-zinc ion complexes mediate nanoparticle assembly and lead to luminescence regeneration. The assembly-induced fluorescence enhancement can be applied for sensitive assay of alkaline phosphatase with a detection limit of 3.4 pM in a continuous and real-time way. This method is capable of screening enzyme inhibitor and analyzing biological samples. [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]