Structural and functional study of a simple,rapid, and label‐free DNAzyme‐based DNA biosensor for optimization activity

Peroxidase‐mimicking DNAzyme has a potential to self‐assemble into a G‐quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase‐mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non‐labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV–Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G‐quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg²⁺. Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.     

Amplified Chemiluminescence Detection of DNA by Strand-Displacement Polymerization Target Recycling and G-quadruplexes/Hemin DNAzyme Catalysis

A new strategy based on strand-displacement polymerization target recycling and G-quadruplex DNAzyme catalysis was developed for amplified chemiluminescence detection of DNA. The amplified detection was achieved by using the system consisted of hairpin DNA probe, G-rich DNA, primer, and polymerase Klenow fragment exo^?. When the target DNA was introduced the system, the hairpin structure of DNA probe was opened by the hybridizing of target DNA with its complementary sequence, the primer hybridized then with DNA probe and initiated polymerase-aided strand-displacement polymerization reaction, resulting in the release of target DNA and G-rich DNA. The released target DNA again hybridizes with another DNA probe to trigger the next polymerase-aided strand-displacement polymerization, generating large numbers of G-rich DNA. The G-rich DNA assembles with hemin to form the G-quadruplexes/hemin DNAzyme, which can catalyze oxidation of luminol by H₂O₂, generating chemiluminescence. This unique amplifying strategy gives a detection limit down to 2.5 pM, which is at least two to three orders of magnitude lower than that of unamplified DNA detection methods.     

A novel homogenous detection method based on the self-assembled DNAzyme labeled DNA probes with SWNT conjugates and its application in detecting pathogen

In this paper, a novel and cost-effective homogeneous detection method was constructed for the detection of genomic DNA and Staphylococcus aureus (S. aureus), based on the noncovalent assembly of DNAzyme-labeled detection probe and single-walled carbon nanotubes (SWNTs). When the target genomic DNA and hemin was existed in the detection solution, the detection probe wrapped on the SWNTs by π-stacking interactions would keep away from SWNTs and form a DNAzyme-self-assembly construction. This DNAzyme construction could catalyze 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2?) and generate a colored product which could lead to the absorbance changes. Hence, according to its catalyzed capacity, the DNAzyme construction could amplify the detection signal. The concentration of target DNA could be quantified by exploiting their optical absorption changes at 414 nm and the concentration limit of detection of the method was 30 nM. And this detection method detected S. aureus quantitatively. In addition, this work proved that the method obtain higher detection sensitivity compared with the method without SWNTs because of the protection profile of SWNTs towards the detection probe.     

Highly effective colorimetric and visual detection of ATP by a DNAzyme-aptamer sensor

A new and simple method was developed to detect adenosine triphosphate (ATP) by using a DNAzyme aptamer sensor. The DNAzyme used was a single‐stranded DNA that could combine with hemin. The aptamer, a single, short nucleic acid sequence that can specifically bind with many targets, was an anti‐ATP aptamer. Two DNA sequences were designed: i) a functional chain (Chain A) consisting of two parts, i.e., the anti‐ATP aptamer (recognition part) and the DNAzyme (signal transduction part) and ii) a blocker chain (Chain B), which could partially hybridize with Chain A. The hybridized chains A and B were unfolded by the addition of ATP and hemin, and the blocker chain and the complex of the functional chain with ATP and hemin were in solution. The DNAzyme in the functional chain formed a G‐quadruplex with hemin and then catalyzed the oxidation by H₂O₂ of 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS²⁻) to the colored ABTS^.− radical. The color change caused by this reaction could be clearly observed by naked eye, and the absorbance was recorded at 414 nm. The detection limit was 1×10⁻⁶ M .     

A one-step electrochemical method for DNA detection that utilizes a peroxidase-mimicking DNAzyme amplified through PCR of target DNA

A novel one-step electrochemical method for DNA detection is described. The procedure utilizes a reaction catalyzed by a peroxidase-mimicking DNAzyme to produce a product, which forms an insoluble precipitation layer on the surface of an electrode. A rationally designed forward primer, conjugated with a peroxidase DNAzyme complementary sequence at its 5′-end, is used for PCR amplification of target DNA. As a result, the DNAzyme sequence is produced by amplification only when the target DNA is present in the sample. The PCR product is then subjected to the precipitation reaction on the electrode surface using an electrolyte assay buffer containing 4-chloronaphthol, hydrogen peroxide, ferrocenemethanol, hemin, and 5′-lambdaexonuclease. Finally, analysis is carried out using Faradaic impedance spectroscopy. The impedance value was found to greatly increase when target DNA is present owing to the formation of a precipitation layer on the electrode surface caused by the catalytic action of the DNAzyme. In contrast, no impedance increase is observed when a control sample not containing target DNA is utilized. By employing this strategy, target DNA from Chlamydia trachomatis was reliably detected within a 10 min period following precipitation without the need for complicated secondary procedures. This effort has led to the development of a highly convenient electrochemical one-step method for DNA detection that utilizes a peroxidase-mimicking DNAzyme, which is specifically designed to undergo amplification during PCR of target DNA.     

Studies of the activity of peroxidase-like DNAzyme by modifying 3'- or 5'-end of aptamers

In the presence of hemin and under appropriate conditions, some modalities of G-quadruplexes can form a peroxidase-like DNAzyme that has been widely used in biology. Structure-function studies on the DNAzyme revealed that its catalytic ability may be dependent on the unimolecular parallel G-quadruplex. In this report, we present the preliminary investigation on the relationship between the structure and function of DNAzymes through a terminal oligo modification in G-quadruplex sequences by adding different lengths of oligo-dT to the 3'- or 5'-end of the aptamers. The results suggested that adding dT(n) to the 5'-end of the DNA sequence of the enzyme improved the ability of hemin to bind with DNA, but the addition of dT(n) to the 3'-end decreased the binding ability of hemin for DNA. The increased stability of the assembled DNAzyme would lead to more favorable binding between the enzyme and substrate (H(2) O(2)), facilitating higher peroxidase activity; on the contrary, with lower stability of the DNAzyme complex, we observed reduced peroxidase activity.     

Immobilization of DNAzyme as a thermostable biocatalyst

Deoxyribozyme (DNAzyme) carrying peroxidase activity was immobilized on two types of particles and the enzymatic activity was measured. The DNA recognizing porphyrin were prepared according to Travascio et al. ([1998] Chem Biol 5:505-517) and the interactions with hemin were investigated by ultraviolet absorbance and circular dichroism spectroscopies. The DNA interacted with hemin and significant conformational change was induced by the interaction. Therefore, the end of this DNA was modified with a thiol group and it was immobilized on thiol-containing polysaccharide beads or on gold particles. The DNA immobilized on the gold particle showed activity catalyzing the peroxidation reaction. No significant reduction of activity was observed even after immobilization. The immobilized DNAzyme could be repeatedly utilized without significant loss of activity. In addition, heat treatment did not reduce the activity, although a protein enzyme, horseradish peroxidase, lost its activity after the heat treatment. The repertoire of DNAzyme is still currently limited. However, in the future the utilization of DNAzyme in the field of biotechnology will be important with the increase of discoveries of new functional DNAzymes.     

不同G的四链体结构对DNAzyme活性的影响

富含鸟嘌呤的DNA序列在金属离子（通常是钠、钾离子）存在的条件下,可以形成稳定的G-四链体（G-quadruplex）。该G 四链体能够结合hemin（氯高铁血红素）形成具有过氧化物酶的活性的G四链体-hemin复合物DNAzyme。将这一原理联合滚环扩增技术可以对核酸进行可视化的检测。本研究旨在探索G-四链体-hemin复合物中,G-四链体结构以及两个G-四链体之间的链接长度与DNAzyme过氧化物酶活性之间的关系。实验分别选取了平行、反平行和混合结构的G-四链体,通过热差异光谱、紫外光谱、圆二色光谱对结构进行分析,不断加长链接序列并测定3种结构形成的DNAzyme活性,发现正平行结构的G-四链体具有更高的DNAzyme活性和更明显的可视化效果。综上所述,平行G-四链体结构可以用来满足裸眼可视化检测的需求,为无需复杂仪器的核酸检测奠定了方法基础。     

Studies of the Activity of Peroxidase‐Like DNAzyme by Modifying 3′‐ or 5′‐End of Aptamers

In the presence of hemin and under appropriate conditions, some modalities of G‐quadruplexes can form a peroxidase‐like DNAzyme that has been widely used in biology. Structure? function studies on the DNAzyme revealed that its catalytic ability may be dependent on the unimolecular parallel G‐quadruplex. In this report, we present the preliminary investigation on the relationship between the structure and function of DNAzymes through a terminal oligo modification in G‐quadruplex sequences by adding different lengths of oligo‐dT to the 3′‐ or 5′‐end of the aptamers. The results suggested that adding dT_n to the 5′‐end of the DNA sequence of the enzyme improved the ability of hemin to bind with DNA, but the addition of dT_n to the 3′‐end decreased the binding ability of hemin for DNA. The increased stability of the assembled DNAzyme would lead to more favorable binding between the enzyme and substrate (H₂O₂), facilitating higher peroxidase activity; on the contrary, with lower stability of the DNAzyme complex, we observed reduced peroxidase activity.     

Interaction of hemin with quadruplex DNA

A DNA enzyme with peroxidase activity is a G-quadruplex-based DNAzyme formed by hemin and G-quadruplex DNA. Activity of peroxide DNAzymes can be influenced by the structure of quadruplex DNA. In this investigation, the interaction of hemin with T30695 G-quadruplex DNA is evaluated. Molecular dynamic simulation indicates that the binding mode of hemin to G-quadruplex DNA is end-stacking, which is consistent with absorption spectroscopy. Based on fluorescence spectroscopy, hemin ejects thiazole orange from bases of four-strand DNA. Circular dichroism spectra showed that no alteration occurs in this type of DNA structure.

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Graphical Abstract Peroxidase DNAzyme is formed by hemin and G-quadruplex DNA.

     

Characterization of the G‐quadruplex structure of a catalytic DNA with peroxidase activity

It has been reported that the complexes formed by hemin and some G‐quadruplexes can be developed as a new class of DNAzyme with peroxidase activity. This kind of DNAzyme has received a great deal of attention. But to date, the actual G‐quadruplex structure that can provide hemin with enhanced peroxidase activity is in doubt. Herein, the G‐quadruplex structure of CatG4, a 21‐nucleotide DNA oligomer which was previously reported to bind hemin and the resulting complex exhibiting enhanced peroxidase activity, was characterized by fluorescence and circular dichroism measurements. The results suggest that the catalytically active form of CatG4 may be a unimolecular parallel quadruplex rather than a unimolecular chair‐type antiparallel quadruplex or a multistranded parallel quadruplex. In addition, the fluorescence analysis of labeled oligonucleotides may be developed as a supplementary tool for the study of DNA conformations. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 331–339, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Zippered G-quadruplex/hemin DNAzyme: exceptional catalyst for universal bioanalytical applications

G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic capacity limits the potential applications. Inspired by nature protease, we conducted a proximity-enhanced cofactor assembly strategy (PECA) to form an exceptional HRP mimic, namely zippered G4/hemin DNAzyme (Z-G4/H). The hybridization of short oligonucleotides induced proximity assembly of the DNA-grafted hemin (DGH) with the complementary G4 sequences (cG4s), mimicking the tight configuration of protease cofactor and apoenzyme. The detailed investigations of catalytic efficiency and mechanism verified the higher activity, more rapid catalytic rate and high environmental tolerance of the Z-G4/H than the classical G4/hemin DNAzymes (C-G4/H). Furthermore, a proximity recognition transducer has been developed based on the PECA for sensitive detection of gene rearrangement and imaging human epidermal growth factor receptor 2 protein (HER2) dimerization on cell surfaces. Our studies demonstrate the high efficiency of Z-G4/H and its universal application potential in clinical diagnostics and biomolecule interaction research. It also may offer significant opportunities and inspiration for the engineering of the protease-free mimic enzyme.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100 mM K⁺, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. ¹D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.     

Efficient silencing of gene expression by an ASON-bulge-DNAzyme complex

Background

DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The “10-23 DNA enzyme” is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs.

Methodology

In this study, we inserted a nonpairing bulge at the 5′ end of the “10–23 DNA enzyme” to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5′ end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme.

Conclusions

Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12–15 bp away from the core catalytic domain.     

Homogeneous time-resolved fluorescence DNA hybridization assay by DNA-mediated formation of an EDTA-Eu(III)-beta-diketonate ternary complex.

A sensitive homogenous time-resolved fluorescence DNA hybridization assay method based on the formation of an EDTA-Eu(3+)-beta-diketonate ternary complex in the DNA hybrid was developed. The new approach combined the use of two DNA probes whose sequences compose the whole complementary strand to the target DNA, in which one probe was labeled with an EDTA-Eu(3+) complex on the 5'-terminus and the other, labeled with a bidentate beta-diketone on the 3'-terminus. After hybridization of two DNA probes with target DNA, EDTA-Eu(3+) and beta-diketone come close to each other, and an EDTA-Eu(3+)-beta-diketonate ternary complex with a strong and long-lived fluorescence was formed; thus the target DNA was detected sensitively with a detection limit of 6 pM (0.6 fmol per assay) by time-resolved fluorescence measurement. In the absence of the target DNA, due to the poor stability of bidentate beta-diketonate-Eu(3+) complex in very diluted solution, only a small amount of ternary fluorescence complex was formed.     

A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification

A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs.     

Microarray gene expression analysis free of reverse transcription and dye labeling

A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250 nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2 pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments.     

Physicochemical perspectives on DNA microarray and biosensor technologies

Detection and sequence-identification of nucleic acid molecules is often performed by binding, or hybridization, of specimen "target" strands to immobilized, complementary "probe" strands. A familiar example is provided by DNA microarrays used to carry out thousands of solid-phase hybridization reactions simultaneously to determine gene expression patterns or to identify genotypes. The underlying molecular process, namely sequence-specific recognition between complementary probe and target molecules, is fairly well understood in bulk solution. However, this knowledge proves insufficient to adequately understand solid-phase hybridization. For example, equilibrium binding constants for solid-phase hybridization can differ by many orders of magnitude relative to solution values. Kinetics of probe-target binding are affected. Surface interactions, electrostatics and polymer phenomena manifest themselves in ways not experienced by hybridizing strands in bulk solution. The emerging fundamental understanding provides important insights into application of DNA microarray and biosensor technologies.     

Label-free and homogeneous DNA hybridization detection using gold nanoparticles-based chemiluiminescence system

We find that the catalytic activity of gold nanoparticles (GNPs) on luminol-H₂O₂ chemiluminescence (CL) system is greatly enhanced after it is aggregated by 0.5 M NaCl. We use this observation to design a CL detection of DNA hybridization. It is based on that the single- and double-stranded oligonucleotides have different propensities to adsorb on GNPs in colloidal solution, and the hybridization occurred between the probe DNA and target DNA can result in aggregation of the GNPs, producing strong CL emission. In the assay, no covalent functionalization of the GNPs, the probe, or the target DNA is required. The assay, including hybridization and detection, occurs in homogenous solution. The detection limit of target DNA (3σ) was estimated to be as low as 1.1 fM. The sensitivity was increased more than 6 orders of magnitude over that of GNPs-based colorimetric method. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.