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Clostridium butyricum ( Cb Ago)-based bioassays are popular due to their programmability and directional cleavage capabilities. However, the relatively compact protein structure of Cb Ago limits its cleavage activity (even at the optimal temperature), thus restricting its wider application. Here, we observed that guide DNA (gDNA) with specific structural features significantly enhanced Cb Ago cleavage efficiency. Then, we invented a novel gDNA containing DNAzyme segments (gDNA zyme ) that substantially enhanced the Cb Ago cleavage efficency (by 100%). Using a molecular dynamics simulation system, we found that the augmented cleavage efficiency might be attributed to the large-scale global movement of the PIWI domain of Cb Ago and an increased number of cleavage sites. Moreover, this gDNA zyme feature allowed us to create a biosensor that simultaneously and sensitively detected three pathogenic bacteria without DNA extraction and amplification. Our work not only dramatically expands applications of the Cb Ago-based biosensor but also provides unique insight into the protein-DNA interactions.

Crohn's disease (CD) is a refractory chronic inflammatory bowel disease (IBD) with unknown etiology. Transmural inflammation, involving the intestine and mesentery, represents a characteristic pathological feature of CD and serves as a critical contributor to its intractability. Here, this study describes an oral pyroptosis nanoinhibitor loaded with tumor necrosis factor-α (TNF-α) deoxyribozymes (DNAzymes) (DNAzymes@degradable silicon nanoparticles@Mannose, Dz@MDSN), which can target macrophages at the site of inflammation and respond to reactive oxygen species (ROS) to release drugs. Dz@MDSN can not only break the inflammatory cycle in macrophages by degrading TNF-α mRNA but also reduce the production of ROS mainly from macrophages. Moreover, Dz@MDSN inhibits excessive pyroptosis in epithelial cells through ROS clearance, thereby repairing the intestinal barrier and reducing the translocation of intestinal bacteria to the mesentery. Consequently, these combined actions synergistically contribute to the suppression of inflammation within both the intestine and the mesentery. This study likely represents the first successful attempt in the field of utilizing nanomaterials to achieve transmural healing for CD, which also provides a promising treatment strategy for CD patients.

Accurate microRNA (miRNA) detection is pivotal in the diagnosis and monitoring of cancer. Entropy-driven catalysis (EDC) has attracted widespread attention as an enzyme-free, isothermal technique for miRNA detection owing to its inherent simplicity and reliability. However, conventional EDC is a single-output mode, limiting the efficiency of signal amplification. In this study, a novel EDC dual-output mode was employed in conjunction with DNAzyme, resulting in the development of an EDC dual-end DNAzyme (EDC-DED) approach for highly sensitive miRNA detection. In this system, miRNA-21 initiated the EDC reaction, producing a large amount of catalytically active dual-end Mg 2+ -dependent DNAzyme. The DNAzyme further cleaved the reporter cyclically, generating a notably amplified fluorescence signal. The proposed method achieved a low detection limit of 2 pM. Compared with the traditional EDC single-end DNAzyme (EDC-SED) strategy, the present method exhibited superior amplification efficiency, enhancing detection sensitivity by approximately 46.5-fold. Furthermore, this platform demonstrated ideal specificity, satisfactory reproducibility and acceptable detection capabilities in clinical serum samples. Therefore, the straightforward and convenient strategy is a potential tool for miRNA analysis, which may provide a new perspective for biological analysis and clinical application., 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.)

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences., 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.)

Cancer is indisputably one of the major threats to mankind, and hence the design of new approaches for the improvement of existing therapeutic strategies is always wanted. Herein, the design of a tumor microenvironment-responsive, DNA-based chemodynamic therapy (CDT) nanoagent with dual Fenton reaction centers for targeted cancer therapy is reported. Self-assembly of DNA amphiphile containing copper complex as the hydrophobic Fenton reaction center results in the formation of CDT-active DNAsome with Cu 2+ -based Fenton catalytic site as the hydrophobic core and hydrophilic ssDNA protrude on the surface. DNA-based surface addressability of the DNAsome is then used for the integration of second Fenton reaction center, which is a peroxidase-mimicking DNAzyme noncovalently loaded with Hemin and Doxorubicin, via DNA hybridization to give a CDT agent having dual Fenton reaction centers. Targeted internalization of the CDT nanoagent and selective generation of • OH inside HeLa cell are also shown. Excellent therapeutic efficiency is observed for the CDT nanoagent both in vitro and in vivo, and the enhanced efficacy is attributed to the combined and synergetic action of CDT and chemotherapy., (© 2024 Wiley‐VCH GmbH.)

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

Pathogens endanger food safety, agricultural productivity, and human health. Those pathogens are spread through direct/indirect contact, airborne transmission and food/waterborne transmission, and some cause severe health consequences. As the population grows and global connections intensify, the transmission of infectious diseases expands. Traditional detection methods for pathogens still have some shortcomings, such as time-consuming procedures and high operational costs. To fulfil the demands for simple and effective detection, numerous biosensors have been developed. DNAzyme, a unique DNA structure with catalytic activity, is gradually being applied in the field of pathogen detection owing to its ease of preparation and use. In this review, we concentrated on the two main types of DNAzyme, hemin/G-quadruplex DNAzyme (HGD) and RNA-cleaving DNAzyme (RCD), explaining their research progress in pathogen detection. Furthermore, we introduced two additional novel DNAzymes, CLICK 17 DNAzyme and Supernova DNAzyme, which showed promising potential in pathogen detection. Finally, we summarize the strengths and weaknesses of these four DNAzymes and offer feasible recommendations for the development of biosensors.

Alpha-foetoprotein (AFP) is taken as a diagnostic tumor marker for the screening and diagnosis of cancer. Nucleic acid-based isothermal amplification strategies are emerging as a potential technology in early screening and clinical diagnosis of AFP. The leakages between hairpins dramatically increase the background and reduce the sensitivity. Thus, it is necessary to develop some strategies to reduce the leakage for isothermal amplification strategies. A DNAzyme-locked leakless enzyme-free amplification system was developed for AFP detection in liver cancer and breast cancer. AFP could open the apt-hairpin and initiate the catalytic hairpin assembly (CHA) reaction to produce a Y-shaped duplex. Two tails of a Y-shaped duplex cleaved the two kinds of leakless hairpins. Then, the third tail of the Y-shaped duplex catalyzed the second CHA between the cleaved leakless hairpins to recover the fluorescent intensity. The limit of detection reached 5 fg/mL by the two levels of signal amplifications. Importantly, the leakless hairpin design effectively reduced leakage between hairpins and weakened the background. In addition, it also showed a great promising potential for AFP detection in early screening and clinical diagnosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

DNAzyme-based assays have found extensive utility in pathogenic bacteria detection but often suffer from limited sensitivity and specificity. The integration of a signal amplification strategy could address this challenge, while the existing combination methods require extensive modification to accommodate various DNAzymes, limiting the wide-spectrum bacteria detection. We introduced a novel hook-like DNAzyme-activated autocatalytic nucleic acid circuit for universal pathogenic bacteria detection. The hook-like connector DNA was employed to seamlessly integrate the recognition element DNAzyme with the isothermal enzyme-free autocatalytic hybridization chain reaction and catalytic hairpin assembly for robust exponential signal amplification. This innovative autocatalytic circuit substantially amplifies the output signals from the DNAzyme recognition module, effectively overcoming DNAzyme's inherent sensitivity constraints in pathogen identification. The biosensor exhibits a strong linear response within a range of 1.5 × 10 3 to 3.7 × 10 7 CFU/mL, achieving a detection limit of 1.3 × 10 3 CFU/mL. Noted that the sensor's adaptability as a universal detection platform is established by simply modifying the hook-like connector module, enabling the detection of various pathogenic bacteria of considerable public health importance reported by the World Health Organization, including Escherichia coli , Staphylococcus aureus , Klebsiella pneumoniae , and Salmonella typhimurium . Additionally, the specificity of DNAzyme in bacterial detection is markedly improved due to the signal amplification process of the autocatalytic circuit. This hook-like DNAzyme-activated autocatalytic platform presents a versatile, sensitive, and specific approach for pathogenic bacteria detection, promising to significantly expand the applications of DNAzyme in bacteria detection.

Heavy metal contamination in food and water is a major public health concern because heavy metals are toxic in minute amounts. DNAzyme sensors are emerging as a promising tool for rapid onsite detection of heavy metals, which can aid in minimizing exposure. However, DNAzyme activity toward its target metal is not absolute and has cross-reactivity with similar metals, which is a major challenge in the wide-scale application of DNAzyme sensors for environmental monitoring. To address this, we constructed a four DNAzyme array (17E, GR-5, EtNA, and NaA43) and used a pattern-based readout to improve sensor accuracy. We measured cross-reactivity between three metal cofactors (Pb 2+ , Ca 2+ , and Na + ) and common interferents (Mg 2+ , Zn 2+ , Mn 2+ , UO 2 2+ , Li + , K + , and Ag + ) and then used t-SNE analysis to identify and quantify the metal ion. We further showed that this method can be used for distinguishing mixtures of metals and detecting Pb 2+ in environmental soil samples at micromolar concentrations.

MicroRNA is regarded as a significant biomarker for cancer diagnosis, disease process evaluation and therapeutic guidance, and dual-parameter measurement may contribute to a more accurate and realistic assessment. To meet the urgent need for simultaneous detection of multiple biomarkers, we combined three-dimensional DNAzyme motors with single molecule imaging technique to construct a convenient, intuitive, and sensitive approach for the simultaneous detection of dual miRNAs in the free state or in extracellular vesicles. Quantification of target miRNAs can be realized through the detection of amplified fluorescence signals generated by the target miRNA-initiated cleavage of fluorescent substrate strands by the DNAzyme motors. The practicability was systematically validated with microRNA-21-5p and microRNA-10b-5p as targets, acquiring a satisfactory sensitivity sufficient to detect low abundance targets at 0.5 or 1 pM to 100 pM. Besides, the extracellular vesicular miRNAs can be conveniently detected without extraction. The clinical applicability was verified with a series of extracellular vesicles from clinical samples, which exhibited good distinguishability between colorectal cancer patients and healthy donors. In addition to the advantages of good specificity and high sensitivity, the system has potential to be easily adapted by minor alteration of the DNA sequences and fluorophore sets for detection of multiple miRNAs and even other types of biomarkers such as proteins. Therefore, it shows promise to be widely applied in various fields such as early diagnosis of cancer and its prognostic assessment.

Apurinic/apyrimidinic endonuclease 1 (APE1), as a vital base excision repair enzyme, is essential for maintaining genomic integrity and stability, and its abnormal expression is closely associated with malignant tumors. Herein, we constructed an electrochemiluminescence (ECL) biosensor for detecting APE1 activity by combining nanoconfined ECL silver nanoclusters (Ag NCs) with X-shaped DNA recognizer-triggered cascade amplification. Specifically, the Ag NCs were prepared and confined in the glutaraldehyde-cross-linked chitosan hydrogel network using the one-pot method, resulting in a strong ECL response and exceptional stability in comparison with discrete Ag NCs. Furthermore, the self-assembled X-shaped DNA recognizers were designed for APE1 detection, which not only improved reaction kinetics due to the ordered arrangement of recognition sites but also achieved high sensitivity by utilizing the recognizer-triggered cascade amplification of strand displacement amplification (SDA) and DNAzyme catalysis. As expected, this biosensor achieved sensitive ECL detection of APE1 in the range of 1.0 × 10 -3 U·μL -1 to 1.0 × 10 -10 U·μL -1 with the detection limit of 2.21 × 10 -11 U·μL -1 , rendering it a desirable approach for biomarker detection.

Both tight and specific binding of folded biological mRNA is required for gene silencing by oligonucleotide gene therapy agents. However, this is fundamentally impossible using the conventional oligonucleotide probes according to the affinity/specificity dilemma. This study addresses this problem for cleaving folded RNA by using multicomponent agents (dubbed 'DNA nanomachine' or DNM). DNMs bind RNA by four short RNA binding arms, which ensure tight and highly selective RNA binding. Along with the improved affinity, DNM maintain the high sequence selectivity of the conventional DNAzymes. DNM enabled up to 3-fold improvement in DNAzymes catalytic efficiency (k cat /K m ) by facilitating both RNA substrate binding and product release steps of the catalytic cycle. This study demonstrates that multicomponent probes organized in sophisticated structures can help to achieve the balance between affinity and selectivity in recognizing folded RNA and thus creates a foundation for applying complex DNA nanostructures derived by DNA nanotechnology in gene therapy., (© 2024 Wiley-VCH GmbH.)

Lipid nanoparticles (LNPs) are emerging as one of the most promising drug delivery systems. The long-circulating effect of intact LNPs (i-LNPs) is the key to efficacy and toxicity in vivo . However, the significant challenge is specific and sensitive detection of i-LNPs. Herein, a dual-recognition fluorescence enzyme-linked immunosorbent assay (DR-FELISA) was developed to directly isolate and detect i-LNPs by combining dual-recognition separation with a one-step signal amplification strategy. The microplates captured and enriched i-LNPs through antibody-antigen reaction. Dual-chol probes were spontaneously introduced into the lipid bilayer of captured i-LNPs, converting the detection of i-LNPs into the detection of double-cholesterol probes. Finally, the end of the dual-chol probes initiated the localized scaffolding autocatalytic DNA circuits (SADC) system for further signal amplification. The SADC system provides a sensitive and efficient amplifier through localized network structures and self-assembled triggers. Simultaneous recognition of i-LNPs surface PEG-lipid and lipid bilayer structures significantly eliminates interference from biological samples. i-LNPs were detected with high selectivity, ranging from 0.2 to 1.25 mg/mL with a limit of detection of 0.1 mg/mL. Moreover, this method allows the isolation and quantitative analysis of different formulations of i-LNPs in serum samples with a satisfactory recovery rate ranging from 94.8 to 116.3%. Thus, the DR-FELISA method provides an advanced platform for the exclusive and sensitive detection of i-LNPs, providing new insights for the study of the quality and intracorporal process of complex formulations.

Long noncoding RNAs (lncRNAs) act as the dynamic regulatory molecules that control the expression of genes and affect numerous biological processes, and their dysregulation is associated with tumor progression. Herein, we develop a fluorescent light-up aptasensor to simultaneously measure multiple lncRNAs in living cells and breast tissue samples based on the DNAzyme-mediated cleavage reaction and transcription-driven synthesis of light-up aptamers. When target lncRNAs are present, they can be recognized by template probes to form the active DNAzyme structures, initiating the T4 PNK-catalyzed dephosphorylation-triggered extension reaction to generate double-strand DNAs with the T7 promoter sequences. The corresponding T7 promoters can initiate the transcription amplification catalyzed by the T7 RNA polymerase to generate abundant Broccoli aptamers and malachite green aptamers, which can bind DFHBI-1T and MG to generate strong fluorescence signals. Taking advantage of the good selectivity of DNAzyme-mediated cleavage of lncRNAs, high amplification efficiency of T7 transcription-driven amplification reaction, and bright fluorescence of the RNA aptamer-fluorophore complex, this method exhibits high sensitivity with a detection limit of 21.4 aM for lncRNA HOTAIR and 18.47 aM for lncRNA MALAT1, and it can accurately measure multiple lncRNAs in both tumor cell lines and breast tissue samples, providing a powerful paradigm for biomedical research and early clinic diagnostics.

Staphylococcus aureus (SA) poses a serious risk to human and animal health, necessitating a low-cost and high-performance analytical platform for point-of-care diagnostics. Cellulose paper-based field-effect transistors (FETs) with RNA-cleaving DNAzymes (RCDs) can fulfill the low-cost requirements, however, its high hydrophilicity and lipophilicity hinder biochemical modification and result in low sensitivity, poor mechanical stability and poor fouling performance. Herein, we proposed a controllable self-cleaning FET to simplify biochemical modification and improve mechanical stability and antifouling performance. Then, we constructed an RCD-based DNA nanotree to significantly enhance the sensitivity for SA detection. For controllable self-cleaning FET, 1 H,1 H,2 H,2 H-perfluorodecyltrimethoxysilane based-polymeric nanoparticles were synthesized to decorate cellulose paper and whole carbon nanofilm wires. O 2 plasma was applied to regulate to reduce fluorocarbon chain density, and then control the hydrophobic-oleophobic property in sensitive areas. Because negatively charged DNA affected the sensitivity of semiconducting FETs, three Y-shaped branches with low-cost were designed and applied to synthesize an RCD-based DNA-Nanotree based on similar DNA-origami technology, which further improved the sensitivity. The trunk of DNA-Nanotree was composed of RCD, and the canopy was self-assembled using multiple Y-shaped branches. The controllable self-cleaning FET biosensor was applied for SA detection without cultivation, which had a wide linear range from 1 to 10 5 CFU/mL and could detect a low value of 1 CFU/mL., (© 2024. The Author(s).)

Background: Osteopontin (OPN) is closely associated with tumorigenesis, growth, invasion, and immune escape and it serves as a plasma biomarker for hepatocellular carcinoma (HCC). Nevertheless, the accurate and rapid detection of low-abundance OPN still poses significant challenges. Currently, the majority of protein detection methods rely heavily on large precision instruments or involve complex procedures. Therefore, developing a simple, enzyme-free, rapid colorimetric analysis method with high sensitivity is imperative., Results: In this study, we have developed a portable colorimetric biosensor by integrating the triple-helix aptamer probe (THAP) and catalytic hairpin assembly (CHA) strategy, named as T-CHA. After binding to the OPN, the trigger probe can be released from THAP, then initiates the CHA reaction and outputs the signal through the formation of a G-quadruplex/Hemin DNAzyme with horseradish peroxidase-like activity. Consequently, this colorimetric sensor achieves visual free-labeled detection without additional fluorophore modification and allows for accurate quantification by measuring the optical density of the solution at 650 nm. Under optimal conditions, the logarithmic values of various OPN concentrations exhibit satisfactory linearity in the range of 5 pg mL -1 to 5 ng mL -1 , with a detection limit of 2.04 pg mL -1 . Compared with the widely used ELISA strategy, the proposed T-CHA strategy is rapid (∼105 min), highly sensitive, and cost-effective., Significance: The T-CHA strategy, leveraging the low background leakage of THAP and the high catalytic efficiency of CHA, has been successfully applied to the detection of OPN in plasma, demonstrating significant promise for the early diagnosis of HCC in point-of-care testing. Given the programmability of DNA and the universality of T-CHA, it can be readily modified for 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.)

Smart drug delivery systems (DDSs) that respond to, interact with, or are actuated by biological signals or pathological abnormalities ( e.g. , the tumor microenvironment) for controllable drug release are appealing therapeutic platforms for cancer treatment. Owing to their inherent self-assembled nature, nucleic acids have emerged as programmable materials for the development of multifunctional structures. In response to external environmental stimuli, DNA response elements can serve as switches to trigger conformational changes in DNA structures. Their stimulus-responsive properties make them promising candidates for constructing smart DDSs, and advancements in DNA response element-based DDSs in the field of biomedicine have been made. This review summarizes different types of DNA response elements, including DNA aptamers, DNAzymes, disulfide bond-modified DNA, pH-responsive DNA motifs, and photocleavable DNA building blocks, and highlights the advancements in DNA response element-based smart DDSs for precise drug release. Finally, future challenges and perspectives in this field are discussed.

Novel hollow AuAg nanoboxes (AuAg NBs) were designed for an innovative electrochemiluminescence (ECL) sensor to ultrasensitively detect Pb 2+ and Hg 2+ with the aid of DNAzyme and "thymine-Hg 2+ -thymine" ("T-Hg 2+ -T") structure. AuAg NBs are employed as an excellent surface plasma resonance (SPR) source, as well as an effective coreaction accelerator for the CoNi NFs/S 2 O 8 2- system to greatly improve ECL performance. To detect Pb 2+ , the DNAzyme catalyzes the cleavage of ribonucleic acid targets into numerous small nucleic acid fragments, leading to an ECL signal. When Hg 2+ is added, the thymine-thymine (T-T) mismatches of the Hg 2+ aptamer bind Hg 2+ to form the "T-Hg 2+ -T" structure, which not only inhibits the SPR process but also produces a large steric hindrance, thus quenching the ECL signal and allowing quantification of Hg 2+ . The novel ECL sensor quantifies Pb 2+ in the range of 0.1 fM to 0.1 μM with a limit of detection of 0.07 fM and Hg 2+ in the range of 10 pM to 1 μM with a LOD of 4.07 pM.

Lumpy skin disease virus (LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV. The method integrated recombinase polymerase amplification (RPA), CRISPR/Cas12a, 10-23 DNAzyme, and Baby Spinach RNA aptamer for triple cascade signal amplification. Based on highly sensitive and specific RPA and CRISPR/Cas12a, DNAzyme achieved a third signal amplification. Additionally, the Baby Spinach RNA aptamer had stronger fluorescence signals and higher quantum yields. The label-free method had ultrahigh sensitivity with the actual detection limit as 1.29 copies·μL -1 . The method was 100-fold more sensitive compared to RPA with Cas12a. Moreover, it had no cross-reactivity with viruses belonging to the Capripoxvirus , such as sheep pox virus and goat pox virus with genetic homology as 97%. Furthermore, the method displayed 100% accuracy in 50 actual samples. Therefore, the method based on RPA, Cas12a, and 10-23 DNAzyme had advantages in LSDV detection and provided a new solution for LSD prevention and control.

Background: MicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis., Results: In this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the "freeze-drying-reduction" method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn 2+ . Without the addition of exogenous Mn 2+ , the dynamically-generated Mn 2+ -powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM., Significance and Novelty: Taking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn 2+ -powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening., 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.)

On-demand engineering of cell membrane receptors to nongenetically intervene in cellular behaviors is still a challenge. Herein, a membraneless enzyme biofuel cell-based self-powered biosensor (EBFC-SPB) was developed for autonomously and precisely releasing Zn 2+ to initiate DNAzyme-based reprogramming of cell membrane receptors, which further mediates signal transduction to regulate cellular behaviors. The critical component of EBFC-SPB is a hydrogel film on a biocathode which is prepared using a Fe 3+ -cross-linked alginate hydrogel film loaded with Zn 2+ ions. In the working mode in the presence of glucose/O 2 , the hydrogel is decomposed due to the reduction of Fe 3+ to Fe 2+ , accompanied by rapid release of Zn 2+ to specifically activate a Zn 2+ -responsive DNAzyme nanodevice on the cell surface, leading to the dimerization of homologous or nonhomologous receptors to promote or inhibit cell proliferation and migration. This EBFC-SPB platform provides a powerful "sensing-actuating-treating" tool for chemically regulating cellular behaviors, which holds great promise in precision biomedicine.

Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg 2+ -dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM - 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis., 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.)

Although DNA probes have attracted increasing interest for precise tumor cell identification by imaging intracellular biomarkers, the requirement of commercial transfection reagents, limited targeting ligands, and/or non-biocompatible inorganic nanostructures has hampered the clinic translation. To circumvent these shortcomings, a reconfigurable ES-NC (Na + -dependent DNAzyme (E)-based substrate (S) cleavage core/shell DNA nanocluster (NC)) entirely from DNA strands is assembled for precise imaging of cancerous cells in a successive dual-stimuli-responsive manner. This nanoprobe is composed of a strung DNA tetrahedral satellites-based protective (DTP) shell, parallelly aligned target-responsive sensing (PTS) interlayer, and hydrophobic cholesterol-packed innermost layer (HCI core). Tetrahedral axial rotation-activated reconfiguration of DTP shell promotes the exposure of interior hydrophobic moieties, enabling cholesterol-mediated cellular internalization without auxiliary elements. Within cells, over-expressed glutathione triggers the disassembly of the DTP protective shell (first stimulus), facilitating target-stimulated signal transduction/amplification process (second stimuli). Target miRNA-21 is detected down to 10.6 fM without interference from coexisting miRNAs. Compared with transfection reagent-mediated counterpart, ES-NC displays a higher imaging ability, resists nuclease degradation, and has no detectable damage to healthy cells. The blind test demonstrates that the ES-NC is suitable for the identification of cancerous cells from healthy cells, indicating a promising tool for early diagnosis and prediction of cancer., (© 2024 Wiley‐VCH GmbH.)

Breast cancer is the most common malignant tumor in women, which accounts for 6.9% of all cancer-related deaths. Early diagnosis is crucial for making the best clinical decision and improving the prognosis of patients. Circulating tumor cells (CTCs) have been regarded as significant tumor biomarkers. Herein, we designed a colorimetric biosensor for breast cancer CTCs quantification based on ladder-branch hybridization chain reaction (HCR) and DNA flowers/gold nanoclusters (DFs/AuNCs) nanozyme. With the assistance of complementary DNA labeled on magnetic beads (MBs), the cleavage products of RNA-cleaving DNAzymes (RCDs) could be rapidly captured, subsequently triggering ladder-branch HCR. In addition, the DFs/AuNCs nanozyme was applied for colorimetric analysis, which further improved the sensitivity for the detection of target CTCs. Benefiting from specific RCDs, ladder-branch HCR and DFs/AuNCs, we achieved a superior detection limit of 3 cells/mL as well as a broad linear range of 10 cells/mL to 10 4  cells/mL. Conclusively, this colorimetric biosensor achieved sensitively and selectively detection of breast cancer CTCs without the participation of enzymes at room temperature, which might provide new insight into the early detection of breast cancer., 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.)

Detecting heavy metal pollution, particularly lead ion (Pb 2 ⁺) contamination, is imperative for safeguarding public health. In this study, we introduced an innovative approach by integrating DNAzyme with rolling circle amplification (RCA) to propose an amplification sensing method termed DNAzyme-based dimeric-G-quadruplex (dimer-G4) RCA. This sensing approach allows for precise and high-fidelity Pb 2 ⁺ detection. Strategically, in the presence of Pb 2 ⁺, the DNAzyme undergoes substrate strand (S-DNA) cleavage, liberating its enzyme strand (E-DNA) to prime isothermal amplification. This initiates the RCA process, producing numerous dimer-G-Quadruplexes (dimer-G4) as the signal reporting transducers. Compared to conventional strategies using monomeric G-quadruplex (mono-G4) as the reporting transducers, these dimer-G4 structures exhibit significantly enhanced fluorescence when bound with Thioflavin T (ThT), offering superior target signaling ability for even detection of Pb 2 ⁺ at low concentration. Conversely, in the absence of Pb 2 ⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb 2 ⁺ detection method combing with the high specificity of DNAzymes for Pb 2 ⁺ recognition ensures highly sensitive detection of Pb 2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental 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.)

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus ( S. aureus ) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

MicroRNAs (miRNAs) have been involved in many biological processes and are regarded as promising biomarkers. The short sequence, low abundance and highly homologous interference sequences greatly hinder the accurate detection of miRNAs. Here, a cascade branch migration-triggered strand displacement amplification (CBM-TSDA) strategy was developed for the first time for specific and sensitive detection of miRNA-155 (miR-155). In the presence of target miR-155, the CBM was initiated and two Y-shaped probes were eventually produced. Next, the Y-shaped probes were transformed into three-way junction (3WJ) structures and triggered the SDA to produce a large number of G-quadruplex (G4) structures. Finally, the increased fluorescence signal of G4/Thioflavin T (ThT) was used to quantify miR-155. Meanwhile, the colorimetric responses of the G4-hemin DNAzyme could be used as supplementary detection to obtain a dual-mode signal readout. This detection strategy showed high detection sensitivity, and the limit of detection was 0.28 pM in the fluorescence detection mode and 0.34 pM in the colorimetric detection mode. Notably, it showed high detection specificity, being able to discriminate the single-base mutations of the target with a high discrimination factor. The strategy also possessed excellent capacity for miR-155 detection in cell lysates and real human blood samples. The developed strategy provides a promising detection platform for miRNA, which may be applied to early clinical diagnosis.

We reported a colorimetric paper-based device by integrating the modified acid RNA-cleaving DNAzymes (MaRCD-EC1) for highly sensitive (detection limit = 10 2 CFU mL -1 ), and rapid (within 30 min) detection of E. coli without amplification. This device exhibited a clinical sensitivity of 100% and a specificity of 100% in identifying E. coli -associated urinary tract infections (UTIs) using the clinical urine samples.

Endowing current artificial chemical reactions (ACRs) with high specificity and intricate activation capabilities is crucial for expanding their applications in accurate bioimaging within living cells. However, most of the reported ACR-based evaluations relied on either single biomarker stimuli or dual activators without obvious biological relevance, still limiting their accuracy and fidelity. Herein, taking the metal-ion-dependent DNAzyme cleavage reaction as a model ACR, two regulators, glutathione (GSH) and telomerase (TE) activated DNAzyme cleavage reactions, were exploited for precise discrimination of cancerous cells from normal cells. DNA probe was self-assembled into the ZIF-90 nanoparticle framework to construct coordination-driven nanoprobes. This approach enhances the stability and specificity of tumor imaging by utilizing biomarkers associated with rapid tumor proliferation and those commonly overexpressed in tumors. In conclusion, the research not only paves the way for new perspectives in cell biology and pathology studies but also lays a solid foundation for the advancement of biomedical imaging and disease diagnostic technologies.

Herein, a dual self-protected DNAzyme-based 3D DNA walker (dSPD walker), composed of activated dual self-protected walking particles (ac-dSPWPs) and track particles (TPs), was constructed for ultrasensitive and ultrahigh-speed fluorescence detection and imaging of microRNAs (miRNAs) in living cells. Impressively, compared with the defect that "one" target miRNA only initiates "one" walking arm of the conventional single self-protected DNAzyme walker, the dSPD walker benefits from the secondary amplification and spatial confinement effect and could guide "one" target miRNA to generate " n " secondary targets, thereby initiating " n " nearby walking strands immediately, realizing the initial rate over one-magnitude-order faster than that of the conventional one. Moreover, in the process of relative motion between ac-dSPWPs and TPs, the ac-dSPWPs could cleave multiple substrate strands simultaneously to speed up movement and reduce the derailment rate, as well as combine with successive TPs to facilitate a large amount of continuous signal accumulation, achieving an ultrafast detection of miRNA-221 within 10 min in vitro and high sensitivity with a low detection limit of 0.84 pM. In addition, the DNA nanospheres obtained by the rolling circle amplification reaction can capture the Cy5 fluorescence dispersed in liquids, which achieves the high-contrast imaging of miRNA-221, resulting in further ultrasensitive imaging of miRNA-221 in cancer cells. The proposed strategy has made a bold innovation in the rapid and sensitive detection as well as intracellular imaging of low-abundance biomarkers, offering promising application in early diagnosis and relevant research of cancer and tumors.

The development of the Point-of-Care Testing (POCT) platform that combines convenience and cost-effectiveness is crucial for enabling the visual detection of disease biomarkers. In this work, a POCT platform for the sensitive in situ detection of prostate specific antigen (PSA) with dual-signal output was constructed by functionalizing the Eppendorf (EP) tube. This was achieved through the modification of aptamer hairpin probes (AHPs) on the lid of the EP tube and the assembly of a nanoenzyme hydrogel film on its inner wall. The target could trigger the release of Ag + by AHP and subsequently activate Ag + -dependent DNAzyme (Ag-DNAzyme). This would initiate the cleavage of the DNA-Au/Pt NP hydrogel network, leading to the release of Au/Pt NPs. The released Au/Pt NPs exhibit both peroxidase (POD)-like and catalase (CAT)-like activity to produce a colorimetric response and induce liquid flow under pressure. Therefore, the target can be measured visually and quantitatively through colorimetric analysis and the measurement of total dissolved solids (TDS) using a pressure-triggered liquid flow device integrated into the platform. The designed platform is distinguished by its simplicity, specificity, cost-effectiveness, and remarkable sensitivity. It allows for the visual detection of PSA within concentration ranges of 0.5-100 ng/L (colorimetric) and 3-100 ng/L (TDS reading), boasting detection limits as low as 0.15 ng/L (colorimetric) and 0.57 ng/L (TDS reading). The strategy of target-triggered nanoenzyme release significantly enhances sensitivity and provides a guiding approach for visual biomarker detection.

Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via D NA- t emplated c lick l igation c hain r eaction (DT-CLCR) catalyzed by a h eterogeneous n anocatalyst made of Cu 2 O (hnCu 2 O). This hnCu 2 O-DT-CLCR method is built on two cross-amplifying hnCu 2 O-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCu 2 O-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCu 2 O-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCu 2 O-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCu 2 O-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.

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. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sensitive identification and effective inactivation of the virus are paramount for the early diagnosis and treatment of viral infections to prevent the risk of secondary transmission of viruses in the environment. Herein, we developed a novel two-step fluorescence immunoassay using antibody/streptavidin dual-labeled polystyrene nanobeads and biotin-labeled G-quadruplex/hemin DNAzymes with peroxidase-mimicking activity for sensitive quantitation and efficient inactivation of living Zika virus (ZIKV). The dual-labeled nanobeads can specifically bind ZIKV through E protein targeting and simultaneously accumulate DNAzymes, leading to the catalytic oxidation of Amplex Red indicators and generation of intensified aggregation-induced emission fluorescence signals, with a detection limit down to 66.3 PFU/mL and 100% accuracy. Furthermore, robust reactive oxygen species generated in situ by oxidized Amplex Red upon irradiation can completely kill the virus. This sensitive and efficient detection-inactivation integrated system will expand the viral diagnostic tools and reduce the risk of virus transmission in the environment.

Hazardous lead ions (Pb 2+ ) even at a minute level can pose side effects on human health, highlighting the need for tools for trace Pb 2+ detection. Herein, we present a DNAzyme-activated CRISPR assay (termed DzCas12T) for sensitive and one-pot detection of lead contamination. Using an extension-bridged strategy eliminates the need for separation to couple the DNAzyme recognition and CRISPR reporting processes. The tandem design endowed the DzCas12T assay with high specificity and sensitivity down to the pM-level. This assay has been used to detect lead contamination in food and water samples, indicating the potential for monitoring lead-associated environmental and food safety.

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

A double 3D DNA walker nanomachine by DNAzyme self-driven positive feedback loop amplification for the detection of miRNA was constructed. This method uses two gold nanoparticles as the reaction core, and because of the spatial confinement effect the local concentration of the reactants increase the collision efficiency was greatly improved. Meanwhile, the introduction of positive feedback loop promotes the conversion efficiency. In presence of miRNA-21, a large amount of DNAzyme was released and hydrolyze the reporter probe, resulting the recovery of fluorescence signal. The linear range for miRNA-21 is 0.5-60 pmol/L, and the detection limit is 0.41 pmol/L (S/N = 3). This nanomachine has been successfully used for accurate detection of miRNA-21 expression levels in cell lysates. At the same time, it can enter cells for intracellular miRNA-21 fluorescence imaging, distinguishing tumor cells from normal cells. This combination of in vitro detection and imaging analysis of living cells can achieve the goal of jointly detecting cancer markers through multiple pathways, providing new ideas for early diagnosis and screening 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 B.V. All rights reserved.)

Assembled gold nanoparticle (AuNP) superstructures can generate unique physicochemical characteristics and be used in various applications, thus becoming an attractive research field. Recently, several DNA-assisted gold nanoparticle assembly methods have been rigorously developed that typically require a non-catalytic equimolar molecular assembly to guarantee the designed assembly. Although efficient and accurate, exploring such non-catalytic nanoparticle assemblies in the complex cellular milieu under low trigger concentrations remains challenging. Therefore, developing a catalytic method that facilitates gold nanoparticle assemblies with relatively low DNA trigger concentrations is desirable. In this report, a catalytic method to program gold nanoparticle assemblies by DNAzyme circuits is presented, where only a small number of DNA triggers are able to induce the production of a large number of the desired nanoparticle assemblies. The feasibility of using logic DNAzyme circuits to control catalytic nanoparticle assemblies is experimentally verified. Additionally, catalytic AuNP assembly systems are established with cascading and feedback functions. The work provides an alternative research direction to enrich the tool library of nanoparticle assembly and their application in biosensing and nanomedicine., (© 2024 Wiley‐VCH GmbH.)

Staphylococcus aureus (S. aureus), as a Gram-positive bacterium, is commonly encountered in various infectious diseases, such as acute skin and soft tissue infections. Despite that many efforts have been made, sensitive and reliable quantitative determination of S. aureus remains a huge challenge. Here, we depict a novel colorimetric approach for sensitive and accurate detection by combining allosteric probe-based target recognition and chain extension-based dual signal recycling. The single-strand DNA (ssDNA) products generated by the chain extension process lead to the liberation of G-quadruplex sequences, which can fold into active DNAzyme under the assistance of hemin. The active DNAzyme can work as peroxidase mimics to catalyze the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS 2- )-H 2 O 2 system, causing the color change of the system. Eventually, the method exhibits a wide detection range from 10 3  cfu/mL to 10 6  cfu/mL. The limit of detection of the approach was determined 232 cfu/mL. Considering the robust capability of the approach in S. aureus detection, we believe that it will be a potential alternative tool for biomedical research and clinical molecular diagnostics., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aptasensors for detection of ochratoxin A (OTA) have been extensively studied, but the majority of them require costly and large-scale equipment as signal readers. Herein, a photothermal aptasensor capable of portable detection of OTA through a thermometer was developed on basis of aptamer structural switching and rolling circle amplification (RCA)-enriched DNAzyme. Oligonucleotides and alkaline phosphatase (ALP) modified magnetic beads were prepared. The binding of aptamers to OTA led to the release of ALP labeled complementary DNA. After magnetic separation, ALP catalyzed the padlock dephosphorylation, inhibiting the subsequent RCA reaction. This process converted the OTA concentration into the amount of the photothermal reagent oxTMB produced from the catalytic reaction induced by RCA-enriched DNAzyme. Under the optimal conditions, the detection limit (LOD) of this aptasensor was 2.28 nM in a clean buffer, while the LOD reached 2.43 nM in 2 % grape juice. The good performance of the photothermal aptasensor makes it possible to measure OTA pollution in low resource environments., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A dual-localized DNAzyme walker (dlDW) was constructed by utilizing multiple split DNAzymes with probes, and their substrates are separately localized on streptavidin and AuNPs, serving as walking pedals and tracks, respectively. Based on dlDW, biosensing platform was successfully constructed and showed great potential application in clinical disease diagnosis.

Manipulating the expression of cellular genes through efficient CRISPR/Cas9 delivery is rapidly evolving into a desirable tumor therapeutics. The exposure of CRISPR/Cas9 to a complex external environment poses challenges for conventional delivery carriers in achieving responsive and accurate release. Here, we report a Trojan horse-like nanocapsule for the on-demand delivery of CRISPR/Cas9 in a microRNA-responsive manner, enabling precise tumor therapy. The nanocapsule comprises a nanoassembled, engineered DNAzyme shell encasing a Cas9/sgRNA complex core. The DNAzyme, functioning as a catalytic unit, undergoes a conformational change in the presence of tumor-associated microRNA, followed by activating a positive feedback-driven autonomous catabolic cycle of the nanocapsule shell. This catabolic cycle is accomplished through chain reactions of DNAzyme "cleavage-hybridization-cleavage", which ensures sensitivity in microRNA recognition and effective release of Cas9/sgRNA. Utilizing this Trojan horse-like nanocapsule, as low as 1.7 pM microRNA-21 can trigger the on-demand release of Cas9/sgRNA, enabling the specific editing of the protumorigenic microRNA coding gene. The resulting upregulation of tumor suppressor genes induces apoptosis in tumor cells, leading to significant inhibition of tumor growth by up to 75.94%. The Trojan horse-like nanocapsule, with superior programmability and biocompatibility, is anticipated to serve as a promising carrier for tailoring responsive gene editing systems, achieving enhanced antitumor specificity and efficacy.

Programming ultrasensitive and stimuli-responsive DNAzyme-based probes holds great potential for on-demand biomarker detection. Here, an optically triggered DNAzyme platform was reported for on-demand activation-sensitive electrochemiluminescence (ECL) c-myc mRNA analysis. In this design, the sensing and recognition function of the split DNAzyme (SDz) probe was silent by engineering a blocking sequence containing a photocleavable linker (PC-linker) group at a defined site that could be indirectly cleaved by 302 nm ultraviolet (UV) light. When the SDz probes were assembled on the Au nanoparticles and potassium (K) element doped graphitic carbon nitride nanosheet (K-doped g-C 3 N 4 ) covered electrode, UV light activation induces the configurational switching and consequently the formation of an active DNAzyme probe with the help of target c-myc mRNA, allowing the cleavage of the substrate strand by magnesium ions (Mg 2+ ). Thus, the release of a ferrocene (Fc)-labeled DNAzyme 2 strand contributed to an extreme ECL signal recovery. In the meantime, the released target c-myc mRNA combined another inactive SDz motif to form active DNAzyme and repeat the cyclic cleavage reaction, resulting in the signal amplification. Furthermore, according to the responses toward two other designed nPC-SDz and m-SDz probes, we demonstrated that controlled UV light mediated photoactivation of the DNAzyme biosensor "on demand" effectively constrained the ECL signal to the mRNA of interest. Moreover, false positive signals could also be avoided due to such a photoactivation design with UV light. Therefore, this study provided a simple methodology that may be broadly applicable for investigating the mRNA-associated physiological events that were difficult to access using traditional DNAzyme probes.

Background: The human immunodeficiency virus (HIV) remains a major worldwide health problem. Nowadays, many methods have been developed for quantitative detecting human immunodeficiency virus DNA (HIV-DNA), such as fluorescence and colorimetry. However, these methods still have the disadvantages of being expensive and requiring professional technicians. Early diagnosis of pathogens is increasingly dependent on portable instruments and simple point-of-care testing (POCT). Therefore, it is meaningful and necessary to develop portable and cheap methods for detecting disease markers., Results: In this work, a label-free chemiluminescence (CL) method was developed for detecting HIV-DNA via a handheld luminometer. To achieve label-free target detection, the CL catalyst, G-triplex-hemin DNAzyme (G3-hemin DNAzyme), was in-situ assembled in the presence of HIV-DNA. For improving sensitivity, HIV-DNA induced the cyclic strand displacement reaction (SDR), which can form three G3-hemin DNAzymes in one cycle. So, the chemiluminescence reaction between luminol and H 2 O 2 was highly effectively catalyzed, and the CL intensity was linearly related with the concentration of HIV-DNA in the range of 0.05-10 nM with a detection limit of 29.0 pM. Due to the high specificity of hairpin DNA, single-base mismatch can be discriminated, which ensured the specific detection of HIV-DNA., Significance: In-situ formation of G3-hemin DNAzyme led to label-free and selective detection without complex synthesis and functionalization. Therefore, it offers a cheap, selective, sensitive and portable method for detecting disease-related genes, which is promising for POCT of clinical diagnosis in resource-limited settings., 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.)

8-17 DNAzymes (8-17, 17E, Mg5, and 17EV1) are in vitro-selected catalytic DNA molecules that are capable of cleaving complementary RNAs. The conserved residues in their similar catalytic cores, together with the metal ions, were suggested to contribute to the catalytic reaction. Based on the contribution of the less conserved residues in the bulge loop residues (W 12 , A 15 , A 15.0 ) and the internal stem, new catalytic cores of 8-17 DNAzymes were programmed. The internal stem CTC-GAG seems to be more favorable for the DNAzymes than CCG-GGC, while an extra W 12.0 led to a significant loss of activity of DNAzymes, which is contrary to the positive effect of A 15.0 , by which a new active DNAzyme 17EM was derived. It conducts a faster reaction than 17E. It is most active in the presence of Pb 2+ , with the metal ion preference of Pb 2+ >> Zn 2+ > Mn 2+ > Ca 2+ ≈ Mg 2+ . In the Pb 2+ and Zn 2+ -mediated reactions of 17EM and 17E, the same Na + - and pH dependence were also observed as what was observed for 17E and other 8-17 DNAzymes. Therefore, 17EM is another member of the 8-17 DNAzymes, and it could be applied as a potential biosensor for RNA and metal ions.

DNAzymes - synthetic enzymes made of DNA - have long attracted attention as RNA-targeting therapeutic agents. Yet, as of now, no DNAzyme-based drug has been approved, partially due to our lacking understanding of their molecular mode of action. In this work we report the solution structure of 8-17 DNAzyme bound to a Zn 2+ ion solved through NMR spectroscopy. Surprisingly, it turned out to be very similar to the previously solved Pb 2+ -bound form (catalytic domain RMSD = 1.28 Å), despite a long-standing literature consensus that Pb 2+ recruits a different DNAzyme fold than other metal ion cofactors. Our follow-up NMR investigations in the presence of other ions - Mg 2+ , Na + , and Pb 2+ - suggest that at DNAzyme concentrations used in NMR all these ions induce a similar tertiary fold. Based on these findings, we propose a model for 8-17 DNAzyme interactions with metal ions postulating the existence of only a single catalytically-active structure, yet populated to a different extent depending on the metal ion cofactor. Our results provide structural information on the 8-17 DNAzyme in presence of non-Pb 2+ cofactors, including the biologically relevant Mg 2+ ion., (© 2024. The Author(s).)

The expression level of human apurinic/apyrimidinic endonuclease 1 (APE1) is closely associated with the onset of various diseases, establishing it as a crucial clinical biomarker and a target in anti-cancer efforts. This study accomplished colorimetric and visual detection of APE1 by harnessing its endonuclease activity through catalytic hairpin self-assembly (CHA) and G-quadruplex/hemin DNAzyme. Optimization of the freedom degrees of the G-rich sequence significantly improved the detection performance of the strategy by influencing DNAzyme formation. Additionally, we replaced the signal reporting system with a molecular beacon to develop a fluorescence detection strategy, which served as an extension of the signal amplification system for validation and signal readout. The fluorescent probe method achieved a detection limit of 3.37 × 10 -4 U/mL, while the colorimetric method yielded a detection limit of 6.5 × 10 -3 U/mL, with a linear range spanning from 0.01 to 0.25 U/mL. Subsequently, the colorimetric approach effectively assessed APE1 activity in biological samples and facilitated the screening of APE1 activity inhibitors. Furthermore, this CHA/G-quadruplex/hemin DNAzyme strategy was adapted for the colorimetric detection of adenosine, showcasing its broad applicability across various biomarkers. The developed colorimetric analytical strategy represents a pivotal biosensing platform for diagnosing and treating 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.)