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As a common heavy metal contaminant, Cd 2+ has adverse effects on food safety and consumer health. It is very important for human health to realize highly sensitive Cd 2+ detection methods. The self-powered sensing system based on enzyme biofuel cells (EBFCs) does not need an external power supply, which can simplify the experimental equipment and has great application value in portable detection. Thus, the biosensor is innovatively integrated into the screen-printed electrode to construct a new type of portable sensor suitable for on-site and real-time Cd 2+ detection. Hybridization chain reaction (HCR) combined with the Cd 2+ -dependent deoxyribose (DNAzyme) signal amplification strategy is used to enhance the detection sensitivity while specifically recognizing the Cd 2+ . Moreover, the self-powered sensor combines with smartphones to realize quantitative Cd 2+ detection without other instruments and has the characteristic of Effectively improving the hazard detection technology is essential to ensure food safety. Portability, simplicity, and speed are suitable for real-time Cd 2+ detection in the field. The dual mechanism and three quantitative modes combining colorimetric and two electrical signals output modes are adopted to realize the visualization and accurate detection. A series of research results confirm that this strategy is of great significance to strengthen the development of intelligent Cd 2+ technology, expand the application of self-powered sensing technology, and improve the safety detection system., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Foodborne bacteria like Escherichia coli threaten global food security, necessitating affordable, on-site detection methods, especially in resource-limited settings. This study optimized loop-mediated isothermal amplification (LAMP) integrated with peroxidase-mimicking G-quadruplex DNA structures (DNAzyme), termed DNAzyme-LAMP which was designed to incorporate two different catalytic DNAzymes per amplification unit, enabling colorimetric detection of E. coli in leafy vegetables and milk samples. Additionally, we introduce a novel electrochemical method that enhances analytical sensitivity. The optimized DNAzyme-LAMP achieved a detection limit below 6.3 CFU per reaction or 0.1 aM gene copies. This system lays the groundwork for the development of on-site biosensors and can be adapted for detecting other foodborne pathogens., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The Hybridization Chain Reaction (HCR) is an isothermal amplification technique widely used for sensing nucleic acids and small molecules. Despite its effectiveness, conventional linear HCR exhibits relatively slow kinetics and insufficient sensitivity. To address this challenge, we have innovatively combined HCR with DNAzyme technology to enhance nucleic acid detection. In this novel approach, the presence of a target molecule triggers the formation of DNAzyme, leading to the cleavage of substrate S, the initiation of HCR, and the production of DNA nanowires and labeled DNAzyme. The newly generated DNAzyme continuously cleaves substrate S, promoting sequential HCR amplification and significantly enhancing the fluorescence signal. This system offers a simple, sensitive, selective, and versatile method for nucleic acid detection, with a detection limit as low as 5 pM. When tested on classical swine fever virus (CSFV) samples, the system demonstrated detection accuracy comparable to RT-qPCR and exhibited superior repeatability.

DNA-templated silver nanoclusters (DNA-AgNCs) are novel nanomaterials with unique fluorescence characteristics. DNAzyme is a functional oligonucleotide that can catalyze the disruption of nucleic acid substrates. In this research, the effect of DNAzyme digestion on the fluorescence property of DNA-AgNCs was explored for the first time. A significant reduction in the fluorescence intensity of DNA-AgNCs after cleavage by DNAzyme was discovered. Further research found that the DNAzyme-catalyzed cleavage reduced the stability of DNA-AgNCs and led to their aggregation, accounting for a decline in fluorescence intensity up to 84%. Inspired by the above finding, a fluorescent aptasensor that integrates the benefits of DNA-AgNCs, exonuclease III (Exo III)-assisted signal amplification and DNAzyme was developed for sensitive detection of adenosine triphosphate (ATP). Under optimal conditions, the linear range was from 25 μM to 1000 μM and the detection limit was estimated to be 4.46 μM. Furthermore, this fluorescent aptasensor was effectively employed to quantify ATP levels in human serum samples, demonstrating its practicality in detecting ATP in biological matrices. The elucidation of DNAzyme-based fluorescence characteristic variation of DNA-AgNCs may provide insights into the interactions between DNAzyme and nanomaterials and has great prospects in the construction of fluorescent biosensors.

Cells use transient membraneless organelles to regulate biological reaction networks. For example, stress granules selectively store mRNA to downregulate protein expression in response to heat or oxidative stress. Models mimicking this active behavior should be established to better understand in vivo regulation involving compartmentalization. Here we use active, complex coacervate droplets as a model for membraneless organelles to spatiotemporally control the activity of a catalytic DNA (DNAzyme). Upon partitioning into these peptide-RNA droplets, the DNAzyme unfolds and loses its ability to catalyze the cleavage of a nucleic acid strand. We can transiently pause the DNAzyme activity upon inducing droplet formation with fuel. After fuel consumption, the DNAzyme activity autonomously restarts. We envision this system could be used to up and downregulate multiple reactions in a network, helping understand the complexity of a cell's pathways. By creating a network where the DNAzyme could reciprocally regulate the droplet properties, we would have a powerful tool for engineering synthetic cells., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Enzymes with critical effects on life systems are regulated by expression and activation to modulate life processes. However, further insights into enzyme functions and mechanisms in various physiological processes are limited to concentration or activation analysis only. Currently, enzyme analysis has received notable attention, particularly simultaneous analysis of their concentration and activation in one system. Herein, N-methyl mesoporphyrin IX (NMM), a specific dye with notable structural selectivity for parallel G-quadruplex nucleic acid enzyme (G4h DNAzyme), is employed for the analysis of its concentration. In addition, the peroxidase activity of G4h DNAzyme is characterized based on G4h DNAzyme-catalyzed decomposition of H 2 O 2 to continuously consume luminol. Accordingly, an increased fluorescence (FL) response of NMM and a decreased FL response of luminol could be simultaneously employed to analyze the concentration and activation of G4h DNAzyme., Result: Herein, a novel concentration and activation biresponsive strategy is proposed using a G4h DNAzyme-based model that simultaneously employs a G4h structure-specific signal probe for enzyme concentration analysis and G4h DNAzyme-catalyzed reactions for enzyme activation analysis. Under optimal conditions, the biresponsive strategy can be effectively used for the simultaneous analysis of G4h DNAzyme concentration and activation, with detection limits of 718.7 pM and 233.4 nM respectively, delivering acceptable performances both in cell and in vitro., Significance: This strategy can not only be applied to concentration and activation analyses of G4h DNAzyme but can also be easily extended to other enzymes by simultaneously combining concentration analysis via target-induced direct reaction and activation analysis via target-induced catalytic reaction, offering deeper insights into various enzymes and enabling their effective implementation in bioanalysis and biochemistry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sensitive imaging of microRNAs (miRNAs) in tumor cells holds great significance in the domains of pathology, drug development, and personalized diagnosis and treatment. DNA nanostructures possess excellent biostability and programmability and are suitable as carriers for intracellular imaging probes. With its highly controllable motion mechanism and remarkable target recognition specificity, the DNA walker is an ideal tool for living cell imaging. Here, we report a DNA nanowire based-DNAzyme Walker (D-Walker), which loads the DNAzyme based-molecular beacon (D-MB) onto DNA nanowires (NWs) functionalized with aptamers. The experimental results demonstrated that the intracellular target miRNA can specifically activate the pre-locked DNAzyme through a strand displacement reaction, thereby triggering the cleavage of its substrate molecular beacon (MB) and subsequent fluorescence emission. NWs decorated with aptamers can effectively prevent the degradation of the D-Walker by nuclease, and can enter target cells without any transfection reagents, which enhances the stability and reliability of cell imaging. Furthermore, the D-Walker exhibited a remarkable sensitivity with a limit of detection (LOD) of 61 pM and was capable of distinguishing miRNA-21 from other closely related family members. This study provides a novel strategy for intracellular miRNA imaging, offering a promising tool for cancer diagnosis and treatment.

Autocatalysis, a self-sustained replication process where at least one of the products functions as a catalyst, plays a pivotal role in life's evolution, from genome duplication to the emergence of autocatalytic subnetworks in cell division and metabolism. Leveraging their programmability, controllability, and rich functionalities, DNA molecules have become a cornerstone for engineering autocatalytic circuits, driving diverse technological applications. In this tutorial review, we offer a comprehensive survey of recent advances in engineering autocatalytic DNA circuits and their practical implementations. We delve into the fundamental principles underlying the construction of these circuits, highlighting their reliance on DNAzyme biocatalysis, enzymatic catalysis, and dynamic hybridization assembly. The discussed autocatalytic DNA circuitry techniques have revolutionized ultrasensitive sensing of biologically significant molecules, encompassing genomic DNAs, RNAs, viruses, and proteins. Furthermore, the amplicons produced by these circuits serve as building blocks for higher-order DNA nanostructures, facilitating biomimetic behaviors such as high-performance intracellular bioimaging and precise algorithmic assembly. We summarize these applications and extensively address the current challenges, potential solutions, and future trajectories of autocatalytic DNA circuits. This review promises novel insights into the advancement and practical utilization of autocatalytic DNA circuits across bioanalysis, biomedicine, and biomimetics.

Dynamic DNA nanodevices, particularly DNA walkers, have proven to be versatile tools for target recognition, signal conversion, and amplification in biosensing. However, their ability to detect low-abundance analytes in complex biological samples is often compromised by limited amplification depth and severe signal leakage. To address these challenges, we developed a simple yet highly efficient strategy to engineer a self-replicating bipedal DNAzyme (SEDY) walker for sensitive and selective electrochemiluminescence (ECL) bioanalysis. Unlike conventional DNA walkers that are typically constructed by catalytic DNA assembly in a single direction, the SEDY walker integrates a self-replicating feedback mechanism that greatly enhances both the selectivity and sensitivity of bioanalysis. First, the SEDY walker is assembled through a target-triggered, enzyme-free, self-replicating catalytic approach, minimizing the risk of undesired side reactions and signal leakage by simplifying reactant complexity. Furthermore, the SEDY walker features newly exposed trigger sequences that facilitate its autonomous replication, leading to a robust and exponential amplification of its products. Our experiments demonstrate that the SEDY walker can sensitively and selectively detect acetamiprid by navigating specific probes within cross-shaped DNA orbits. The ECL biosensor offers a linear detection range from 1 × 10 -15 M to 1 × 10 -9 M, with a limit of detection as low as 5.8 × 10 -16 M. We anticipate that the SEDY walker will be a powerful tool for detecting various analytes in biological applications.

Methods for modifying intact peptides are useful but can be unselective with regard to amino acid position and sequence context. In this work, we used in vitro selection to identify DNAzymes that acylate a Lys residue of a short peptide in sequence-dependent fashion. The DNAzymes do not acylate Lys when placed at other residues in the peptide, and the acylation activity depends on the Lys sequence context. A high acylation yield is observed on the preparative nanomole scale. These findings are promising for further development of DNAzymes for broader application to top-down Lys acylation of peptide and protein substrates., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

In this work, we reported an electrochemical biosensor with target-assisted self-cleavage DNAzyme function for signal amplified detection of miRNA. The target-recycling amplification led to significant signal enhancement and thus offers high detection sensitivity.

Background: Rapid industrial development has generated serious pollution, including the presence of toxic and harmful heavy metal ions. Among them, trivalent chromium ion (Cr 3+ ) is a very important element that poses a threat to life and health in our industrial wastewater pollution. Thus, it is important to develop efficient fluorescence methods for Cr 3+ detection. In this study, an upconversion luminescence biosensor for detecting Cr 3+ was constructed based on a DNAzyme, strand displacement reaction (SDR), and DNA-functionalized upconversion nanoparticles (UCNPs)., Results: The sulfonate-rich poly (sodium 4-styrene sulfonate) (PSS) was modified onto the surface of UCNPs, forming UCNPs@PSS. Then, NH 2 -Capture probe DNA (NH 2 -Cp) was further modified onto the UCNPs@PSS surface through sulfonylation, resulting in UCNPs@PSS@NH 2 -Cp. The DNAzyme activated by Cr 3+ triggered the release of the primer probe (Pp), which initiated the SDR system cycle, thereby releasing a tetramethylrhodamine (TAMRA)-modified signal probe (TAMRA-Sp). Finally, UCNPs@PSS@NH 2 -Cp bound to TAMRA-Sp through complementary base pairing, causing UCNPs and TAMRA to approach each other. Because of the luminescence resonance energy transfer (LRET) mechanism, the upconversion luminescence (UCL) signal of the UCNPs was quenched by TAMRA, enabling the detection of Cr 3+ by the change of I 585 /I 545 ratio. This biosensor has good stability, selectivity, and sensitivity, with a linear range of 0.5-75 nM and a detection limit of 0.135 nM for Cr 3+ ., Significance and Novelty: Firstly, based on LRET between UCNPs and TAMRA, the quantitative analysis of Cr 3+ is achieved through the changes of ratio fluorescence. Secondly, the specificity of the biosensor is improved by utilizing the specific recognition of DNA enzymes. Thirdly, the signal amplification technology of the SDR cycle greatly improves the sensitivity of biosensor. This biosensor will be useful for future environmental safety monitoring and biopsy of biological fluids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As a kind of mycotoxin, aflatoxin B1 (AFB1), which is often found in agricultural products, poses a threat to human health. Developing a simple sensitive method for AFB1 detection is in great demand. Here, we reported an aptamer-based fluorescence assay for AFB1 detection by using DNAzyme to generate and amplify a signal. We redesigned a pair of DNA sequences, which originated from the anti-AFB1 aptamer and RNA-cleaving DNAzyme 10-23. In the absence of AFB1, the aptamer hybridized with the region of the substrate-binding arm of the DNAzyme, inhibiting the activity of the DNAzyme. In the presence of AFB1, the binding of AFB1 to the aptamer led to the displacement of the DNAzyme from the aptamer. The substrate-binding arm was unblocked, and the activity of the DNAzyme was restored for the hydrolysis of the fluorophore and quencher-labeled substrate, causing a significant fluorescence increase. This assay could detect AFB1 in the dynamic range from 0.98 to 2000 nmol/L with high selectivity, and the detection limit was 0.98 nmol/L. Moreover, the assay was able to detect AFB1 in a complex sample matrix. This work provides a useful tool for the analysis of AFB1., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Monitoring biomarkers secreted by cardiomyocytes is critical to evaluate anticancer drug-induced myocardial injury (MI). Cardiac troponin I (cTnI) is considered the gold standard biomarker for MI. Herein, an electrochemical aptasensor is engineered for cTnI detection based on lanthanide europium metal-organic frameworks (Eu-MOFs) and a hybridization chain reaction-directed DNAzyme strategy. Three types of Eu-MOF morphologies were easily synthesized by changing the solvent, and the Eu-MOF modulated by mixing the solvent of dimethylformamide and H 2 O (D-Eu-MOF) exhibited the best performance compared to other morphologies of the Eu-MOFs. Multifunctional nanoprobes were constructed from D-Eu-MOF@Pt loaded with natural horseradish peroxidase and combined with an aptamer-initiated nuclear acid hybridization chain reaction to form G-quadruplex/hemin DNAzymes for signal amplification. A novel capture probe is constructed on the basis of DNA nanotetrahedrons modified on screen-printed gold electrodes to enhance the capture of the target and multifunctional nanoprobes for signal amplification. It exhibits a detection limit of 0.17 pg mL -1 and a linear range from 0.5 pg mL -1 to 15 ng mL -1 . The practicality of the platform is evaluated by measuring cTnI in real samples and secreted by cardiomyocytes after drug treatment, which provides great potential in drug-induced MI evaluation for clinical application.

Catalytic DNA has gained significant attention in recent decades as a highly efficient and tunable catalyst, thanks to its flexible structures, exceptional specificity, and ease of optimization. Despite being composed of just four monomers, DNA's complex conformational intricacies enable a wide range of nuanced functions, including scaffolding, electrocatalysis, enantioselectivity, and mechano-electro spin coupling. DNA catalysts, ranging from traditional DNAzymes to innovative DNAzyme hybrids, highlight the remarkable potential of DNA in catalysis. Recent advancements in spectroscopic techniques have deepened our mechanistic understanding of catalytic DNA, paving the way for rational structural optimization. This review will summarize the latest studies on the performance and optimization of traditional DNAzymes and provide an in-depth analysis of DNAzyme hybrid catalysts and their unique and promising properties.

Dipeptidyl peptidase IV (DPPIV) is an enzyme belonging to the type II transmembrane serine protease family that has gained wide interest in the fields of hematology, immunology, and cancer biology. Moreover, DPPIV has emerged as a promising target for therapeutic intervention in type II diabetes. Due to its biological limitations, traditional strategies cannot meet the requirements of low abundance DPPIV analysis in complex environments. In this work, combining the high programmability of DNA and the chemical diversity of peptides, we designed DNA-peptide conjugates that can be specifically recognized, polypeptides as specific substrates for target DPPIV and DNA probes as primers for catalytic hairpin assembly (CHA), recycling a large amount of DNAzymes by triggering CHA amplification. The DNAzyme substrate modified with the FAM fluorescent group was immobilized on the surface of gold nanoparticles by S-Au chemical bonds to form a signal output probe. The DNAzymes enzyme cleaved the substrate of the signal outputs probe, yielding a double-amplified fluorescence signal. This method has a detection limit as low as 0.18 mU mL -1 and a linear range of 0-5 mU mL -1 in serum samples, showing high stability and good potential for practical applications.

The development of highly sensitive and precise imaging techniques capable of visualizing crucial molecules at the subcellular level is essential for elucidating mitochondrial functions and uncovering novel mechanisms in biological processes. However, traditional molecular imaging strategies are still limited by off-mitochondria signal leakage because of the "always-active" sensing mode. To address this limitation, we have developed a light-triggered activation sequence activated plasmonic DNAzyme walker (PDW) for accurate subcellular molecular imaging by the combination of an organelle localized strategy, upconversion nanotechnology, and a plasmon enhanced fluorescence (PEF) technique. Exploiting the advantage of light activation enables precise control over when and where to activate the probe's sensing function, effectively reducing off-mitochondria signal leakage as validated by the dynamic monitoring of changes in off-mitochondria signals during the mitochondrial entry process. Furthermore, by leveraging the PEF capability of triangular gold nanoprisms (Au NPRs), the fluorescence intensity can be enhanced by approximately 11.9 times, ensuring highly sensitive and accurate subcellular molecular imaging.

DNA walkers have attracted considerable attention in biosensing and bioimaging. Compared with the conventional single leg-based DNA walker, the bipedal DNA walker has remarkable advantages, with improved sensitivity and fast kinetics, and can work efficiently in a crowded cellular environment. However, most reported bipedal DNA walkers are powered by exogenous supplementation, and elaborate DNA sequence designs, auxiliary additives or extra carriers are often needed. A highly integrated bipedal DNA walker that can address robustness, sensitivity and consistency issues in a single system is highly desirable but remains a great challenge. We herein report a novel bipedal DNA nanowalker system through simple assembly of a DNA substrate, hairpin functionalized-AuNPs (AuNPs-H2), and a blocked Mn 2+ -dependent DNAzyme hairpin (H1) on degradable MnO 2 nanosheets, which holds great potential for living cell operation. Highly integrated features enable the simultaneous delivery of core components of the bipedal DNA walker, including a walking track (AuNPs-H2), a walking strand (H1 cleaved by APE1), and a driving force (Mn 2+ -dependent DNAzyme cleavage) as a whole, thereby enhancing the control of the spatiotemporal distribution of these components at the intracellular target sites. The redox reaction between the MnO 2 nanosheets and GSH inside the cells not only consumed the intracellular GSH to improve the biostability of the walking track but also generated abundant Mn 2+ as a cofactor of the DNAzyme. As a proof of concept, the developed nanowalker was demonstrated to work efficiently for monitoring base excision repair (BER)-related human apurinic/apyrimidinic endonuclease 1 (APE1) in living cells, highlighting the great potential of the bipedal DNA nanowalker in biological systems., (© 2024. The Author(s).)

CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage efficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate to Cas12a but also result in an exceptionally high signal-to-noise ratio by mediating enzymatic click reaction. Combined with a functional nucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose sensitivity is approximately 54-fold higher than that of TaqMan probe-mediated CRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory recoveries in food matrices ranging from 80% to 101%. The DNA extraction- and preamplification-free detection format as well as the potent detection performance highlight its tremendous potential as a next-generation analysis tool.

The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We have systematically investigated and found surprising superior catalytic activities of very short DNAzymes for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), both in solution and on surface. As a key reaction of the "click chemistry" class, CuAAC is a highly efficient and specific covalent conjugation tool with demonstrated applications in organic synthesis, bioconjugation, and surface functionalization; however, it requires the presence of the Cu(I) catalyst, which is an unstable species in aqueous solutions. We show here that one ultrashort, 14-nucleotide-truncated fragment of an earlier in vitro selected DNAzyme (CLICK-17) shows a striking and superior catalytic activity toward the in trans CuAAC reaction in solution and on surface in the presence of either Cu(I) or Cu(II), at significantly lowered concentrations. These results obviate the need for long-sequence DNAzymes, selected out of the homogeneous solution phase, for application in complex surface environments.

Multiplex detection can enhance diagnostic precision and improve diagnostic efficiency, providing important assistance for epidemiological investigation and epidemic prevention. There is a great need for multi-detection sensing platforms to accurately diagnose diseases. Herein, we reported a μPAD-based chemiluminescence (CL) assay for ultrasensitive multiplex detection of AIV biomarkers, based on three DNAzyme/Lum/PEI/CaCO 3 . Three time-resolved CL signals were sequentially generated with detection limits of 0.32, 0.34, and 0.29 pM for H1N1, H7N9, and H5N1, respectively, and with excellent selectivity against interfering DNA. The recovery test in human serum displayed satisfactory analysis capabilities for complex biological samples. The μPAD-based CL assay achieved multiplex detection within 70 s, with a high time resolution of 20 s. The proposed strategy has the advantages of low cost, high sensitivity, good selectivity, and wide time resolution, the μPAD-based CL assay has shown great potential in the early and accurate diagnosis of diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Effective isolation and sensitive detection of Pseudomonas aeruginosa ( P. aeruginosa ) is crucial for the early diagnosis and prognosis of various diseases, such as urinary tract infections. However, efficient isolation and simultaneous detection of P. aeruginosa remains a huge challenge. Herein, we depict a novel fluorescence assay for sensitive, enzyme-free detection of P. aeruginosa by integrating DNAzyme cascade-induced DNA tweezers and magnetic nanoparticles (MNPs)-based separation. The capture probe@MNPs is capable of accurately identifying target bacteria and transporting the bacteria signal to nucleic acid signals. Based on the DNAzyme cascade-induced DNA tweezers, the nucleic acid signals are extensively amplified, endowing the method with a high sensitivity and a low detection limit of 1 cfu/mL. In addition, the method also exhibits a wide detection of six orders of magnitudes. The proposed method could be extended to other bacteria detection by simply changing the aptamer sequence. Taking the merit of the high sensitivity, greatly minimized detection time (less than 1.5 h), enzyme-free characteristics, and stability, the proposed method could be potentially applied to diagnosing and preventing diseases caused by pathogenic bacteria.

DNAzymes, a class of single-stranded catalytic DNA with good stability, high catalytic activity, and easy synthesis, functionalization and modification properties, have garnered significant interest in the realm of biosensing and bioimaging. Their integration with fluorescent dyes or chemiluminescent moieties has led to remarkable bioimaging outcomes, while DNAzyme-based biosensors have demonstrated robust sensitivity and selectivity in detecting metal ions, nucleic acids, proteins, enzyme activities, exosomes, bacteria and microorganisms. In addition, by delivering DNAzymes into tumor cells, the mRNA therein can be cleaved to regulate the expression of corresponding proteins, which has further propelled the application of DNAzymes in cancer gene therapy and synergistic therapy. This paper reviews the strategies for screening attractive DNAzymes such as SELEX and high-throughput sequencing, and briefly describes the amplification strategies of DNAzymes, which mainly include catalytic hairpin assembly (CHA), DNA walker, hybridization chain reaction (HCR), DNA origami, CRISPR-Cas12a, rolling circle amplification (RCA), and aptamers. In addition, applications of DNAzymes in bioimaging, biosensing, and cancer therapy are also highlighted. Subsequently, the possible challenges of these DNAzymes in practical applications are further pointed out, and future research directions are suggested.

Background: Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative., Results: Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS 2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM., Significance: The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5'-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca 2+ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs.

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg 2+ -dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted Dk1sub and Dk1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis., (© 2024 Wiley-VCH GmbH.)

Hemin/G-quadruplex (hG4) complexes are frequently used as artificial peroxidase-like enzymatic systems (termed G4 DNAzymes) in many biosensing applications, in spite of a rather low efficiency, notably in terms of detection limits. To tackle this issue, we report herein a strategy in which hemin is chemically modified with the amino acids found in the active site of parent horseradish peroxidase (HRP), with the aim of recreating an environment conducive to high catalytic activity. When hemin is conjugated with a single arginine, it associates with G4 to create an arginine-hemin/G4 (R-hG4) DNAzyme that exhibits improved catalytic performances, characterized by kinetic analysis and DFT calculations. The practical relevance of this system was demonstrated with the implementation of biosensing assays enabling the chemiluminescent detection of G4-containing DNA and colorimetry detection of the flap endonuclease 1 (FEN1) enzyme with a high efficiency and sensitivity. Our results thus provide a guide for future enzyme engineering campaigns to create ever more efficient peroxidase-mimicking DNA-based systems.

Developing sensors with high selectivity and sensitivity is of great significance for pesticide analysis in environmental assessment. Herein, a versatile three-way sensor array was designed for the detection of the pesticide atrazine, based on the integration of catalytic hairpin assembly (CHA) amplification and three-mode signal transducers. With atrazine, CHA was triggered to generate abundant G-quadruplex. The produced G-quadruplex hybrid could assemble with thioflavin T (TFT) or hemin to mimic enzyme and induce the fluorescence enhancement by TFT, or the colorimetric increase by the oxidized chromogenic substrate and the naked-eye color change by inhibiting the L-cysteine-mediated aggregation of gold nanoparticles. A distinctive three-mode array was successfully constructed with convenience, on-site accessibility and high sensitivity for enzyme-free practical analysis of atrazine. It is also effective and reliable for analyzing real samples including paddy water, paddy soil and polished rice. The detection limits for atrazine were as low as 7.4 pg/mL by colorimetric observation and 0.25 pg/mL by fluorescent detection. Furthermore, the array was exploited to monitor the residue, distribution and bioaccumulation of atrazine in maize and rice for food security and environmental assessment. Hence, this work presented a versatile example for sensitive and on-site all-in-one pesticide analysis arrays with multiple signal report modes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A substantial development in nanoscale materials possessing catalytic activities comparable with natural enzymes has been accomplished. Their advantages were owing to the excellent sturdiness in an extreme environment, possibilities of their large-scale production resulting in higher profitability, and easy manipulation for modification. Despite these advantages, the main challenge for artificial enzyme mimetics is the lack of substrate selectivity where natural enzymes flourish. This review addresses this vital problem by introducing substrate selectivity strategies to three classes of artificial enzymes: molecularly imprinted polymers, nanozymes (NZs), and DNAzymes. These rationally designed strategies enhance the substrate selectivity and are discussed and exemplified throughout the review. Various functional mechanisms associated with applying enzyme mimetics in biosensing and bioassays are also given. Eventually, future directives toward enhancing the substrate selectivity of biomimetics and related challenges are discussed and evaluated based on their efficiency and convenience in biosensing and bioassays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg 2+ -stabilized DNA duplex (Hg 2+ -dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg 2+ in Hg 2+ -dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Modern cryptography based on computational complexity theory is mainly constructed with silicon-based circuits. As DNA nanotechnology penetrates the molecular domain, utilizing molecular cryptography for data access protection in the biomolecular domain becomes a unique approach to information security. However, building security devices and strategies with robust security and compatibility is still challenging. Here, this study reports a time-controlled molecular authentication strategy using DNAzyme and DNA strand displacement as the basic framework. A time limit exists for authorization and access, and this spontaneous shutdown design further protects secure access. Multiple hierarchical authentications, temporal Boolean logic authentication, and enzyme authentication strategies are constructed based on DNA networks'good compatibility and programmability. This study gives proof of concept for the detection and protection of bioinformation about single nucleotide variants and miRNA, highlighting their potential in biosensing and security protection., (© 2024 Wiley‐VCH GmbH.)

Herein, an electrochemical aptasensor for highly sensitive detection of acetamiprid (ACE) was constructed based on a one-step cascade amplification strategy. This innovative strategy integrated DNA walker containing DNAzyme sequence into entropy-driven catalysis (EDC) system. The trigger strand was released by aptamer-specific binding to ACE, initiating the EDC amplification circuit and delivering DNA walker strands. The dangling DNA walker continuously bound and cleaved hairpin substrate to form G-quadruplex fragments with the assistance of Mg 2+ . The G-quadruplex fragments folded and captured hemin to form multitudinous G-quadruplex/hemin complexes in the presence of K + , generating significantly enhanced current, enabling enzyme-free, label-free and highly sensitive detection of ACE, with a linear detection range of 100 fM to 50 nM and a detection limit of 68.36 fM (S/N = 3). The constructed aptasensor achieved the reliable detection of ACE in vegetable soil and cucumber samples, demonstrating its potential application prospects in environmental protection and food supervision., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Three-dimensional (3D) DNA walking nanomachines have been widely applied in molecular diagnostics and cancer therapy. Specially, DNA dendrimer with 3D nanostructure has gained increasing attention in biomedical applications because of its high biostability, excellent programmability, and outstanding biocompatibility. In this chapter, we developed a 3D DNA walking nanomachine by assembling all nanomachine components (DNAzyme and substrate) onto a DNA dendrimer for miRNA imaging in living cells. In this design, the number of DNAzyme and substrate on the surface of the DNA dendrimer could be precisely regulated to fully implement the function of the DNA nanomachine., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

There is an urgent need for novel strategies to accurately and reliably detect pathogenic bacteria to address the global epidemic of antibiotic resistance. This study proposes an innovative approach combining dual aptamer-based target recognition and proximity ligation assay (PLA) triggered DNAzyme recycling cleavage. This method allows for the precise identification and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA). The fluorescence probe labeled with a fluorophore is modified on gold nanoparticles (AuNPs), resulting in the quenching of the fluorescent signal by the AuNPs. The interaction between MRSA and two aptamers leads to forming a Mg 2+ -dependent DNAzyme. The DNAzyme cleaves the fluorescence probe, causing the fluorescent fragment to detach from the surface of the AuNPs, in which the quenched fluorescence signal in the fluorescence probe reappears. The DNAzyme-assisted cleavage and rebinding process generates a processive strolling along the surface track of AuNPs. Consequently, the fluorescence intensity experiences a substantial recovery. A strong linear correlation is observed between the fluorescence intensity and MRSA concentration within 50 cfu/mL to 10 6  cfu/mL. We believe that implementing the novel integrated strategy will broaden the range of bacterial detection methods in the battlefield environment and stimulate the creation of potential new drugs in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The concentration elevation of myocardial microRNA (miRNA) biomarker is associated with the pathogenic process of acute myocardial infarction (AMI), and sensitive quantification of myocardial miRNA biomarker plays an important role for early AMI diagnosis and its treatment. In response, this work describes an ultrasensitive and non-label electrochemical biosensor for the assay of myocardial miRNA based on cascade signal amplifications integrated by DNAzyme walker and hemin/G-quadruplex nanowires. The DNAzyme walker is activated by presence of target miRNAs to move along the electrode surface to cyclically cleave the substrate hairpins to release G-quadruplex segments, which further trigger the in situ formation of many hemin/G-quadruplex nanowires. The large amounts of hemin intercalated into the DNA nanowires subsequently generate drastically magnified electrochemical current signals for highly sensitive label-free assay of myocardial miRNAs down to 15.7 fM within dynamic range of 100 fM to 10 nM. Such a biosensor also has high selectivity and can monitor myocardial miRNAs in diluted serums at low levels, providing a sensitive and reliable platform for diagnosing infarct-associated cardiovascular diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N 6 -methyladenosine (m 6 A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m 6 A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m 6 A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m 6 A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m 6 A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m 6 A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m 6 A fractions in real biological samples, even in low abundance RNA biomarkers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In situ monitoring microRNA (miRNA) expression in vivo holds immense potential for directly visualizing the occurrence and progression of tumors. However, the significant barrier to developing a probe that can overcome the low abundance of miRNAs while providing an output signal with unlimited tissue penetration depth remains formidable. In this study, we developed a DNA machine-based magnetic resonance imaging nanoprobe (MRINP) for amplified detection of miR-21 in vivo. The MRINP was constructed with superparamagnetic Fe 3 O 4 nanoparticles (NPs), paramagnetic Gd-DOTA complexes, and miR-21-activated DNA machines; the DNA machine was composed of hairpin DNAzyme (HD) strands serving as the DNAzyme walker and hairpin substrate (HS) strands serving as the track. Once uptake into tumor cells, the intracellular miR-21 specifically recognized and hybridized with the HD strand, restoring the activity of DNAzyme. Subsequently, the DNAzyme walker autonomously traveled on the surface of MRINP, and each step movement of the DNAzyme walker resulted in the cleavage of its substrate strands and the ensued release of the Gd-DOTA complex-labeled oligonucleotides, turning on the T 1 signal of Gd-DOTA complexes for in situ imaging of miR-21 in tumor-bearing mice. This strategy would offer a promising approach for mapping tumor-specific biomarkers in vivo with unlimited penetration depth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The development of synthetic molecular tools responsive to biological cues is crucial for advancing targeted cellular regulation. A significant challenge is the regulation of cellular processes in response to gaseous signaling molecules such as hydrogen sulfide (H 2 S). To address this, we present the design of Gas signaling molecule-Responsive Artificial DNAzyme-based Switches (GRAS) to manipulate cellular functions via H 2 S-sensitive synthetic DNAzymes. By incorporating stimuli-responsive moieties to the phosphorothioate backbone, DNAzymes are strategically designed with H 2 S-responsive azide groups at cofactor binding locations within the catalytic core region. These modifications enable their activation through H 2 S-reducing decaging, thereby initiating substrate cleavage activity. Our approach allows for the flexible customization of various DNAzymes to regulate distinct cellular processes in diverse scenarios. Intracellularly, the enzymatic activity of GRAS promotes H 2 S-induced cleavage of specific mRNA sequences, enabling targeted gene silencing and inducing apoptosis in cancer cells. Moreover, integrating GRAS with dynamic DNA assembly allows for grafting these functional switches onto cell surface receptors, facilitating H 2 S-triggered receptor dimerization. This extracellular activation transmits signals intracellularly to regulate cellular behaviors such as migration and proliferation. Collectively, synthetic switches are capable of rewiring cellular functions in response to gaseous cues, offering a promising avenue for advanced targeted cellular engineering., (© 2024 Wiley-VCH GmbH.)

Sensitive monitoring of human 8-oxyguanine DNA glycosylase (hOGG1) activity in living cells is helpful to understand its function in damage repair and evaluate its role in disease diagnosis. Herein, a functional DNA-Zn 2+ coordination nanospheres was proposed for sensitive imaging of hOGG1 in living cells. The nanospheres were constructed through the coordination-driven self-assembly of the entropy driven reaction (EDR) -deoxyribozyme (DNAzyme) system with Zn 2+ , where DNAzyme was designed to split structure and assembled into the EDR system. When the nanospheres entered the cell, the competitive coordination between phosphate in the cell and Zn 2+ leaded to the disintegration of the nanospheres, releasing DNA and some Zn 2+ . The released Zn 2+ acted as a cofactor of DNAzyme. In the presence of hOGG1, the EDR was completed, accompanied by fluorescence recovery and the generation of a complete DNAzyme. With the assistance of Zn 2+ , DNAzyme continuously cleaved substrates to produce plenty of fluorescence signals, thus achieving sensitive imaging of hOGG1 activity. The nanospheres successfully achieved sensitive imaging of hOGG1 in human cervical cancer cells (HeLa), human non-small cell lung cancer cells and human normal colonic epithelial cells, and assayed changes in hOGG1 activity in HeLa cells. This nanospheres may provide a new tool for intracellular hOGG1 imaging and related biomedical studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

While the intracellular imaging of miRNA biomarkers is of significant importance for the diagnosis and treatment of human cancers, DNA assembled nanoprobe has recently attracted considerable attention for imaging intracellular biomolecules. However, the complex construction process, intrinsic vulnerability to nuclease degradation and the limited signal transduction efficiency hamper its widespread application. In this contribution, based on persistent autonomous molecular motion of DNAzyme walker along a nano-substrate track, a DNA nanosphere probe (PNLD) is developed for the sensitive intracellular miR-21 imaging. Specifically, DNA nanosphere (called PN, single-molecule nano-track) is assembled from only one palindromic substrate, into which the locking strand-silenced DNAzymes (LD) are installed in a controlled manner. PNLD (made of PN and LD) can protect all DNA components against nuclease attack and maintain its structural integrity in serum solution over 24 h. Upon the activation by target miRNA, DNAzyme walker can move on the substrate scattered within PNLD (or on the surface) and between different PNLD objects and cleave many DNA substrates, generating an amplified signal. As a result, miR-21 can be detected down to 6.83 pM without the detectable interference from co-existing nontarget miRNAs. Moreover, PNLD system can accurately screen the different expression levels of miR-21 within the same type of cells and different types of cells, which is consistent with gold standard polymerase chain reaction (PCR) assay. Via changing the target recognition sequence, the PNLD system can be suitable for the intracellular imaging of miR-155, exhibiting the desirable universality. In addition, the DNAzyme walker-based PNLD system can be used to distinguish cancer cells from healthy cells, implying the potential application in cancer diagnosis and prognosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biosensors, devices capable of detecting biomolecules or bioactive substances, have recently become one of the important tools in the fields of bioanalysis and medical diagnostics. A biosensor is an analytical system composed of biosensitive elements and signal-processing elements used to detect various biological and chemical substances. Biomimetic elements are key to biosensor technology and are the components in a sensor that are responsible for identifying the target analyte. The construction methods and working principles of biosensors based on synthetic biomimetic elements, such as DNAzyme, molecular imprinted polymers and aptamers, and their updated applications in biomedical analysis are summarised. Finally, the technical bottlenecks and future development prospects for biomedical analysis are summarised and discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this work, we proposed a sensitive and selective colorimetric assay for single nucleotide mutation (SNM) detection combining rolling circle amplification (RCA) and G-quadruplex/hemin DNAzyme complex formation. In the detection principle, the first step involves ssDNA hybridization with a padlock probe (PLP) DNA, which can discriminate a single base mismatch. The successful ligation is followed by an RCA event to generate an abundance of G-quadruplexes (GQ-RCA) which are then transformed into a DNAzyme (G-quadruplex/hemin complex) by the addition of hemin. The color change from colorless 3,3',5,5'-tetramethylbenzidine (TMB) into colored oxTMB when hydrogen peroxide (H 2 O 2 ) is added indicated the presence of a mutation. The assay had a limit of detection (LOD) of 2.14 pM. Mutations in samples from breast cancer patients were successfully detected with an accuracy of 100% when compared to Sanger sequencing results. The method is easily applicable even in resource poor setting regions given that it doesn't require any sophisticated or expensive instruments, and the signal readout is detectable simply by the naked eye. Our assay might be a useful tool for genetic analysis and clinical molecular diagnosis for breast cancer risk assessment and early detection.

Degradation and interference of the nucleic acid probes in complex biological environments like cytoplasm or body fluid can cause obvious false-positive signals and inefficient bioregulation in biosensing and biomedicine. To solve this problem, here, we proposed a universal strategy, termed L-DNA assembly mirror-image box-based environment resistance (L-AMBER), to protect nucleic acid probes from degradation and maintain their responsive activity in complex biological environments. Strand displacement reaction (SDR), aptamer, or DNAzyme-based D-DNA probes were encapsulated into an L-DNA box by using an L-D-L block DNA carrier strand to construct different kinds of L-AMBER probes. We proved that the L-DNA box could effectively protect the encapsulated D-DNA probes by shielding the interference of complex biological environments and only allowing small target molecules to enter for recognition. Compared with the D-AMBER probes, the L-AMBER probes can realize DNase I-assisted amplification detection of biological samples, low false-positive bioimaging, and highly efficient miRNA silence in living cells. Therefore, L-AMBER provided a universal and effective strategy for enhancing the resistance to environmental interference of nucleic acid probes in biosensing and biomedicine applications.

Fluorescence facilitates the detection, visualization, and tracking of molecules with high sensitivity and specificity. A functional DNA molecule that generates a robust fluorescent signal would offer significant advantages for many applications compared to intrinsically fluorescent proteins, which are expensive and labor intensive to synthesize, and fluorescent RNA aptamers, which are unstable under most conditions. Here, we describe a novel deoxyriboyzme that rapidly and efficiently generates a stable fluorescent product using a readily available coumarin substrate. An engineered version can detect picomolar concentrations of ribonucleases in a simple homogenous assay, and was used to rapidly identify novel inhibitors of the SARS-CoV-2 ribonuclease Nsp15 in a high-throughput screen. Our work adds an important new component to the toolkit of functional DNA parts, and also demonstrates how catalytic DNA motifs can be used to solve real-world problems., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Colorimetric assays in which the color of a solution changes in the presence of an input provide a simple and inexpensive way to monitor experimental readouts. In this study we used in vitro selection to identify a self-phosphorylating kinase deoxyribozyme that produces a colorimetric signal by converting the colorless substrate pNPP into the yellow product pNP. The minimized catalytic core, sequence requirements, secondary structure, and buffer requirements of this deoxyribozyme, which we named Apollon, were characterized using a variety of techniques including reselection experiments, high-throughput sequencing, comparative analysis, biochemical activity assays, and NMR. A bimolecular version of Apollon catalyzed multiple turnover phosphorylation and amplified the colorimetric signal. Engineered versions of Apollon could detect oligonucleotides with specific sequences as well as several different types of nucleases in homogenous assays that can be performed in a single tube without the need for washes or purifications. We anticipate that Apollon will be particularly useful to reduce costs in high-throughput screens and for applications in which specialized equipment is not available., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA and RNA nucleobase modifications are biologically relevant and valuable in fundamental biochemical and biophysical investigations of nucleic acids. However, directly introducing site-specific nucleobase modifications into long unprotected oligonucleotides is a substantial challenge. In this study, we used in vitro selection to identify DNAzymes that site-specifically N-alkylate the exocyclic nucleobase amines of particular cytidine, guanosine, and adenosine (C, G and A) nucleotides in DNA substrates, by reductive amination using a 5'-benzaldehyde oligonucleotide as the reaction partner. The new DNAzymes each require one or more of Mg2+, Mn2+, and Zn2+ as metal ion cofactors and have kobs from 0.04 to 0.3 h-1, with rate enhancement as high as ∼104 above the splinted background reaction. Several of the new DNAzymes are catalytically active when an RNA substrate is provided in place of DNA. Similarly, several new DNAzymes function when a small-molecule benzaldehyde compound replaces the 5'-benzaldehyde oligonucleotide. These findings expand the scope of DNAzyme catalysis to include nucleobase N-alkylation by reductive amination. Further development of this new class of DNAzymes is anticipated to facilitate practical covalent modification and labeling of DNA and RNA substrates., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The 10-23 DNAzyme, a catalytic DNA molecule with RNA-cleaving activity, has garnered significant interest for its potential therapeutic applications as a gene-silencing agent. However, the lack of a detailed understanding about its mechanism has hampered progress. A recent structural analysis has revealed a highly organized conformation thanks to the stabilization of specific interactions within the catalytic core of the 10-23 DNAzyme, which facilitate the cleavage of RNA. In this configuration, it has been shown that G14 is in good proximity to the cleavage site which suggests its role as a general base, by activating the 2'-OH nucleophile, in the catalysis of the 10-23 DNAzyme. Also, the possibility of a hydrated metal acting as a general acid has been proposed. In this study, through activity assays, we offer evidence of the involvement of general acid-base catalysis in the mechanism of the 10-23 DNAzyme by analyzing its pH-rate profiles and the role of G14, and metal cofactors like Mg 2+ and Pb 2+ . By substituting G14 with its analogue 2-aminopurine and examining the resultant pH-rate profiles, we propose the participation of G14 in a catalytically relevant proton transfer event, acting as a general base. Further analysis, using Pb 2+ as a cofactor, suggests the capability of the hydrated metal ion to act as a general acid. These functional results provide critical insights into the catalytic strategies of RNA-cleaving DNAzymes, revealing common mechanisms among nucleic acid enzymes that cleave RNA.