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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]

Non‐invasive cell regulation represents a promising approach for on‐site cell modulation, but is confronted with inaccuracy or poor cell selectivity. Herein, by virtue of the interfacial‐activated concatenate DNA circuit, an activated recombination‐to‐modulation (ARM) machinery is developed to achieve accurate membrane imaging and effective cell modulation. Bioorthogonal signal transduction through the catalytic hairpin assembly circuit can continuously regenerate triggers for activating the subsequent circuit, with no activator consumption and occupation. The ARM strategy utilizes the amplified hybridization chain reaction to sensitively rearrange membrane receptors for blocking the related signaling pathways and indirectly modulating cell behaviors. Based on these two membrane receptor inputs, the controllably‐activated ARM strategy enables the identification of target cells among similar interfering cells with high accuracy and sensitivity. This machinery has demonstrated good performance in the efficient inhibition of tumor metastasis via the disruption of HGF/c‐Met‐signaling pathway. The modular‐designed ARM machinery provides a general platform for non‐invasive and specific cell modulation, suggesting a broad opportunity for advanced exploration of cellular metabolism behaviors. [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]

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 direct visualization of vaccine fate is important to investigate its immunoactivation process to elucidate the detailed molecular reaction process at single‐molecular level. Yet, visualization of the spatiotemporal trafficking of vaccines remains poorly explored. Here, we show that quantum dot (QD) nanomaterials allow for monitoring vaccine dynamics and for amplified immune response. Synthetic QDs enable efficient conjugation of antigen and adjuvants to target tissues and cells, and non‐invasive imaging the trafficking dynamics to lymph nodes and cellular compartments. The nanoparticle vaccine elicits potent immune responses and anti‐tumor efficacy alone or in combination with programmed cell death protein 1 blockade. The synthetic QDs showed high fluorescence quantum yield and superior photostability, and the reliable and long‐term spatiotemporal tracking of vaccine dynamics was realized for the first time by using the synthetic QDs, providing a powerful strategy for studying immune response and evaluating vaccine efficacy. [ABSTRACT FROM AUTHOR]

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]

DNAzymes are synthetic deoxynucleotide oligomers with catalytic properties selected through in vitro systematic evolution of ligands by exponential enrichment (SELEX) technique. RNA‐cleaving DNAzymes have attracted increasing attentions in bioanalysis and biomedical applications due to their simple synthesis and modification, stable structure, and high catalytic activity. Especially, their different modes of activity regulation enable their extensive theranostic development. This review introduces the in vitro selection of DNAzymes and some typical ones, followed by the description of various stimuli‐responsive regulation strategies on DNAzyme activity, including metal ion cofactors, small molecules, protease, light, and heat. Then the recent advances of DNAzyme‐based biosensors, therapeutic applications, cell‐cell interaction regulation, and logic operation are summarized. At last, the current challenges and possible development of DNAzyme‐based biosensors are discussed. [ABSTRACT FROM AUTHOR]