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.

Engineering of a DNAzyme-Based dimeric G-quadruplex rolling circle amplification for robust analysis of lead ion.

Detecting heavy metal pollution, particularly lead ion (Pb2⁺) 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 Pb2⁺ detection. Strategically, in the presence of Pb2⁺, 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 Pb2⁺ at low concentration. Conversely, in the absence of Pb2⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb2⁺ detection method combing with the high specificity of DNAzymes for Pb2⁺ recognition ensures highly sensitive detection of Pb2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental monitoring.

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.

Catalytic hairpin assembly triggering amplified DNAzyme-feedback for sensitive detection of hepatitis C virus genotype 1b.

Developing a simple, reliable, and sensitive hepatitis C virus (HCV) genetic sensing platform is of great significance for diagnosing diseases and selecting appropriate antiviral treatments. Herein, a tandem nucleic acid amplification strategy for sensitive detection of HCV genotype 1b (HCV-1b) was developed by stringing the catalytic hairpin assembly (CHA) and the triggered DNAzyme amplifier. The hairpin reactants were initiated by the target to produce lots of triggering double-stranded DNA sequences which can efficiently activate the subsequent blocked DNAzyme. Thereby, the continuous cleavage of substrate was realized, resulting in the fluorescence signal amplification. The DNA-based isothermal CHA-DNAzyme (CDz) sensing platform was successfully applied for sensitive detection of HCV-1b with the limit of detection (84 pM) and showed good selectivity. Moreover, the practical detection of target DNA in the complex biologic matrix indicated that the developing strategy had good potential for early HCV infection diagnosis.

High-affinity truncated aptamers for detection of Cronobacter spp with magnetic separation-assisted DNAzyme-driven 3D DNA walker.

After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL-1, with a low detection limit of 2 CFU mL-1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine  C. sakazakii in real samples with recoveries of 92.86%-108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation.

Improving the Sensitivity of a Mn(II)-Specific DNAzyme for Cellular Imaging Sensor through Sequence Mutations.

Detection of Mn2+ in living cells is important in understanding the roles of Mn2+ in cellular processes and investigating its potential implications in various diseases and disorders. Toward this goal, we have previously selected a Mn2+-specific 11-5 DNAzyme through an in vitro selection method and converted it into a fluorescence sensor for intracellular Mn2+ sensing. Despite the progress, the nucleotides responsible for the activity are unclear, and the performance of the DNAzyme needs to be improved in order for more effective applications in biological systems. To address these issues, we herein report site-specific mutations within the catalytic domain of the selected 11-5 DNAzyme. As a result, we successfully identified a variant DNAzyme, designated as Mn5V, which exhibited a twofold increase in activity compared to the original 11-5 DNAzyme. Importantly, Mn5V DNAzyme maintained its high selectivity for Mn2+ over other competing metal ions. Upon the addition of Mn2+, Mn5V DNAzyme exhibited a higher fluorescence signal within the tumor cells compared to that of the 11-5 DNAzyme. This study therefore provides a better understanding of how the DNAzyme functions and a more sensitive probe for investigating Mn2+ in biological systems.

Generation of DNAzyme in Bacterial Cells by a Bacterial Retron System.

DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10-23 (Dz 10-23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequence-specific mRNA cleavage. However, the in vivo application of Dz 10-23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10-23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10-23 by cloning the Dz 10-23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cfp encoding cyan fluorescence protein and other functional genes such as mreB and ftsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10-23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10-23 but also demonstrated that Dz 10-23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.

DNAzyme-based faithful probing and pulldown to identify candidate biomarkers of low abundance.

Biomarker discovery is essential for the understanding, diagnosis, targeted therapy and prognosis assessment of malignant diseases. However, it remains a huge challenge due to the lack of sensitive methods to identify disease-specific rare molecules. Here we present MORAC, molecular recognition based on affinity and catalysis, which enables the effective identification of candidate biomarkers with low abundance. MORAC relies on a class of DNAzymes, each cleaving a sole RNA linkage embedded in their DNA chain upon specifically sensing a complex system with no prior knowledge of the system's molecular content. We show that signal amplification from catalysis ensures the DNAzymes high sensitivity (for target probing); meanwhile, a simple RNA-to-DNA mutation can shut down their RNA cleavage ability and turn them into a pure affinity tool (for target pulldown). Using MORAC, we identify previously unknown, low-abundance candidate biomarkers with clear clinical value, including apolipoprotein L6 in breast cancer and seryl-tRNA synthetase 1 in polyps preceding colon cancer.

DNAzyme-Catalyzed Site-Specific N-Acylation of DNA Oligonucleotide Nucleobases.

We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6-tetrafluorophenyl ester (TFPE) of a 5'-carboxyl oligonucleotide. Yields are as high as >95 % in 6 h. Several of the N-acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site-specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site-specific N-acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N-acylation, toward a universal approach for site-specific oligonucleotide modification.

Biomimetic Construction of Degradable DNAzyme-Loaded Nanocapsules for Self-Sufficient Gene Therapy of Pulmonary Metastatic Breast Cancer.

Pulmonary metastasis of breast cancer is the major cause of deaths of breast cancer patients, but the effective treatment of pulmonary metastases is still lacking at present. Herein, a degradable biomimetic DNAzyme biocapsule is developed with the poly(ethylenimine) (PEI)-DNAzyme complex encapsulated in a Mn2+/Zn2+-coordinated inositol hexaphosphate (IP6) capsule modified with the cRGD targeting peptide for high-efficiency gene therapy of both primary and pulmonary metastatic breast tumors. This DNAzyme biocapsule is degradable inside acidic lysosomes, leading to the release of DNAzyme and abundant Mn2+/Zn2+ for catalytic cleavage of EGR-1 mRNA. We find that PEI promotes the lysosomal escape of the released DNAzyme. Both in vitro and in vivo experiments demonstrate the apparent downregulation of EGR-1 and Bcl-2 protein expression after treatment with the DNAzyme biocapsule, thereby inducing apoptotic death of tumor cells. We further verify that the DNAzyme biocapsule exhibits potent therapeutic efficacy against both primary and pulmonary metastatic breast tumors with significant inhibition of peri-pulmonary metastasis. This study provides a promising effective strategy for constructing degradable DNAzyme-based platforms with self-supply of abundant metal ion cofactors for high-efficiency gene therapy of metastatic breast cancer.

A Cascade Signal Amplification Strategy for the Ultrasensitive Fluorescence Detection of Cu2+ via λ-Exonuclease-Assisted Target Recycling with Mismatched Catalytic Hairpin Assembly.

Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu2+ was designed using the cascade signal amplification strategy, coupling λ-exonuclease-assisted target recycling and mismatched catalytic hairpin assembly (MCHA). In the designed detection system, the target, Cu2+, can activate the Cu2+-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (tDNA). Then, tDNA binds to hairpin DNA (H0) with an overhanging 5'-phosphorylated terminus to form dsDNA with a blunt 5'-phosphorylated terminus, which activates the dsDNA to be digested by λ-Exo and releases tDNA along with another ssDNA (iDNA). Subsequently, the iDNA initiates MCHA, which can restore the fluorescence of carboxyfluorescein (FAM) previously quenched by tetramethylrhodamine (TAMRA), resulting in a strong fluorescent signal. Furthermore, MCHA efficiently improves the signal-to-noise ratio of the detection system. More importantly, tDNA recycling can be achieved with the λ-Exo digestion reaction to release more iDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+ with a detection limit of 60 fM. The practical application of the developed biosensor was also demonstrated by detecting Cu2+ in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.

Strategy of functional nucleic acids-mediated isothermal amplification for detection of foodborne microbial contaminants: A review.

Foodborne microbial contamination (FMC) is the leading cause of food poisoning and foodborne illness. The foodborne microbial detection methods based on isothermal amplification have high sensitivity and short detection time, and functional nucleic acids (FNAs) could extend the detectable object of isothermal amplification to mycotoxins. Therefore, the strategy of FNAs-mediated isothermal amplification has been emergingly applied in biosensors for foodborne microbial contaminants detection, making biosensors more sensitive with lower cost and less dependent on nanomaterials for signal output. Here, the mechanism of six isothermal amplification technologies and their application in detecting FMC is firstly introduced. Then the strategy of FNAs-mediated isothermal amplification is systematically discussed from perspectives of FNAs' versatility including recognition elements (Aptamer, DNAzyme), programming tools (DNA tweezer, DNA walker and CRISPR-Cas) and signal units (G-quadruplex, FNAs-based nanomaterials). Finally, challenges and prospects are presented in terms of addressing the issue of nonspecific amplification reaction, developing better FNAs-based sensing elements and eliminating food matrix effects.

Fluorescence biosensor for ultrasensitive detection of the available lead based on target biorecognition-induced DNA cyclic assembly.

A fluorescence biosensor was developed for the ultrasensitive detection of the available lead in soil samples by coupling with DNAzyme and hairpin DNA cyclic assembly. The biorecognition between lead and 8-17 DNAzyme will cleave the substrate strands (DNA2) and release the trigger DNA (T), which can be used to initiate the DNA assembly reactions among the hairpins (H1, H2, and H3). The formed Y-shaped sensing scaffold (H1-H2-H3) contains active Mg2+-DNAyzmes at three directions. In the presence of Mg2+, the BHQ and FAM modified H4 will be cleaved by the Mg2+-DNAyzme to generate a high fluorescence signal for lead monitoring. The linear range of the fluorescence biosensor is from 1 pM to 100 nM and the detection limit is 0.2 pM. The biosensor also exhibited high selectivity and the nontarget competing heavy metals did not interfere with the detection results. Compare with the traditional method (DTPA+ICP-MS) for the available lead detection, the relative error (Re) is in the range from -8.3 % to 9.5 %. The results indicated that our constructed fluorescence biosensor is robust, accurate, and reliable, and can be applied directly to the detection of the available lead in soil samples without complex extraction steps.

An RNA-Cleaving DNAzyme That Requires an Organic Solvent to Function.

Engineering functional nucleic acids that are active under unusual conditions will not only reveal their hidden abilities but also lay the groundwork for pursuing them for unique applications. Although many DNAzymes have been derived to catalyze diverse chemical reactions in aqueous solutions, no prior study has been set up to purposely derive DNAzymes that require an organic solvent to function. Herein, we utilized in vitro selection to isolate RNA-cleaving DNAzymes from a random-sequence DNA pool that were "compelled" to accept 35 % dimethyl sulfoxide (DMSO) as a cosolvent, via counter selection in a purely aqueous solution followed by positive selection in the same solution containing 35 % DMSO. This experiment led to the discovery of a new DNAzyme that requires 35 % DMSO for its catalytic activity and exhibits drastically reduced activity without DMSO. This DNAzyme also requires divalent metal ions for catalysis, and its activity is enhanced by monovalent ions. A minimized, more efficient DNAzyme was also derived. This work demonstrates that highly functional, organic solvent-dependent DNAzymes can be isolated from random-sequence DNA libraries via forced in vitro selection, thus expanding the capability and potential utility of catalytic DNA.

Enzyme-free dual amplification biosensor based on functional nucleic acid and CDs/CoOOH for detection of leukemia fusion gene.

Acute promyelocytic leukemia (APL) is an acute myeloid leukemia (AML) with a specific fusion gene target, PML/RARα fusion gene (PML/RARα), which is formed by the translocation of chromosomes 15 and 17. Detection of PML/RARα is the most reliable parameter for the diagnosis, treatment adjustment, efficacy evaluation, prognosis analysis and relapse prediction of APL. In this study, a novel biosensor was constructed for rapid enzyme-free detection of PML/RARα using DNAzyme and carbon dots/cobalt oxhydroxide nanosheet complexs (CDs/CoOOH). In the detection system, the separated DNAzymes could specifically recognize and bind together by the PML/RARα to form a complete DNAzyme for shearing hairpin probe (HP), then generated trigger, which was the first signal amplification. Then, trigger could hybridize with the capture probe (CP) anchored to streptavidin (SA) modified microplate as well as fluorescence quenching signal probe (SP@CDs/CoOOH). Finally, ascorbic acid (AA) was added to decompose CoOOH and the fluorescence of CDs was released, which was the second signal amplification. Through the dual signal amplification of DNAzyme and CDs/CoOOH, PML/RARα could be detected quickly and sensitively, which overcame the limitation of protein enzyme in traditional fluorescence methods, showing potential clinical application value in the diagnosis and treatment of leukemia.

mRNA-activated DNAzyme nanoprobe for tumor cell precise imaging and gene therapy.

A novel Au-nucleic acid nanoprobe, catalyzed by mRNA, has been developed for live cell imaging and precise treatment of tumor cells. This nanoprobe exhibits the remarkable ability to differentiate between tumor cells and normal cells through live cell mRNA imaging, while selectively inducing apoptosis in tumor cells.

Site-Specific Bioorthogonal Activation of DNAzymes for On-Demand Gene Therapy.

RNA-cleaving DNAzymes hold great promise as gene silencers, and spatiotemporal control of their activity through site-specific reactions is crucial but challenging for on-demand therapy. We herein report a novel design of a bioorthogonally inducible DNAzyme that is deactivated by site-specific installation of bioorthogonal caging groups on the designated backbone sites but restores the activity via a phosphine-triggered Staudinger reduction. We perform a systematical screening for installing the caging groups on each backbone site in the catalytic core of 10-23 DNAzyme and identify an inducible DNAzyme with very low leakage activity. This design is demonstrated to achieve bioorthogonally controlled cleavage of exogenous and endogenous mRNA in live cells. It is further extended to photoactivation and endogenous stimuli activation for spatiotemporal or targeted control of gene silencing. The bioorthogonally inducible DNAzyme is applied to a triple-negative breast cancer mouse model using a lipid nanoparticle delivery system, demonstrating high efficiency in knockdown of Lcn2 oncogenes and substantial suppression of tumor growth, thus highlighting the potential of precisely controlling the DNAzyme functions for on-demand gene therapy.

A Smart Deoxyribozyme-Programmable Catalytic DNA Circuit for High-Contrast MicroRNA Imaging.

Synthetic catalytic DNA circuits have been recognized as a promising signal amplification toolbox for sensitive intracellular imaging, yet their selectivity and efficiency are always constrained by uncontrolled off-site signal leakage and inefficient on-site circuitry activation. Thus, the endogenously controllable on-site exposure/activation of DNA circuits is highly desirable for achieving the selective imaging of live cells. Herein, an endogenously activated DNAzyme strategy was facilely integrated with a catalytic DNA circuit for guiding the selective and efficient microRNA imaging in vivo. To prevent the off-site activation, the circuitry constitute was initially caged without sensing functions, which could be selectively liberated by DNAzyme amplifier to guarantee the high-contrast microRNA imaging in target cells. This intelligent on-site modulation strategy can tremendously expand these molecularly engineered circuits in biological systems.

A triple-cycle amplification biosensor for colorimetric detection of mutant PIK3CAE545K based on cascade-driven DNA walker and branched hybridization strand reaction.

Circulating tumor DNA (ctDNA) is an ideal candidate for liquid biopsy biomarkers. Therefore, detecting a low abundance of ctDNA is essential for early cancer diagnosis. Here, we developed a novel triple circulation amplification system integrating entropy and enzyme cascade-driven three-dimensional (3D) DNA walker and branched hybridization strand reaction (B-HCR) for ultrasensitive detection of breast cancer-related ctDNA. In this study, the 3D DNA walker was constructed by inner track probes (NH) and complex S on a microsphere. Once the DNA walker was triggered by the target, the strand replacement reaction ran first and kept circulating to rapidly displace the DNA walker containing 8-17 DNAzyme. Secondly, the DNA walker could repeatedly cleave NH autonomously along the inner track, generating numerous initiators, and then promoting B-HCR to activate the third cycle. Subsequently, the split G-rich fragments were brought in close to form the G-quadruplex/hemin DNAzyme by adding hemin, with the addition of H2O2 and ABTS, the target could be observed. Benefiting from triplex cycles, the PIK3CAE545K mutation detection possesses a good linear range from 1-103 fM, and the limit of detection was 0.65 fM. Due to the low cost and high sensitivity, the proposed strategy has great potential in early diagnosis of breast cancer.

Label-Free Sequence-Specific Visualization of LAMP Amplified Salmonella via DNA Machine Produces G-Quadruplex DNAzyme.

Salmonella is one of four key global causes of diarrhea, and in humans, it is generally contracted through the consumption of contaminated food. It is necessary to develop an accurate, simple, and rapid method to monitor Salmonella in the early phase. Herein, we developed a sequence-specific visualization method based on loop-mediated isothermal amplification (LAMP) for the detection of Salmonella in milk. With restriction endonuclease and nicking endonuclease, amplicons were produced into single-stranded triggers, which further promoted the generation of a G-quadruplex by a DNA machine. The G-quadruplex DNAzyme possesses peroxidase-like activity and catalyzes the color development of 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) as the readouts. The feasibility for real samples analysis was also confirmed with Salmonella spiked milk, and the sensitivity was 800 CFU/mL when observed with the naked eye. Using this method, the detection of Salmonella in milk can be completed within 1.5 h. Without the involvement of any sophisticated instrument, this specific colorimetric method can be a useful tool in resource-limited areas.