Real-time monitoring of DNAzyme cleavage process using fluorescent assay

Detection of deoxyribozyme(DNAzyme) cleavage process usually needs complex and time-consuming radial labeling,gel electrophoresis and autoradiography.This paper reported an approach to detect DNAzyme cleavage process in real time using a fluorescence probe.The probe was employed as DNAzyme substrate to convert directly the cleavage information into fluorescence signal in real time.Compared with traditional approach,this non-isotope method not only brought a convenient means to monitor the DNAzyme cleavag...     

Assistant deoxyribonucleic acid recycling with Zn and molecular beacon for electrochemical detection of deoxyribonucleic acid via target-triggered assembly of mutated DNAzyme

A novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn²⁺ assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn²⁺. After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35 fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn²⁺-based amplification. The Zn²⁺ assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon(MB),we developed a versatile sensing strategy for amplified detection of adenosine.In this strategy,the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme.A short sequence,denoted as inhibitor,is designed to form a duplex spanning the aptamer–DNAzyme junction,which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine,the aptamer binds to adenosine,thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme.The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+,making the fluorophore separate from the quencher and resulting in fluorescence signal.The results showed that the detection method has a dynamic range from 10 nmol/L to 1 nmol/L,with a detection limit of 10 nmol/L.     

Adenosine Triphosphate Sensing by Electrocatalysis with DNAzyme

Electrocatalysis of redox enzymes shows wide application for biosensing. DNAzymes exhibiting specific catalytic activities have aroused great interest recently. However, there are few studies on the electrocatalysis between DNAzyme and electron mediator. In this paper, based on the electrocatalysis of methylene blue (MB) and horseradish peroxidase mimicking DNAzyme (HRP‐DNAzyme), an amplified electrochemical biosensor for the detection of adenosine triphosphate (ATP) was designed. In the present system, by means of the ATP‐aptamer interaction, two guanine‐rich DNA sequences, one of which was labeled with MB at the 5′ end, were assembled on the gold electrode. In the presence of K⁺ and hemin, the guanine‐rich DNA sequences transferred to HRP‐DNAzyme. The conformational change of the structure resulted in the approaching of MB and HRP‐DNAzyme which made the electrocatalytic process between MB and HRP‐DNAzyme possible. We used cyclic voltammetry and electrochemical impedance spectroscopy to study the electrocatalytic process. The system was therefore utilized for amplified detection of ATP without imposing any new constraints to the platform which showed satisfactory result.     

Fabrication of a Colorimetric Carcino-Embryonic Antigen Sensor Using High-Activity DNAzyme as a Catalytic Label

Detection of biomarkers in a biologically complex mixture remains a major challenge. Herein, an ultrasensitive colorimetric sandwich sensor for carcino-embryonic antigen (CEA) detection is introduced. The DNAzyme was tethered to biotinylated monoclonal antibodies (McAbs) which serve as the sensing element to recognize the target protein and was then introduced on to the CEA-McAbs assembled micro plate. The CEA was captured in a sandwich assay by the McAbs. The peroxidase-like DNAzyme catalyzed the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), which generated a blue-green colorimetric signal. This method detected CEA in a serum-containing medium at a concentration as low as 10 nM. This strategy is a promising tool for bioanalytical and clinical applications.     

Amplified fluorescence detection of mercury(II) ions (Hg2+) using target-induced DNAzyme cascade with catalytic and molecular beacons

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg(2+)) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg(2+)-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg(2+), the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction induced the two fragments to produce the activated Mg(2+)-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg(2+) sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg(2+) to the T-T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg(2+) detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg(2+) detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg(2+) from a wide range of real samples.     

Colorimetric Cu2+ detection with a ligation DNAzyme and nanoparticles

Using a Cu(2+)-dependent DNA ligation DNAzyme, a colorimetric sensor for Cu2+ has been developed based on directed assembly of DNA-functionalized gold nanoparticles by the ligation product, and such ligation DNAzyme-based sensors are intrinsically more sensitive than cleavage DNAzyme systems due to the lack of background.     

A SERS DNAzyme biosensor for lead ion detection

SERS biosensor for sensitive and selective detection of lead ions (Pb(2+)) based on DNAzyme was developed by taking advantage of the specific catalytic reaction of DNAzyme upon binding to Pb(2+) ions. Detection was accomplished by SERS nanoprobe labeled with DNA and Raman reporters for signal amplification.     

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol?L^?1 and detect ATP specifically with a detection limit as low as 13 nmol?L^?1. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.     

Near-Infrared Photothermally Activated DNAzyme–Gold Nanoshells for Imaging Metal Ions in Living Cells

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal-dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near-infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three-stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal-ion-dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn²⁺ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.     

Near‐Infrared Photothermally Activated DNAzyme–Gold Nanoshells for Imaging Metal Ions in Living Cells

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal‐dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near‐infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three‐stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal‐ion‐dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn²⁺ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.     

Combing DNAzyme with single-walled carbon nanotubes for detection of Pb(II) in water

A sensitive and simple assay for the detection of Pb(2+) in aqueous solutions is reported. It takes advantage of the high affinity between single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWCNT) as well as the capability of SWCNT in fluorescence quenching. Lead(II) catalyzes the cleavage of a fluorescently labeled DNA substrate by a DNAzyme, which releases the single-stranded product to be adsorbed onto a SWCNT. The decrease in fluorescence is proportional to the Pb(2+) concentration. Concentrations as low as 1 nM Pb(2+) in water could be detected and the detection range spans over 5 orders of magnitude. The unique combination of Pb-specific DNAzyme with SWCNT produces a universal, facile and cost-effective sensing platform for lead ions. The concept can be applied to the design of detection assays for other metal ions or small molecules.     

A methylation-stimulated DNA machine: an autonomous isothermal route to methyltransferase activity and inhibition analysis

The operation of DNA nanomachines is generally triggered by either conformational changes of DNA nanostructure or external environmental stimuli. In the present study, we demonstrate an alternative driving force, DNA methylation, to stimulate DNA machine operation. DNA methylation changes neither DNA sequence and conformation nor external environment, however, blocks its cleavage by corresponding methylation-sensitive restriction endonuclease. We thus designed a strand displacement amplification DNA machine, which could be stimulated upon DNA methylation and then autonomously generates accumulated amounts of peroxidase-mimicking DNAzyme signaling machine products in an isothermal manner. The machine product DNAzyme could catalyze the H₂O₂-mediated oxidation of 2,2′-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS²⁻) to a colored product ABTS^·−. This methylation-stimulated DNA machine was further used as a colorimetric assay for analysis of methyltransferases activities and screening of methylation inhibitors. As compared with classical methylation assay, this facile isothermal DNA machine avoids the introduction of methylation-specific polymerase chain reaction and radioactive labels, which might be employed as an effective tool for DNA methylation analysis.     

A novel strategy of chemical modification for rate enhancement of 10-23 DNAzyme: a combination of A9 position and 8-aza-7-deaza-2'-deoxyadenosine analogs

With the help of a divalent-metal ion, 10-23 DNAzyme cleaves RNA. Chemical modification of its catalytic loop to make a more efficient enzyme has been a challenge. Our strategy started from its five 2'-deoxyadenosine residues (A5, A9, A11, A12, and A15) in the loop based on the capability of the N7 atom to form hydrogen bonds in tertiary structures. 8-Aza-7-deaza-2'-deoxyadenosine and its analogs with 7-substituents (3-aminopropyl, 3-hydroxylpropyl, or phenethyl) were each used to replace five dA residues, respectively, and their effect on cleavage rate were evaluated under single-turnover conditions. The results indicated that the N7 atom of five dA residues were necessary for catalytic activity, and the N8 atom and 7-substituents were detrimental to the catalytic behavior of 10-23 DNAzyme, except that all these modifications at A9 were favourable for the activity. Especially, DZ-3-9 with 7-(3-aminopropyl)-8-aza-7-deaza-2'-deoxyadenosine (3) at A9 position gave a 12- fold increase of k(obs), compared to the corresponding parent 10-23 DNAzyme. DZ-3-9 was supposed to catalyze the cleavage reaction with the same mechanism as 10-23 DNAzyme based on their very similar pH-dependent and divalent metal ions-dependent cleavage patterns. Introduction of functional groups at A9 position was demonstrated to be a successful and feasible approach for more efficient 10-23 DNAzyme analogs.     

Zn(2+)-ligation DNAzyme-driven enzymatic and nonenzymatic cascades for the amplified detection of DNA

A generic fluorescence sensing platform for analyzing DNA by the Zn(2+)-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the target DNA induced ligation of two substrate subunits and the subsequent opening of a beacon hairpin probe by the ligated product. The strand displacement of the ligated product by the beacon hairpin is, however, of limited efficiency. Two strategies are implemented to overcome this limitation. By one method, a "helper" nucleic acid sequence is introduced into the system, and this hybridizes with the DNAzyme components and releases the ligated product for opening of the hairpin. By the second method, a nicking enzyme (Nt.BspQI) is added to the system, and this nicks the duplex between the beacon and ligated product while recycling the free ligation product. By combining the two coadded components ("helper" sequence and nicking enzyme), the sensitive detection of the analyte is demonstrated (detection limit, 20 pM). The enzyme-free amplified fluorescence detection of the target DNA is further presented by the Zn(2+)-dependent ligation DNAzyme-driven activation of the Mg(2+)-dependent DNAzyme. According to this method, the Mg(2+)-dependent DNAzyme subunits displace the ligated product, and the resulting assembled DNAzyme cleaves a fluorophore/quencher-modified substrate to yield fluorescence. The method enabled the detection of the target DNA with a detection limit corresponding to 10 pM. The different sensing platforms are implemented to detect the Tay-Sachs genetic disorder mutant.     

基于单壁碳纳米管-DNA酶复合结构的电化学生物传感器

结合DNA酶优异的氧化还原催化特性和碳纳米管的电化学特性, 制备了单壁碳纳米管-DNA酶复合材料, 并通过壳聚糖将其固定到玻碳电极表面构建了电化学生物传感界面. 研究了单壁碳纳米管-DNA酶复合结构的氧化还原反应催化特性, 并以此为传感平台构建了葡萄糖氧化酶电化学生物传感器. 结果表明, 单壁碳纳米管-DNA酶复合材料修饰的电极对过氧化氢的响应具有较宽的线性范围(5×10^-6~1×10^-2 mol/L)和良好的检测灵敏度(检出限为1×10^-6 mol/L). 采用制备的葡萄糖氧化酶传感器实现了对葡萄糖的快速灵敏检测.     

Quadruple signal amplification strategy based on hybridization chain reaction and an immunoelectrode modified with graphene sheets,a hemin/G-quadruplex DNAzyme concatamer,and alcohol dehydrogenase: ultrasensitive determination of influenza virus subtype H7N9

We report on a new amplification strategy for use in an immunoassay for influenza virus subtype H7N9. Graphene sheets were first placed on a glassy carbon electrode (GCE), and gold nanoparticles were then electrodeposited as a support for a layer of alcohol dehydrogenase (ADH) in a sol–gel containing thiol groups. Protein A was used to properly orientate immobilized antibody against H7N9 on the sol–gel, and this is shown to result in strongly improved specificity of the antigen-antibody binding. Thus, a sensitive and specific immunosensor was obtained in which a quadruple signal amplification strategy is employed, viz. (a) via the use of graphene sheets, (b) via a hybridization chain reaction, (c) the use of hemin/G-quadruplex DNAzyme concatamers, and (d) the use of ADH. The hemin/G-quadruplex is a typical DNAzyme, which simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme. The hybridization chain reaction-based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system. Sandwich immunoreactions occurred between the capture antibody on the electrode and the secondary antibody labeled with MWCNTs. Positively charged Methylene Blue (MB) was then used as an intercalator to detect the DNAzyme concatamer formed. The differential pulse voltammetric signals for MB are related to the concentration of H7N9 in the range from 8 to 60 pg · mL⁻¹, and the detection limit is 0.81 pg · mL⁻¹ (at an S/N ratio of 3). This immunoassay is very sensitive, specific and robust.

An electrochemical sandwich immunosensor has been developed for sensitive and specific detection of influenza virus subtype H7N9. Protein A was used to properly orientate antibody. The hybridization chain reaction based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system.

     

LNAzymes: incorporation of LNA-type monomers into DNAzymes markedly increases RNA cleavage

Incorporation of two alpha-L-LNA/LNA nucleotides into each of the two binding arms of a "10-23" DNAzyme has been accomplished and the RNA cleavage with these novel LNAzymes studied. In comparison with the unmodified DNAzyme, the LNAzymes show significantly improved cleavage of the phosphodiester backbone at the target nucleotide in a small RNA substrate (58n RNA) under single-turnover conditions. The LNAzymes show efficient multiple turnover. With the LNAzymes, efficient cleavage was accomplished also of a naturally occurring ribosomal RNA at a target site within a highly structured region. The reference DNAzyme was ineffective at cleaving the ribosomal RNA target.     

A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzyme

In this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H₂O₂-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV–visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from 0.5 to 20 nM and the detection limit of thrombin was 0.5 nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins.