A DNAzyme-enhanced nonlinear hybridization chain reaction for sensitive detection of microRNA.

As a typical biomarker, the expression of microRNA is closely related to the occurrence of cancer. However, in recent years, the detection methods have had some limitations in the research and application of microRNAs. In this paper, an autocatalytic platform was constructed through the combination of a nonlinear hybridization chain reaction and DNAzyme to achieve efficient detection of microRNA-21. Fluorescently labeled fuel probes can form branched nanostructures and new DNAzyme under the action of the target, and the newly formed DNAzyme can trigger a new round of reactions, resulting in enhanced fluorescence signals. This platform is a simple, efficient, fast, low-cost, and selective method for the detection of microRNA-21, which can detect microRNA-21 at concentrations as low as 0.004 nM and can distinguish sequence differences by single-base differences. In tissue samples from patients with liver cancer, the platform shows the same detection accuracy as real-time PCR but with better reproducibility. In addition, through the flexible design of the trigger chain, our method could be adapted to detect other nucleic acid biomarkers.

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

The sensing performance of DNAzymes in live cells is tremendously hampered by the inefficient and inhomogeneous delivery of DNAzyme probes and their incontrollable off-site activation, originating from their susceptibility to nuclease digestion. This requires the development of a more compact and robust DNAzyme-delivering system with site-specific DNAzyme activation property. Herein, a highly compact and robust Zn@DDz nanoplatform is constructed by integrating the unimolecular microRNA-responsive DNA-cleaving DNAzyme (DDz) probe with the requisite DNAzyme Zn2+ -ion cofactors, and the amplified intracellular imaging of microRNA via the spatiotemporally programmed disassembly of Zn@DDz nanoparticles is achieved. The multifunctional Zn@DDz nanoplatform is simply composed of a structurally blocked self-hydrolysis DDz probe and the inorganic Zn2+ -ion bridge, with high loading capacity, and can effectively deliver the initially catalytic inert DDz probe and Zn2+ into living cells with enhanced stabilities. Upon their entry into the acidic microenvironment of living cells, the self-sufficient Zn@DDz nanoparticle is disassembled to release DDz probe and simultaneously supply Zn2+ -ion cofactors. Then, endogenous microRNA-21 catalyzes the reconfiguration and activation of DDz for generating the amplified readout signal with multiply guaranteed imaging performance. Thus, this work paves an effective way for promoting DNAzyme-based biosensing systems in living cells, and shows great promise in clinical diagnosis.

Catalytic Gold Nanoparticle Assembly Programmed by DNAzyme Circuits.

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.

Time-Controlled Authentication Strategies for Molecular Information Transfer.

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.

G-Quadruplex-Programmed Versatile Nanorobot Combined with Chemotherapy and Gene Therapy for Synergistic Targeted Therapy.

Developing synergistic targeted therapeutics to improve treatment efficacy while reducing side effects has proven promising for anticancer therapies, but how to conveniently modulate multidrug cooperation remains a challenge. Here, a novel synergistic strategy using a G-quadruplex-programmed versatile nanorobot (G4VN) containing two subunits of DNAzyme (DzG4) and ligand-drug conjugates (LDCs) is proposed to precisely target tumors and then execute both gene silencing and chemotherapy. As the core module of this nanorobot, a well-designed G4 responding to a high level of K+ in tumor microenvironment smartly kills three birds with one stone, which makes two TfR aptamers proximate to improve their efficiency of targeting tumor cells, and in situ activates a split 10-23 DNAzyme to downregulate target mRNA expression, meanwhile promotes the cell uptake of a GSH-responsive LDCs to enhance drug efficacy. Such a design enables a potently synergistic anticancer therapy with low side effects in vivo, showing great promise for broad applications in precision disease treatment.

Proximity-Dependent Activation of Split DNAzyme Kinase.

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.

In-vitro Clinical Diagnostics using RNA-Cleaving DNAzymes.

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.

Enhancing Sensitivity in Nucleic Acid Detection via Collaborative Multiple Catalytic Cores in DNAzyme Nanomachines.

We introduce a novel multicore DNA nanomachine (MDNM), utilizing four binary DNAzymes for nucleic acid detection without the need for a preamplification step. This innovation remarkably yields a reduction in limit of detection (LOD), over 5-fold, as compared to single-core systems. This reduces the required test time, highlighting the potential of MDNM in advancing nucleic acid detection.

Cleaving Folded RNA by Multifunctional DNAzyme Nanomachines.

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 (kcat/Km) 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.

Exonuclease-iii -propelled DNAzyme cascade for sensitive and reliable cervical cancer related miRNA analysis.

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.

Microfluidic bead-based biosensor: Ultrasensitive ctDNA detection based on duplex-functional split-DNAzyme and dendritic enzyme-free signal amplification.

Circulating tumor DNA (ctDNA) is a crucial cancer biomarker for early or noninvasive monitoring, which is essential for developing ultrasensitive and selective assays in cancer diagnosis and treatment. Herein, a cascade signal amplification of duplex-functional split-DNAzyme and dendritic probes was proposed for ultrasensitive and specific detection of nasopharyngeal carcinoma-associated Epstein-Barr virus (EBV) DNA on microfluidic microbead array chips. With the assistance of Pb2+, the duplex-functional split-DNAzyme recognizes EBV DNA and then rapidly cleaves the substrate strand. Subsequently, the released target could be recycled, and its exposed capture probe, triggered the dendritic enzyme-free signal amplification. As the enhanced mass transfer capability, target recycling, and dendritic DNA structure signal amplification inherent to microfluidic bead arrays were integrated, it achieved an excellent detection limit of 0.36 fM and a wide linear range of 1 fM∼103 fM. Further, it was applied to content detect simulated samples of EBV DNA, recovery ranged from 97.2 % to 108.1 %, and relative standard deviation (RSD) from 3.3 % to 5.9 %, exhibiting satisfactory recovery results. The developed microfluidic biosensor was a high-sensitivity and anti-interference system for ctDNA analysis, with minimal reagent volumes (microlitres) required. Thus, it is a promising platform for ctDNA at the lowest level at their earliest incidence.

Proximity ligation-triggered DNAzyme for selective fluorescent aptasensing of methicillin-resistant Staphylococcus aureus.

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 Mg2+-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 106 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.

Dual Detection of microRNAs by a Signal-Off Colorimetric Nanobiosensor Based on Novel Split DNAzyme Nanostructure.

Breast cancer is the most common cancer and the second cause of cancer-related death in women, especially in the age of 20-59 years. It is very important to diagnose it in the early stages of development due to high chance of survival. In this research, the early detection of two microRNAs involved in breast cancer including miR-21 and miR-155 was performed simultaneously using a nanobiosensor based on a special G-quadruplex structure and a colorimetric manner. This nanobiosensor contains two probes (p1, p2) that play a role in the formation of a special structure called DNA-G4. This structure has peroxidase-like properties and can oxidize TMB and produce a blue color. The diagnostic method is designed as a signal off, where the hybridization of probes with microRNA sequences, no DNA-G4 structure is formed and no color change is observed. The results of this study showed that the linear range of response is in the range of 2 to 10 nm and limit of detection in buffer, blood and urine samples was calculated as 0.43 nM, 0.54 nM, and 0.62 nM (R2 = 0.98), respectively. Evaluation using the method for cancer monitoring can be a simple, fast and cost-effective technique.

Fluorescence assay for aflatoxin B1 based on aptamer-binding triggered DNAzyme activity.

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.

Multicomponent DNAzyme Nanomachines: Structure, Applications, and Prospects.

Nucleic acids (NAs) are important components of living organisms responsible for the storage and transmission of hereditary information. They form complex structures that can self-assemble and bind to various biological molecules. DNAzymes are NAs capable of performing simple chemical reactions, which makes them potentially useful elements for creating DNA nanomachines with required functions. This review focuses on multicomponent DNA-based nanomachines, in particular on DNAzymes as their main functional elements, as well as on the structure of DNAzyme nanomachines and their application in the diagnostics and treatment of diseases. The article also discusses the advantages and disadvantages of DNAzyme-based nanomachines and prospects for their future applications. The review provides information about new technologies and the possibilities of using NAs in medicine.

A dual amplified gold nanoparticle-based biosensor for ultrasensitive and selective detection of fibrin.

Ultrasensitive, selective, and non-invasive detection of fibrin in human serum is critical for disease diagnosis. So far, the development of high-performance and ultrasensitive biosensors maintains core challenges for biosensing. Herein, we designed a novel ribbon nanoprobe for ultrasensitive detection of fibrin. The probe contains gold nanoparticles (AuNPs) that can not only link with homing peptide Cys-Arg-Glu-Lys-Ala (CREKA) to recognize fibrin but also carry long DNA belts to form G-quadruplex-based DNAzyme, catalyzing the chemiluminescence of luminol-hydrogen peroxide (H2O2) reaction. Combined with the second amplification procedure of rolling circle amplification (RCA), the assay exhibits excellent sensitivity with a detection limit of 0.04 fmol L-1 fibrin based on the 3-sigma. Furthermore, the biosensor shows high specificity on fibrin in samples because the structure of antibody-fibrin-homing peptide was employed to double recognize fibrin. Altogether, the simple and inexpensive approach may present a great potential for reliable detection of biomarkers.

Exonuclease III-propelled DNAzyme walker: an electrochemical strategy for microRNA diagnostics.

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100 pM and achieves a detection limit of 5 fM (3σ criterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics.

Transient Dynamic Operation of G-Quadruplex-Gated Glucose Oxidase-Loaded ZIF-90 Metal-Organic Framework Nanoparticle Bioreactors.

Glucose oxidase-loaded ZIF-90 metal-organic framework nanoparticles conjugated to hemin-G-quadruplexes act as functional bioreactor hybrids operating transient dissipative biocatalytic cascaded transformations consisting of the glucose-driven H2O2-mediated oxidation of Amplex-Red to resorufin or the glucose-driven generation of chemiluminescence by the H2O2-mediated oxidation of luminol. One system involves the fueled activation of a reaction module leading to the temporal formation and depletion of the bioreactor conjugate operating the nickase-guided transient biocatalytic cascades. The second system demonstrates the fueled activation of a reaction module yielding a bioreactor conjugate operating the exonuclease III-dictated transient operation of the two biocatalytic cascades. The temporal operations of the bioreactor circuits are accompanied by kinetic models and computational simulations enabling us to predict the dynamic behavior of the systems subjected to different auxiliary conditions.

Bioorthogonal Disassembly of Hierarchical DNAzyme Nanogel for High-Performance Intracellular microRNA Imaging.

Rolling circle amplification (RCA) enables the facile construction of compact and versatile DNA nanoassemblies which are yet rarely explored for intracellular analysis. This is might be ascribed to the uncontrollable and inefficient probe integration/activation. Herein, by encoding with tandem allosteric deoxyribozyme (DNA-cleaving DNAzyme), a multifunctional RCA nanogel was established for realizing the efficient intracellular microRNA imaging via the successive activation of the RCA-disassembly module and signal amplification module. The endogenous microRNA stimulates the precise degradation of DNA nanocarriers, thus leading to the efficient exposure of RCA-entrapped DNAzyme biocatalyst for an amplified readout signal. Our bioorthogonal DNAzyme disassembly strategy achieved the robust analysis of intracellular biomolecules, thus showing more prospects in clinical diagnosis.

The Effect of 8,5'-Cyclo 2'-deoxyadenosine on the Activity of 10-23 DNAzyme: Experimental and Theoretical Study.

The in vivo effectiveness of DNAzymes 10-23 (Dz10-23) is limited due to the low concentration of divalent cations. Modifications of the catalytic loop are being sought to increase the activity of Dz10-23 in physiological conditions. We investigated the effect of 5'S or 5'R 5',8-cyclo-2'deoxyadenosine (cdA) on the activity of Dz10-23. The activity of Dz10-23 was measured in a cleavage assay using radiolabeled RNA. The Density Functional Tight Binding methodology with the self-consistent redistribution of Mulliken charge modification was used to explain different activities of DNAzymes. The substitution of 2'-deoxyadenosine with cdA in the catalytic loop decreased the activity of DNAzymes. Inhibition was dependent on the position of cdA and its absolute configuration. The order of activity of DNAzymes was as follows: wt-Dz > ScdA5-Dz ≈ RcdA15-Dz ≈ ScdA15-Dz > RcdA5-Dz. Theoretical studies revealed that the distance between phosphate groups at position 5 in RcdA5-Dz was significantly increased compared to wt-Dz, while the distance between O4 of dT4 and nonbonding oxygen of PO2 attached to 3'O of dG2 was much shorter. The strong inhibitory effect of RcdA5 may result from hampering the flexibility of the catalytic loop (increased rigidity), which is required for the proper positioning of Me2+ and optimal activity.