Peroxidase-mimicking DNAzyme Research Articles

Bimetallic DNAsome Decorated with G4-DNA as a Nanozyme for Targeted and Enhanced Chemo/Chemodynamic Cancer Therapy.

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 Cu2+-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.

Amplified and Specific Staining of Protein Dimerization on Cell Membrane Catalyzed by Responsively Installed DNA Nanomachines for Cancer Diagnosis.

In situ staining of protein dimerization on cell membrane has an important significance in accurate diagnosis during perioperative period, yet facile integration of specific recognition function and local signal conversion/amplification abilities on membrane surface remains a great challenge. Herein, a two-stage catalytic strategy is developed by installing DNA nanomachines and employing. Specifically, dual-aptamer-assisted DNA scaffold perform a "bispecific recognition-then-computing" operation and the output signal initiate a membrane-anchored biocatalysis for self-assembly of DNA catalytic converters, that is, G-quadruplex nanowire/hemin DNAzyme. Then, localized-deposition of chromogenic polydopamine is chemically catalyzed by horseradish peroxidase-mimicking DNAzyme and guided by supramolecular interactions between conjugate rigid plane of G-tetrad and polydopamine oligomer. The catalytic products exhibit nanofiber morphology with a diameter of 80-120nm and a length of 1-10µm, and one-to-one localize on DNA scaffold for amplified and specific staining of protein dimers. The bispecific staining leads to a higher (≈3.4-fold) signal intensity than traditional immunohistochemistry, which is beneficial for direct visualization. Moreover, an efficient discrimination ability of the bispecific staining strategy is observed in co-culture model staining. This study provides a novel catalytic method for controlling deposition of chromogens and paves a new avenue to sensitively stain of protein-protein interactions in disease diagnosis.

Rapid Colorimetric Determination of microRNA (miRNA) Using an Autocatalytic Entropy-Driven Circuit (AEDC)

A colorimetric and rapid signal amplified microRNA (miRNA) detection system based on autocatalytic entropy-driven circuit (AEDC) is reported. The target miRNA-21 (miR-21) was firstly hybridized with the sticky end of the triple-stranded hybrid complex (S, which consisting of a reporter strand (P), a byproduct strand (R) and an assistant strand (Q)), and P strand, which contained the G-quadruplex sequence that was released through the toehold-mediated strand displacement reaction. The G-quadruplex sequence interacted with hemin to form the peroxidase-mimicking DNAzymes and catalyzed the H2O2-mediated oxidation of colorless 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS2−) to the green ABTS•−. Meanwhile, fuel strand (F) hybridized with S to release the target miR-21 and the waste duplex DNA product F-Q was formed, which brings the two split mimic target DNA sequences into proximity to form the intact mimic target DNA sequence (analogue of miR-21). The obtained mimic target DNA as well as the released miR-21 can be cyclically used to trigger autocatalytic EDC reaction, leading to the rapidly enhanced formation of mimic target DNA with the concomitant generation of a higher colorimetric signal. Finally, a colorimetric and rapid miR-21 system based on the hairpin-free AEDC reaction was successfully fabricated. This system also has been applied for the miRNA determination in serum, exhibiting the potential application in the clinical diagnosis.

Self-Assembly of Designed Peptides with DNA to Accelerate the DNA Strand Displacement Process for Dynamic Regulation of DNAzymes.

Toehold-mediated DNA strand displacement (TMSD) is a powerful tool for controlling DNA-based molecular reactions and devices. However, the slow kinetics of TMSD reactions often limit their efficiency and practical applications. Inspired by the chemical structures of natural DNA-operating enzymes (e.g., helicase), we designed lysine-rich peptides to self-assemble with DNA-based systems. Our approach allows for accelerating the TMSD reactions, even during multiple displacement events, enhancing their overall efficiency and utility. We found that the acceleration is dependent on the peptide's sequence, length, and concentration as well as the length of the DNA toehold domain. Molecular dynamics simulations revealed that the peptides promote toehold binding between the double-stranded target and the single-stranded invader, thereby facilitating strand displacement. Furthermore, we integrated our approach into a horseradish peroxidase-mimicking DNAzyme, enabling the dynamic modulation of enzymatic functions on and off. We anticipate that the established acceleration of strand displacement reactions and the modulation of enzymatic activities offer enhanced functionality and control in the design of programmable DNA-based nanodevices.

A highly sensitive signal-on biosensor based on restriction enzyme-mediated molecular switch for detection of TET1

Ten-eleven translocation 1 (TET1) is a member of the TET enzyme family of dioxygenases, which plays an important role in active DNA demethylation. Therefore, the sensitive TET1 detection could help us better understand DNA methylation-demethylation in epigenetics. Here we report a detection method that consists of electrode fabrication, TET1 modification, DNA digestion, signal-on oxidoreduction, and current peak monitoring. An exquisitely designed 5′end-G-rich oligodeoxynucleotide was synthesized bearing a methylated cytosine (5-mC), which formed into hairpin dsDNA with the MspI recognition sequence (CmCGG/GGCC). Then hairpin dsDNA was fabricated onto gold nanoparticles modified glassy carbon electrode (DNA/AuNPs/GCE) via Au-S bond. The combination uses of restriction enzyme MspI and hemin converted fabricated-dsDNA into peroxidase-mimicking DNAzyme, thereby promoting the reduction of H2O2 with a current peak. However, the current peak was extremely decreased once TET1 and T4 β-GT were used in advance. We confirmed a delicately linear relationship matching between the current difference and TET1 activity from 0.7 to 10.5 ng μL−1 with a detection limit of 0.027 ng μL−1, which outcompeted the former methods at least one order of magnitudes. The TET1 activity evaluation in the existence of Bobcat339 was also tested as the proof of concept of inhibitors screening. Our strategy provides a novel, label-free, and sensitive electrochemical approach that enables us to complete both TET1 activity evaluation and potential TET1 inhibitors screening.

Peroxidase-Mimicking DNAzymes as Receptors for Label-Free Discriminating Heavy Metal Ions by Chemiluminescence Sensor Arrays.

Receptors are crucial to the analytical performance of sensor arrays. Different from the previous receptors in sensor arrays, herein, peroxidase-mimicking DNAzymes were innovatively used as receptors to develop a label-free chemiluminescence sensor array for discriminating various heavy metal ions in complex samples. The peroxidase-mimicking DNAzymes are composed of functional oligonucleotides and hemin, including G-triplex-hemin DNAzyme (G3-DNAzyme), G-quadruplex-hemin DNAzyme (G4-DNAzyme), and the dimer of G-quadruplex-hemin DNAzyme (dG4-DNAzyme). Circular dichroism (CD) spectroscopy demonstrated that different metal ions diversely affect the conformation of G-quadruplex and G-triplex, resulting in a change in the activity of peroxidase-mimicking DNAzyme. Thus, the unique fingerprints formed to easily discriminate seven kinds of heavy metal ions by principal component analysis (PCA) within 20 min. The discrimination of unknown metal ions in tap water further confirmed its ability for discriminating multiple heavy metal ions. Moreover, it will not bring water pollution due to the good biocompatibility of DNA. Therefore, it not only merely offers a label-free, rapid, environment-friendly, and cheap (1.49 $) sensor assay for discriminating metal ions but also comes up with an innovative way for developing sensor arrays.

Self-assembly of Dendrimer-DNA amphiphiles and their catalysis as G-quadruplex/hemin DNAzymes

Nanomaterials self-assembled by DNA amphiphiles have great potential application in catalysis, bioimaging and biomedicine. Here, we constructed two kinds of DNA-dendrimer amphiphiles including the G-quadruplex-dendrimer hybrid (G20-Den) and single-stranded DNA-dendrimer hybrid (D20-Den) as a control. Both of them self-assembled into nanofibers with a length of several micrometers in MES buffer, and the diameter of G20-Den nanofibers was smaller than that of D20-Den nanofibers due to the compact stacking of G-quadruplex formed in G20-Den hybrids. At the same time, the catalytic properties of G20-Den nanofiber/hemin, as horseradish peroxidase-mimicking DNAzymes, were investigated. Our results showed that G20-Den nanofiber/hemin, as well as the single-stranded G20/hemin system, exhibited higher catalytic peroxidation activity than D20-Den nanofiber/hemin. Furthermore, through simple ultrafiltration, the recycle of these nanofibers can be simply achieved. This research has provided a new solution for the construction of functional DNA nanomaterials.

CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity.

Food authenticity is a critical issue associated with the economy, religion, and food safety. Herein, we report a label-free and colorimetric nucleic acid assay for detecting DNA barcodes, enabling the determination of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR associated protein) to specifically recognize the polymerase chain reaction (PCR) products for further trans-cleavaging the peroxidase-mimicking G-quadruplex DNAzyme. Based on this principle, the presence of the cytochrome oxidase subunit I gene could be directly observed with the naked eye via the color change of 3,3',5,5'-tetramethylbenzidine sulfate (TMB). The whole detection process, including PCR amplification and TMB colorimetric analysis, can be completed within 90 min. The proposed assay can detect pufferfish concentrations diluted to 0.1% (w/w) in a raw pufferfish mixture, making it one of the most sensitive methods for food authenticity. The robustness of the assay was verified by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay is advantageous in easy signal readout, high sensitivity, and general applicability and thus could be a competitive candidate for food authenticity.

Inactivation Kinetics of G-Quadruplex/Hemin Complex and Optimization for More Reliable Catalysis.

Reliable catalysis is critical for the synthesis of various chemicals, molecular sensing and biomedicine. G-quadruplex/Hemin (GQH) complex, a peroxidase-mimicking DNAzyme, has been widely used in various publications. However, a concern exists about the unstable kinetics of GQH-catalyzed peroxidation. This work investigates several factors that result in the inactivation of GQH and the signal degradation during long reaction periods, including pH, buffer component, the selection of substrate and the oxidation damage of cofactor. Using colorimetric and fluorescent assays, GQH was found to be highly unstable under basic conditions with 50 % of GQH activity lost within 2 minutes at high H2 O2 concentrations. Appropriate conditions and substrates are suggested for accurately characterizing GQH-catalyzed reactions, as well as optimization to improve the catalytic reliability, such as the use of polyhistidine and cascade reactions. These results could be useful for GQH-related applications.

Enhancement of Activity for Peroxidase-Mimicking DNAzyme by Covalent Attachment of Hemin to G-Quadruplex-Forming Oligonucleotide Using Click Chemistry.

Peroxidase-mimicking DNAzymes are used in the development of new bioanalytical assays due to their advantages like thermal stability, simple synthesis and purification, and ability to hybridize with the complementary strand of nucleic acid. Here, we describe the method of covalent attachment of hemin to DNA oligonucleotide using click chemistry that allows good yield (60-70%) of the final conjugate product. The activity of obtained DNAzymes is monitored using chromogenic and fluorogenic substrates.

Sensitive electrochemical detection of microRNA based on DNA walkers and hyperbranched HCR-DNAzyme cascade signal amplification strategy

The designable and configurable DNA nanostructures with various biological functions have been widely applied in biosensors especially for detection of nucleic acid. In this work, a cascade signal amplification strategy based on DNAzyme-driven 3D DNA walkers combined with hyperbranched HCR and DNAzyme catalyzation were applied for sensitive electrochemical detection of microRNA-141. First, the Mg2+-dependent cleavage of DNAzyme trigger the autonomous DNA walking process, inducing the generation of mediate strand output, thereby leading the construction of hyperbranched HCR and subsequently providing more embedding sites for electroactive molecules. Meanwhile, the G-quadruplex domains are introduced by the terminal of DNA strand in constructing hyperbranched nanostructure. Finally, the formed hemin/G-quadruplex DNAzyme (a horseradish peroxidase-mimicking DNAzyme) could catalyze the decomposition of H2O2, greatly increasing the intensity of electrochemical signal. Combined with the DNAzyme-driven 3D DNA walkers, hyperbranched HCR and DNAzyme catalyst assisted amplification strategies, the electrochemical biosensor showed a wide linear range for the detection of microRNA-141 with favorable sensitivity and selectivity. The performance of this biosensor in different cell lysates were evaluated, which showed great potential in assay of microRNAs at low expression levels in complex samples.

Analyzing Criteria Affecting the Functionality of G-Quadruplex-Based DNA Aptazymes as Colorimetric Biosensors and Development of Quinine-Binding Aptazymes.

A DNA aptazyme consists of an aptamer domain and a DNAzyme module, in which the DNAzyme activity can be regulated by the aptamer-target interaction. The complex of G-quadruplex (GQ) and hemin is a peroxidase-mimicking DNAzyme and has become increasingly popular as a reporter system for biosensing applications. The development of GQ-based aptazymes is of high interest as they can be used as label-free biosensors for the real-time detection of pathogens. Herein, we rationally designed ca. 200 GQ-based aptazyme candidates and evaluated the suitability of 14 aptamers targeting quinine, Protein A, Staphylococcus enterotoxin B, and ATP for this detection concept. As a result, six novel aptazymes were developed for the specific detection of quinine based on two quinine-binding aptamers. The rest of designed probes, however, hardly showed significant functionality. To uncover the reasons, we performed enzyme-linked oligonucleotide assays to find how the affinity of aptamers is affected once conjugated to the DNAzyme sequence or upon integration into the aptazyme probe. Furthermore, we investigated the impact of the structure-switching functionality in the parent aptamer and the effect of the reaction matrix on the efficiency of probes.

DNAzymes as key components of biosensing systems for the detection of biological targets

DNAzymes are synthetic functional nucleic acids that have found widespread use in biosensing applications both for molecular recognition and signal generation. Two classes of DNAzymes have proved particularly effective for use in proof-of-concept biosensing systems, namely RNA-cleaving DNAzymes (RCDs) and peroxidase mimicking DNAzymes (PMDs). RCDs catalyze the site-specific cleavage reaction of an RNA dinucleotide junction, generating two cleavage fragments. PMDs are capable of catalyzing peroxidation reactions of chromophores, thereby generating a measurable signal. Herein, we review the use of these DNAzymes in biomedical assays and diagnostics, and show that this emerging field should have great promise for biosensor development over the next few decades.

Rolling circle amplified DNAzyme followed with covalent organic frameworks: Cascade signal amplification of electrochemical ELISA for alfatoxin M1 sensing

A sensitive, selective and high-throughput electrochemical enzyme-linked immunosorbent assay (EC-ELISA) platform was constructed for determination of alfatoxin M1 (AFM1). Rolling circle amplified (RCA) DNAzyme coupled with covalent organic frameworks (COFs) modified electrode was used as signal amplification strategies. The immunoreaction was performed on a microplate via specific recognition of AFM1 by primer-AuNPs-aptamer and anti-AFM1 antibody. The primer triggered RCA reaction produced a long single-stranded DNA, which can fold into a peroxidase-mimicking DNAzyme with the presence of hemin and K+. In the presence of H2O2, DNAzyme catalyzed the oxidation of 2-aminophenol and the oxidation product 3-aminophenylhydrazine was enriched as a signal molecule on the TpBD modified electrode with a significant current response. Under optimized conditions, the sensor exhibited high selectivity and sensitivity towards AFM1 with a detection limit of 0.15 ng/mL. In addition, the AFM1 can also be quantified by the naked eye. This EC-ELISA platform was successfully applied for the determination of AFM1 in milk samples with recoveries from 93.37% to 104.01%.

A specific, rapid and high-throughput cascade catalytic method for determination of plasma uric acid by using uricase and trivalent peroxidase-mimicking DNAzyme

Abstract In this study, we report a facile and cost-effective system for selective measurement of uric acid (UA) using a cascade mechanism composed of Uricase and peroxidase-mimicking trivalent DNAzyme. A three G-quadruplex-hemin complex was engaged as the peroxidase-mimicking DNAzyme. In this cascade catalysis system, UA was oxidized by uricase to produce hydrogen peroxide, which was also acted as a substrate for the oxidation of 3, 3′, 5, 5′ tetramethylbenzidine (TMB) by a peroxidase-mimicking trivalent DNAzyme. TMB oxidation showed a straight relationship with UA concentration. The cascade catalysis system displayed high selectivity toward UA because of the natural selectivity of uricase. This cascade uric acid sensing system revealed the best activity at 30 °C and pH 7.0, which indicated its capacity to uric acid sensing near physiological conditions. The linear regression equation for UA was quantified as A = 0.0072C + 0.0197, with a correlation coefficient of 0.9906. Results suggested that this system could identify as low as 0.66 μM of uric acid with a linear range from 2.5 to 40 μM. Results exhibited that the interfering public components had no noticeable absorbance values. Overall, these results indicated that this facile system would be valuable for uric acid measurement in the medical laboratory.

Rapid and colorimetric detection of nucleic acids based on entropy-driven circuit and DNAzyme-mediated autocatalytic reactions.

In this work, a novel, rapid and enzyme-free colorimetric biosensor for the detection of nucleic acids has been developed based on entropy-driven (EDC) circuit and DNAzyme-mediated autocatalytic reactions. On sensing the target DNA, the EDC reaction could be initiated and the intact Mg2+-dependent DNAzyme was formed in the reaction product; then, a "mimic target" DNA was generated during the cleavage process of DNAzyme, which in turn catalyzed the EDC reaction corresponding to an autocatalytic process. Meanwhile, numerous G-quadruplex structures were released and further interacted with hemin to form peroxidase-mimicking DNAzyme, inducing a remarkably amplified colorimetric signal. This autocatalytic EDC (AEDC) sensing system exhibited a linear relationship in the range from 20 pM to 10 nM with a detection limit of 10.2 pM. More importantly, benefitting from the Mg2+-dependent DNAzyme-mediated autocatalytic reaction, the detection time (20 min) was significantly reduced compared to that for the reported EDC strategies. In addition, this sensing system has been applied for the detection of target DNA in human serum samples, indicating that it is promising for the on-site and real-time detection of nucleic acids in biomedical research and disease diagnosis.

Detection of breast cancer-derived exosomes using the horseradish peroxidase-mimicking DNAzyme as an aptasensor.

Exosomes are membrane-enclosed phospholipid extracellular vesicles with a variety of tumor antigens which can be applied in the diagnosis and treatment of cancer due to the high secretion on the surface of cancer cells. Until now, many research studies on exosomes have been reported, but convenient and low-cost detection methods still need to be developed. Recently, we have developed a sensitive, simple and low-cost colorimetric aptasensor by designing a hairpin-like structure, which combined the highly specific MUC1 aptamer with a hemin/G-quadruplex for the detection of breast cancer exosomes. The hemin/G-quadruplex toward H2O2 reduction was used to generate an evidently strong colorimetric response owing to acting as a HRP-mimicking DNAzyme. The aptasensor is regarded as an "on-off" type switch, which strictly controls the process of reaction responding to the existence or not of exosomes. In our study, associated exosome detection limits are 3.94 × 105 particles per mL, which showed a higher sensitivity compared to commercial ELISA. Our method not only exhibited the advantages of being convenient and time-saving, with few instruments used, but also the signal generated using this method can be easily observed by the naked eye. Furthermore, the proposed strategy can well differentiate breast cancer patients from healthy individuals, demonstrating potential application in the analysis of clinical specimens.

Proximity Binding-Triggered Assembly of Two MNAzymes for Catalyzed Release of G-Quadruplex DNAzymes and an Ultrasensitive Homogeneous Bioassay of Platelet-Derived Growth Factor.

When the target biorecognition-triggered assembly of two Mg2+-dependent DNAzymes (MNAzymes) is employed for dually catalytic release of peroxidase-mimicking G-quadruplex DNAzymes (G-DNAzymes), this work develops a novel homogeneous colorimetric method for an ultrasensitive bioassay of platelet-derived growth factor-BB (PDGF-BB). The first MNAzyme assembly is realized through a highly specific aptamer biorecognition-driven proximity ligation reaction. Its catalytic cleavage toward the two designed hairpin substrates not only releases a large amount of G-DNAzymes for colorimetric signal transduction but also enables the spontaneous assembly of another MNAzyme for signal amplification. This leads to the successful detection of PDGF-BB in a wide linear range from 2.0 pg mL-1 to 20 ng mL-1 with a very low detection down to 0.088 pg mL-1. As the whole reactions including aptamer biorecognitions, DNA hybridizations, and catalytic cleavages of MNAzymes are conducted in a homogeneous solution, this method has very simple manipulations and also has high repeatability. In addition, the high specificity of the aptamer biorecognition-triggered signal transduction decides the excellent selectivity of the method. This bioassay does not require an expensive instrument and nucleic acid labeling for signal readout or any nanomaterial, enzyme, or nuclease for signal amplification. Thus, it displays an extensive potential for clinical diagnostic applications.

Biosensors Made of Synthetic Functional Nucleic Acids Toward Better Human Health.

The invention of the in vitro selection technique, or SELEX (Systematic Evolution of Ligands by EXponential enrichment), in the early 1990s, identified single-stranded (ss) nucleic acid molecules with catalytic or binding properties from random-sequence nucleic acid libraries.1–3 This technique has given rise to intensive examinations of synthetic functional nucleic acids (aptamers and nucleic acid enzymes) for wide-ranging applications including biosensors, diagnostics and therapeutics.4–9After nearly three decades of research by many groups around the world, many functional nucleic acids have been isolated and characterized. A few well-known examples are provided in Figure 1, including two RNA-cleaving DNAzymes (8-17 and 10-23),10 a peroxidase-mimicking DNAzyme (PS2.M),11 two DNA aptamers that bind human α-thrombin (TBA15 and TBA29),12,13 a DNA aptamer that binds ATP,14 and a modified nucleic acid aptamer that binds human VEGF-165 (Pegaptanib).15 Thousands of papers have been published describing the us...

One-step label-free chemiluminescent assay for determination of exonuclease III activity towards hairpin oligonucleotides

Fast label-free chemiluminescent assay for determination of exonuclease III (ExoIII) activity measured towards hairpin oligonucleotide substrates was developed. The designed substrates consisted of EAD2 aptamer to hemin which was associated with DNA sequence complementary to 5’-terminus fragment of EAD2. In the presence of ExoIII the associated sequence of the hairpin stem was digested, producing EAD2 aptamer which reacted with hemin with the formation of peroxidase-mimicking DNAzyme (PMDNAzyme). The catalytic activity of the produced PMDNAzyme was measured towards luminol/H2O2. Under the optimized conditions the limit of detection and sensitivity of the one-step chemiluminescent assay of ExoIII were 7.3 nM and 1.7 × 108 M−1, respectively. The coefficient of variation (CV) was lower than 6%.