A Versatile DNAzyme-Amplified Protease-Sensing Platform for Accurate Diagnosis of SARS-CoV-2 and Reliable Classification of Colorectal Cancer.

Peptide-based biosensors are widely used for in vitro detection of protease activity but often suffer from the limited sensitivity, poor accuracy, and incompatibility with point-of-care testing (POCT) devices. Herein, we developed a versatile deoxyribozyme (DNAzyme)-amplified protease-sensing (DP) platform that integrates the positively charged oligopeptides with a negatively charged DNAzyme biocatalyst for highly-sensitive protease detection. The system leverages the electrostatic peptide-DNAzyme interactions to inhibit DNAzyme catalytic activity, which is reactivated upon the protease-triggered peptide hydrolysis, thus enabling an efficient signal amplification via the successive cleavage of DNAzyme substrate.

Endogenous Enzyme Stimulation of Localized Catalytic DNA Assembly for High-Contrast microRNA Imaging and Reliable Evaluation of Drug Resistance.

Localized DNA circuits have emerged as a powerful platform for real-time visualization of disease-associated biomolecules in living cells, enabling deeper insights into complex biological processes. However, their extensive applications have been limited by a low probe utilization efficiency and nonspecific background signals. Here, we present a cell-specifically triggered localized catalytic assembly (TCA) circuit designed for high-contrast, reliable imaging of microRNAs (miRNAs).

Leveraging Demethylase Activation in DNA Circuits to Overcome Signal Leakage for Reliable MicroRNA Bioimaging.

DNA circuits show great potential in monitoring intracellular biomarkers based on their high programmability, predictability, and unique signal amplification capabilities, yet face challenges from uncontrollable signal leakage caused by the complex intracellular environment. Herein, a demethylase-activated DNA-assembly (DAD) circuit is designed for the reliable and robust imaging of cellular microRNA, by incorporating the sequential activation of a hybridization chain reaction (HCR) amplifier system. The DAD circuit consists of a demethylase-activated DNAzyme module and a microRNA-recognizing HCR signal-amplifying module.

Redox-stimulated catalytic DNA circuit for high-fidelity imaging of microRNA and in situ interpretation of the relevant regulatory pathway.

Biomolecules play essential roles in regulating the orderly progression of biochemical reaction networks. DNA-based biocircuits supplement an attractive and ideal approach for the visual imaging of endogenous biomolecules, yet their sensing performance is commonly encumbered by the undesired signal leakage. To solve this issue, here we proposed a glutathione (GSH)-activated DNA circuit for achieving the spatio-selective microRNA imaging through the successive response of a GSH-specific activation procedure and a non-enzymatic catalytic signal amplification procedure.

Self-Adaptive Activation of DNAzyme Nanoassembly for Synergistically Combined Gene Therapy.

DNAzyme represents a promising gene silencing toolbox yet is obstructed by the poor substrate accessibility in specific cells. Herein, a compact DNA nanoassembly, incorporating multimeric therapeutic DNAzyme, was prepared for selective delivery of gene-silencing DNAzyme with requisite cofactors and auxiliary chemo-drugs. By virtue of the sequence-conservative duplex-specific nuclease, the endogenous miRNA catalyzes the successive and site-specific cleavage of DNA nanoassembly substrate (nominated as the localized RNA walking machine) and thus ensures the liberation/activation of therapeutic agents with high accuracy and efficacy.

On-Site Multiply Stimulated Self-Confinement of an Integrated DNA Cascade Circuit for Highly Reliable Intracellular Imaging of miRNA and In Situ Interrogation of the Relevant Regulatory Pathway.

Artificial DNA circuits represent a versatile yet promising toolbox for in situ monitoring and concomitant regulation of diverse biological events within live cells. Nonetheless, their performance is significantly impeded by the diffusion-dominated slow reaction kinetics and the uncontrollable off-target activation. Herein, a self-localized cascade (SLC) circuit is reported for the robust and efficient microRNA (miRNA) analysis in living cells.

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.

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.

Enzyme-Free Dynamic DNA Reaction Networks for On-Demand Bioanalysis and Bioimaging.

ConspectusThe pursuit of in-depth studying the nature and law of life activity has been dominating current research fields, ranging from fundamental biological studies to applications that concern synthetic biology, bioanalysis, and clinical diagnosis. Motivated by this intention, the spatiotemporally controlled and in situ analysis of living cells has been a prospective branch by virtue of high-sensitivity imaging of key biomolecules, such as biomarkers. The past decades have attested that deoxyribonucleic acid (DNA), with biocompatibility, programmability, and customizable features, is a competitive biomaterial for constructing high-performance molecular sensing tools.

Integration of Isothermal Enzyme-Free Nucleic Acid Circuits for High-Performance Biosensing Applications.

The isothermal enzyme-free nucleic acid amplification method plays an indispensable role in biosensing by virtue of its simple, robust, and highly efficient properties without the assistance of temperature cycling or/and enzymatic biocatalysis. Up to now, enzyme-free nucleic acid amplification has been extensively utilized for biological assays and has achieved the highly sensitive detection of various biological targets, including DNAs, RNAs, small molecules, proteins, and even cells. In this Review, the mechanisms of entropy-driven reaction, hybridization chain reaction, catalytic hairpin assembly and DNAzyme are concisely described and their recent application as biosensors is comprehensively summarized.

Spatiotemporally Programmed Disassembly of Multifunctional Integrated DNAzyme Nanoplatfrom for Amplified Intracellular MicroRNA Imaging.

The sensing performance of DNAzymes in live cells is tremendously hampered by the inefficient and inhomogeneous delivery of DNAzyme probes and their incontrollable off-site activation, originating from their susceptibility to nuclease digestion. This requires the development of a more compact and robust DNAzyme-delivering system with site-specific DNAzyme activation property. Herein, a highly compact and robust Zn@DDz nanoplatform is constructed by integrating the unimolecular microRNA-responsive DNA-cleaving DNAzyme (DDz) probe with the requisite DNAzyme Zn -ion cofactors, and the amplified intracellular imaging of microRNA via the spatiotemporally programmed disassembly of Zn@DDz nanoparticles is achieved.

Nano- and micro-plastic transport in soil and groundwater environments: Sources, behaviors, theories, and models.

With the increasing use of plastics, nano- and micro-plastic (NMP) pollution has become a hot topic in the scientific community. Ubiquitous NMPs, as emerging contaminants, are becoming a global issue owing to their persistence and potential toxicity. Compared with studies of marine and freshwater environments, investigations into the sources, transport properties, and fate of NMPs in soil and groundwater environments remain at a primary stage.

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.

The compact integration of a cascaded HCR circuit for highly reliable cancer cell discrimination.

The accurate identification of multiple biomarkers involved in disease plays a vital role in effectively distinguishing cancer cells from normal cells, facilitating reliable cancer diagnosis. Motivated by this knowledge, we have engineered a compact and clamped cascaded DNA circuit for specifically discriminating cancer cells from normal cells the amplified multi-microRNA imaging strategy. The proposed DNA circuit combines the traditional cascaded DNA circuit with multiply localized responsive character through the elaboration of two super-hairpin reactants, thus concurrently streamlining the circuit components and realizing localization-intensified cascaded signal amplification.

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.

Tailoring a Minimal Self-Replicate DNA Circuit for Highly Efficient Intracellular Imaging of microRNA.

Trace analyte detection in complex intracellular environment requires the development of simple yet robust self-sufficient molecular circuits with high signal-gain and anti-interference features. Herein, a minimal non-enzymatic self-replicate DNA circuitry (SDC) system is proposed with high-signal-gain for highly efficient biosensing in living cells. It is facilely engineered through the self-stacking of only one elementary cascade hybridization reaction (CHR), thus is encoding with more economic yet effective amplification pathways and reactants.

Sensitive Autocatalytic Hybridization Circuit for Reliable 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 intracellular imaging of PNK with high reliability.

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.

An Autocatalytic DNA Circuit Based on Hybridization Chain Assembly for Intracellular Imaging of Polynucleotide Kinase.

Polynucleotide kinase (PNK) plays an essential role in various cellular events by regulating phosphorylation processes, and abnormal homeostasis of PNK could cause many human diseases. Herein, we proposed an autocatalytic hybridization system (AHS) through the elaborate integration of hybridization chain assembly (HCA) and catalytic DNA assembly (CDA) that enables a highly efficient positive feedback amplification. The PNK-targeting AHS biosensor is composed of three modules: a recognition module, an HCA amplification module, and a CDA autocatalytic module.

Modular Assembly of a Concatenated DNA Circuit for In Vivo Amplified Aptasensing.

Probing endogenous molecular profiles in living entities is of fundamental significance to decipher biological functions and exploit novel theranostics. Despite programmable nucleic acid-based aptasensing systems across the breadth of molecular imaging, an aptasensing system enabling in vivo imaging with high sensitivity, accuracy, and adaptability is highly required yet is still in its infancy. Artificial catalytic DNA circuits that can modularly integrate to generate multiple outputs from a single input in an isothermal autonomous manner, have supplemented powerful toolkits for intracellular biosensing research.

An Isothermal Autocatalytic Hybridization Reaction Circuit for Sensitive Detection of DNA Methyltransferase and Inhibitors Assay.

Abnormal DNA methylation contributes to the annoying tumorigenesis and the elevated expression of methylation-related methyltransferase (MTase) is associated with many diseases. Hence DNA MTase could serve as a promising biomarker for cancer-specific diagnosis as well as a potential therapeutic target. Herein, we developed an isothermal autocatalytic hybridization reaction (AHR) circuit for the sensitive detection of MTase and its inhibitors by integrating the catalytic hairpin assembly (CHA) converter with the hybridization chain reaction (HCR) amplifier.

Enzyme-Free Autocatalysis-Driven Feedback DNA Circuits for Amplified Aptasensing of Living Cells.

Aptasensors with high specificity have emerged as powerful tools for understanding various biological processes, thus providing tremendous opportunities for clinical diagnosis and prognosis. However, their applications in intracellular molecular imaging are largely impeded due to the low anti-interference capacity in biological environments and the moderate sensitivity to targets. Herein, a robust enzyme-free autocatalysis-driven feedback DNA circuit is devised for amplified aptasensing, for example, adenosine triphosphate (ATP) and thrombin, with a significantly improved sensitivity in living cells.

Bioorthogonal regulation of DNA circuits for smart intracellular microRNA imaging.

Catalytic DNA circuits represent a versatile toolbox for tracking intracellular biomarkers yet are constrained with low anti-interference capacity originating from their severe off-site activation. Herein, by introducing an unprecedented endogenous DNA repairing enzyme-powered pre-selection strategy, we develop a sequential and specific on-site activated catalytic DNA circuit for achieving the cancer cell-selective imaging of microRNA with high anti-interference capacity. Initially, the circuitry reactant is firmly caged by an elongated stabilizing duplex segment with a recognition/cleavage site of a cell-specific DNA repairing enzyme, which can prevent undesired signal leakage prior to its exposure to target cells.