Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase

A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL⁻¹. This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.     

水溶性碳纳米粒子的制备及其在DNA和单核苷酸多态性分析中的应用

利用电化学氧化的方法制备了水溶性好、粒径为7～12nm的碳纳米粒子,该碳纳米粒子通过π-π相互作用吸附荧光标记的单链DNA探针,并能有效地猝灭其荧光．当单链DNA探针与匹配的DNA目标分子杂交形成双链DNA时,猝灭的荧光被恢复,由此可以检测1-200nmol／L的DNA目标分子。此外,在碳纳米粒子存在时,由荧光标记的DNA探针和DNA目标分子形成的双链DNA的熔解温度可以简便地被测定,当双链DNA有错配碱基时,其熔解温度降低,由此可方便、快速地分析单核苷酸多态性．     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

灿烂甲酚蓝在DNA修饰金电极上的电化学行为

利用自组装技术将巯基乙醇固定在金电极表面形成巯基乙醇自组装膜修饰金电极, 用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为偶联试剂, 分别将鲱鱼精单链DNA(ssDNA)和双链DNA(dsDNA)固定于金电极表面形成ssDNA和dsDNA 修饰电极. 考察了灿烂甲酚蓝(BCB)在不同DNA 修饰电极上的电化学行为,结果表明, BCB 在ssDNA 和dsDNA 修饰电极上的吸附常数分别为1.67×10^4和3.22×10^4 L·mol-1, BCB 与ssDNA 主要以静电作用结合, 而与dsDNA作用存在静电和嵌插两种模式. dsDNA 对BCB 具有更高的亲和力, 使BCB 可以作为一种有效的电化学杂交指示剂.     

Hairpin Probe for Sequence-specific Recognition of Double-stranded DNA on Simian Virus 40

Simian virus 40(SV40) is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine·homopurine duplex DNA(dsDNA) of SV40(4424—4440, gp6) was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5'-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3'(HP) containing G-rich sequence and 17-bp triplex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structure to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.     

Orthogonal Protein Assembly on DNA Nanostructures Using Relaxases

DNA‐binding proteins are promising reagents for the sequence‐specific modification of DNA‐based nanostructures. Here, we investigate the utility of a series of relaxase proteins—TrwC, TraI, and MobA—for nanofunctionalization. Relaxases are involved in the conjugative transfer of plasmids between bacteria, and bind to their DNA target sites via a covalent phosphotyrosine linkage. We study the binding of the relaxases to two standard DNA origami structures—rodlike six‐helix bundles and flat rectangular origami sheets. We find highly orthogonal binding of the proteins with binding yields of 40–50 % per binding site, which is comparable to other functionalization methods. The yields differ for the two origami structures and also depend on the position of the binding sites. Due to their specificity for a single‐stranded DNA target, their orthogonality, and their binding properties, relaxases are a uniquely useful addition to the toolbox available for the modification of DNA nanostructures with proteins.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

Label-free and sequence-specific DNA detection down to a picomolar level with carbon nanotubes as support for probe DNA

This study describes a simple and label-free electrochemical impedance spectroscopic (EIS) method for sequence-specific detection of DNA by using single-walled carbon nanotubes (SWNTs) as the support for probe DNA. SWNTs are confined onto gold electrodes with mixed self-assembly monolayers of thioethanol and cysteamine. Single-stranded DNA (ssDNA) probe is anchored onto the SWNT support through covalent binding between carboxyl groups at the nanotubes and amino groups at 5′ ends of ssDNA. Hybridization of target DNA with the anchored probe DNA greatly increases the interfacial electron-transfer resistance (R_et) at the double-stranded DNA (dsDNA)-modified electrodes for the redox couple of Fe(CN)₆^3−/4−, which could be used for label-free and sequence-specific DNA detection. EIS results demonstrate that the utilization of SWNTs as the support for probe DNA substantially increases the surface loading of probe DNA onto electrode surface and thus remarkably lowers the detection limit for target DNA. Under the conditions employed here, R_et is linear with the concentration of target DNA within a concentration range from 1 to 10 pM with a detection limit down to 0.8 pM (S/N = 3). This study may offer a novel and label-free electrochemical approach to sensitive sequence-specific DNA detection.     

Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping

We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.     

萘普生和酵母DNA相互作用的荧光光谱研究

使用紫外和荧光光谱法研究了萘普生和酵母DNA之间的相互作用。酵母DNA对萘普生的荧光存在强烈的猝灭作用，其作用方式随DNA浓度的变化而发生转变。用Stern-Volmer方程与Scatchard方程两种方法得到相同结果：在较低的DNA浓度下，萘普生与DNA间的作用较弱，而在较高DNA浓度时，萘普生与DNA的作用较强，键合位点数也随着酵母DNA浓度的升高而在临界酵母DNA浓度100 mmol/L附近出现转变。紫外光谱、离子强度的影响和I^-猝灭等研究表明，DNA浓度的变化并不改变两者间的作用方式，它们之间始终是一种沟槽作用模式。     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.     

A Competitor-switched Electrochemical Sensor for Detection of DNA

A competitor‐switched electrochemical sensor based on a generic displacement strategy was designed for DNA detection. In this strategy, an unmodified single‐stranded DNA (cDNA) completely complementary to the target DNA served as the molecular recognition element, while a hairpin DNA (hDNA) labeled with a ferrocene (Fc) and a thiol group at its terminals served as both the competitor element and the probe. This electrochemical sensor was fabricated by self‐assembling a dsDNA onto a gold electrode surface. The dsDNA was pre‐formed through the hybridization of Fc‐labeled hDNA and cDNA with their part complementary sequences. Initially, the labeled ferrocene in the dsDNA was far from surface of the electrode, the electrochemical sensor exhibited a "switch‐off" mode due to unfavorable electron transfer of Fc label. However, in the presence of target DNA, cDNA was released from hDNA by target DNA, the hairpin‐open hDNA restored its original hairpin structure and the ferrocene approached onto the electrode surface, thus the electrochemical sensor exhibited a "switch‐on" mode accompanying with a change in the current response. The experimental results showed that as low as 4.4×10⁻¹⁰ mol/L target DNA could be distinguishingly detected, and this method had obvious advantages such as facile operation, low cost and reagentless procedure.     

Paranemic cohesion of topologically-closed DNA molecules

Specific cohesion of DNA molecules is key to the success of work in biotechnology, DNA nanotechnology and DNA-based computation. The most common form of intermolecular cohesion between double helices is by sticky ends, but sticky ends generated by naturally occurring restriction enzymes may often be too short to bind large constructs together. An alternative form of binding is available through the paranemic crossover (PX) motif. Each of the two components of a PX motif can be a DNA dumbbell or other topologically closed species. Alternate half-turns of the dumbbell are paired intramolecularly. The intervening half-turns are paired with those of the opposite component. We demonstrate the efficacy of PX cohesion by showing that it can result in the 1:1 binding of two triangle motifs, each containing nearly 500 nucleotides. The cohesion goes to completion, demonstrating an alternative to binding nucleic acid molecules through sticky ends.     

Protein p53 Binding to Cisplatin‐modified DNA Targets Evaluated by Modification‐specific Electrochemical Immunoprecipitation Assay

Studies of protein interactions with chemically modified nucleic acids are of importance in various areas of biomolecular and biomedical research, including investigations of the binding of proteins important in medicine with DNA modified with drugs and diagnostic applications of modified DNAs in biosensing and bioanalysis. Chemical modification of DNA substrates with various species inside or outside specific protein binding sites can affect the protein‐DNA recognition. In this paper we present a simple electrochemical immunoprecipitation technique designed for evaluation of the effects of antitumor drug cisplatin on the p53‐DNA binding. The cisplatin‐DNA adducts are utilized as electroactive labels allowing a facile determination of the p53‐bound modified DNA. Effects observed using this technique accord with results of previous biochemical assays. This approach is potentially applicable in studies that deal with the influence of any electroactive DNA modifications on the protein‐DNA binding.     

Capture of cationic ligands bound diffusely to base pairs during DNA refolding

We obtained the intrinsic binding affinity for metal ions, polyamines, and oligolysine peptides diffusely bound to base-paired sites in DNA by monitoring the shift of the hairpin-duplex equilibrium of the self-complementary DNA sequences, which can be widely used for capturing cationic ligands bound diffusely to nucleotide base pairs.     

Site-specific discrimination of Cytosine and 5-methylcytosine in duplex DNA by Peptide nucleic acids

For site-specific discrimination of cytosine (C) and 5-methylcytosine (mC) in duplex DNA, we developed a new method using peptide nucleic acids (PNAs). The combination of a PNA-assisted DNA displacement complex and a fluorescein-labeled probe oligomer allowed the detection of mC at the defined sites in target DNA using a restriction enzyme. After treatment of the complex with a restriction enzyme, strong fluorescence emission was observed for the complex containing C at the target site, whereas the fluorescence intensity for the complex containing mC was extremely weak.     

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiements showed that the elongation temperature of 50 °C and the Mg²⁺ concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.     

Stability analysis for double-stranded DNA oligomers and their noncovalent complexes with drugs by laser spray

Laser spray, which is a newly developed ionization technique, can characterize the stability of noncovalent complexes in the solution phase. By using this advantage, laser spray has been applied to probe the intrinsic stability of double-stranded DNA (dsDNA) sequences and their binding affinities with various drugs in the solution phase. Systematic experiments were carried out using six 16-mer and three 22-mer dsDNA oligomers, together with the complexes of the 16-mer dsDNA with minor groove binders: berenil, Hoechst 33342, DAPI, and netropsin. Dissociation curves for each dsDNA or each complex were plotted as a function of laser power. The laser power (E50%), where 50% of each dsDNA or each complex was dissociated, was compared with its melting temperature (Tm) determined by UV spectroscopy. Linear correlations between E50% and Tm were obtained not only for the dsDNA oligomers (correlation factor r = 0.9835) but also for the 16-mer dsDNA complexes with minor groove binders (r = 0.9966). In addition, laser spray has successfully clarified the binding affinities of a 16-mer dsDNA with two intercalators: daunomycin and nogalamycin. In the case of the dsDNA-daunomycin complex, by changing the molar ratio of dsDNA : drug from 1 : 1 to 1 : 5, the concentration-dependent stability of the complex was confirmed by laser spray. The present results demonstrate that laser spray mass spectrometry can be a powerful and convenient method to investigate the relative binding affinities of dsDNA-ligand complexes in the solution phase, which could be applied to the early stage of high-throughput screening of drugs targeting for dsDNA.     

Fluorescent Silver Nanoclusters in Condensed DNA

We study the formation and fluorescent properties of silver nanoclusters encapsulated in condensed DNA nanoparticles. Fluorescent globular DNA nanoparticles are formed using a dsDNA–cluster complex and polyallylamine as condensing agents. The fluorescence emission spectrum of single DNA nanoparticles is obtained using tip‐enhanced fluorescence microscopy. Fluorescent clusters in condensed DNA nanoparticles appear to be more protected against destructive damage in solution compared to clusters synthesized on a linear polymer chain. The fluorescent clusters on both dsDNA and ssDNA exhibit the same emission bands (at 590 and 680 nm) and the same formation efficiency, which suggests the same binding sites. By using density functional theory, we show that the clusters may bind to the Watson–Crick guanine–cytosine base pairs and to single DNA bases with about the same affinity.