Ligase-based ultrasensitive detection of DNAzyme cleavage product using molecular beacon

Detection for deoxyribozyme(DNAzyme) cleavage usually needs complex and time-consuming radial labeling,gel electrophoresis and autoradiography.A new approach was reported for detection DNAzyme cleavage product based on molecular beacon (MB).Part of the loop of MB was designed to complementary to DNAzyme cleavage product.MB was employed to monitor ligation process of RNA/DNA complex and to convert directly cleavage product information into fluorescence signal.Detection limit of the assay is 0.02 nmol/L.The cleavage product of 8 -17 DNAzyme against HCV-RNA was detected perfectly based on this assay.The method is fast,simple and ultrasensitive,which might hold great promise in DNAzyme reaction and DNAzyme gene therapy.     

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.     

Towards single-spot multianalyte molecular beacon biosensors

The separate developments of microarray patterning of DNA oligonucleotides, and of DNA hairpins as sensitive probes for oligonucleotide identification in solution, have had a tremendous impact on basic biological research and clinical applications. Herein, we will discuss several successful efforts to develop oligonucleotide sensors based on the surface immobilization of functionalized DNA hairpins. We also will discuss the development of prototypical single-spot multianalyte “Molecular Beacon” biosensors. Importantly, we show that organic fluorophores will likely be inadequate in moving this technology forward and new approaches, such as the use of nanotechnology, will be needed.     

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.     

Real-time PCR detection of telomerase activity using specific molecular beacon probes

Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg²⁺) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg²⁺, the specific coordination between Hg²⁺ and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H₂O₂ in the presence of dissolved O₂. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H₂O₂, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg²⁺ detection with the dynamic concentration range spanning from 1.0 ng L⁻¹ to 10 mg L⁻¹ Hg²⁺ and a detection limit of 0.5 ng L⁻¹ (2.5 pM) at the 3S_blank level, and it also demonstrated excellent selectivity against other interferential metal ions.     

Detection of T4 DNA ligase using a solid-state electrochemiluminescence biosensing switch based on ferrocene-labeled molecular beacon

A solid-state electrochemiluminescence (ECL) biosensing switch based on special ferrocene-labeled molecular beacon (Fc-MB) has been successfully developed for T4 DNA ligase detection. Such special switch system consisted of two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)₃²⁺-AuNPs) onto Au electrode. A molecular beacon labeled by ferrocene as the ECL intensity switch. The molecular beacon is designed with special base sequence, which could combine with its target biomolecule via the reaction of the repair and recombination of nucleic acids by DNA ligase. During the reaction, the molecular beacon opened its stem-loop, and the labeled Fc was consequently kept away from the ECL substrate. Such structural change resulted in an obvious increment in ECL intensity due to the decreased Fc quenching effect to the ECL substrate. The analysis results are sensitive and specific.     

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer–dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method. Principle of the U-MB real-time PCR method for HBV DNAdetection     

Amplified electrochemical DNA sensor using peroxidase-like DNAzyme

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H₂O₂, which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.     

Protein analysis based on molecular beacon probes and biofunctionalized nanoparticles

With the completion of the human genome-sequencing project, there has been a resulting change in the focus of studies from genomics to proteomics. By utilizing the inherent advantages of molecular beacon probes and biofunctionalized nanoparticles, a series of novel principles, methods and techniques have been exploited for bioanalytical and biomedical studies. This review mainly discusses the applications of molecular beacon probes and biofunctionalized nanoparticles-based technologies for realtime, in-situ...     

A novel sandwich assay with molecular beacon as report probe for nucleic acids detection on one-dimensional microfluidic beads array

A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of ∼0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.     

基于分子信标末端现场标记三聚氰胺-Cu~(2+)电活性分子的高灵敏电化学传感器

本文构建了一种基于分子信标自由末端现场标记电活性信号分子的新型DNA传感器.首先将3′修饰巯基的分子信标通过Au–S键自组装到金电极表面,然后在修饰有羧基的5′自由末端通过共价偶合和配位作用依次组装上三聚氰胺(Mel)和铜离子(Cu2+),得到以Mel-Cu2+配合物为电活性信号源的分子信标.该方法简单实现了电活性分子信标的标记、分离和纯化.以[Fe(CN)6]3-/4-为电化学探针,采用循环伏安和电化学阻抗法对层层自组装过程进行了表征.杂交实验表明,Mel-Cu2+信号源所对应的峰电流强度随着杂交液浓度的增大逐渐降低,且氧化峰电流与互补序列浓度对数在1.0×10-15~1.0×10-9 mol/L范围内呈良好的线性关系.根据3σ计算得到检测限为2.4×10-16 mol/L.另外,由于分子信标特殊的茎环结构特征和Mel-Cu2+信号源稳定的无机配位组成,传感器显示了很高的特异性、再生性和稳定性.     

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.     

RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.     

A label-free, quadruplex-based functional molecular beacon (LFG4-MB) for fluorescence turn-on detection of DNA and nuclease

We demonstrate a novel concept for the construction of a label-free, quadruplex-based functional molecular beacon (LFG4-MB) by using G-quadruplex motif as a substitute for Watson-Crick base pairing in the MB stem and a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM) as a reporter. It shows high sensitivity in assays for UDG activity/inhibition and detection of DNA sequence based on the unique fluorescence increase that occurs as a result of the strong interaction between NMM and the folded quadruplex upon removal of uracil by UDG or displacement of block sequence by target DNA. The LFG4-MB is simple in design, fast in operation and could be easily transposed to other biological relevant target analysis by simply changing the recognition portion. The LFG4-MB does not require any chemical modification for DNA, which offers the advantages of simplicity and cost efficiency and obviates the possible interference with the affinity and specificity of the MB as well as the kinetic behavior of the catalysts caused by the bulky fluorescent groups. More importantly, the LFG4-MB offers great extent of freedom to tune the experimental conditions for the general applicability in bioanalysis.     

Rapid determination of total hardness in water using fluorescent molecular aptamer beacon

A double-labelled synthetic oligonucleotide is used as a fluorescent molecular aptamer beacon for the reagentless determination of total hardness in tap and bottled waters. Modified thrombin binding aptamer (5′-NH-C3-GGTTGGTGTGGTTGG-C3-SH-3′) carrying 6-carboxyfluorescein (FAM) and 7-amino-4-methyl-coumarin labels at 5′ and 3′, respectively, was used for the simultaneous combined measurement of Mg²⁺ and Ca²⁺ cations. Interference from the K⁺ cation is eliminated via selective tuning of the assay conditions, increasing the temperature beyond the melting point of the potassium-stabilised quadruplex facilitating its liberation from the quadruplex, whilst maintaining the integrity of the magnesium/calcium-stabilised structure. No interference from other cations found in tap or bottled water was observed. The detection limit of the aptamer beacon is 0.04 mmol L⁻¹, with a dynamic linear range of 0-0.5 μM and is very reproducible, with an R.S.D. = 8%, n = 3. The fluorescent molecular beacon is applied to the determination of total hardness in tap and bottled waters and its’ performance compared to that of the standard method of complexiometric titration and atomic absorption spectroscopy, with an excellent correlation observed. Further work is focused on the immobilization of the aptamer for the development of a re-usable fluorescent/electrochemical aptasensor, for the determination of water hardness.     

TaqMan-分子灯标：一种新型的荧光基因检测探针

在TaqMan及分子灯标的基础上开发了一类新型的均相荧光检测探针—— TaqMan-分子灯标(TaqMan-MB)，该探针集合了分子灯标的发夹结构及TaqMan探针降 解作用的工作原理，使检测效果更好．与实时PCR仪联用，可用于靶基因的定量检 测．     

Quencher-free molecular beacon systems with two pyrene units in the stem region

We appended pyrene units covalently onto adenosine and uridine nucleosides (forming A^P and U^P units, respectively) and then incorporated them into oligonucleotides such that they were positioned in complementary locations in opposite strands in the middle positions of hairpin stems. Systems 1 (A^PU^P) and 3 (A^PA^P) individually exhibit aromatic stacking between the opposing pyrene units in the stems of their hairpins and display in their spectra the photophysical properties of strongly red-shifted bands; in contrast, the U^PU^P system 2 exhibits quenching spectra. Systems 1 (A^PU^P) and 3 (A^PA^P) behave as effective molecular beacons (MBs) that change color from green to blue upon duplex formation, whereas 2 (U^PU^P) is an effective MB that changes the intensity of its fluorescence upon forming its perfectly matched duplex.     

基于分子信标对hOGG1活性的定量分析

设计了一种含有8-oxoG碱基的新型分子信标, 结合酶促反应发展了一种非同位素标记的人8-oxoG-鸟嘌呤糖苷酶1(hOGG1)的活性分析新方法, 检出限可达0.0125 U/mL. 此外, 该方法还可用于快速考察金属离子对酶促反应的影响和肿瘤细胞中hOGG1活性水平的定量检测. 实验结果表明, 该方法简单、 灵敏, 有望用于肿瘤样品中hOGG1活性的高通量分析和hOGG1抑制剂的筛选.