One Mismatch Detection of DNA Hybridization Using Fluorescence Polarization

Abstract

The potential of fluorescent polarization analysis as a method for detection of mismatch DNA hybridization was investigated. The dependency of DNA hybridization rate on salt concentration was surveyed. In greater than 0.1 M NaCl, the hybridization of probe and target DNA proceeds rapidly and the reaction is complete within 3 min. Furthermore, the hybridization of probe DNA and one mismatch target DNAs was investigated. It was successfully shown that even one mismatch could be detected using fluorescence polarization analysis if the mismatch position was on the base that pairs with the probe DNA at the 5′ terminus where fluorescein isothiocyanate (FITC) is attached.     

Synthesis,characterization, and DNA binding of two copper(II) complexes as DNA fluorescent probes

[Cu(DAPT)₂Cl]Cl·H₂O and [Cu(DBM)(DAPT)Cl] [DAPT = 2,4-diamine-6-(pyrazin-2-yl)-1,3,5-triazine] were synthesized and characterized by IR and UV spectroscopy, elemental analysis, TG–DTA, molar conductivity, and LC–MS. The interaction with calf thymus DNA (ct-DNA) of the two complexes has been studied using UV spectra, fluorescent spectra, cyclic voltammetry, and viscosity measurements. The complexes interact with ct-DNA through classical intercalation. Fluorescence intensity changes of 1 and 2 in the absence and presence of ct-DNA have been investigated for quantitative determination of ct-DNA with the limit of detection of 3.8 and 7.7 ng mL^?1, respectively. From the result, the two complexes are potentially sensitive DNA fluorescent probes.     

Synthesis,crystal structure,DNA binding,and DNA cleavage of a zinc complex containing N,N-bis(2-pyridylmethyl)amine

A water-soluble zinc complex, [Zn(bpea)Cl₂] (1) (bpea?=?N,N-bis(2-pyridylmethyl)ethylamine), was prepared to serve as a nuclease mimic. The complex was characterized by X-ray, infrared, and UV spectroscopy. Interactions of the complex with calf thymus-DNA (ct-DNA) have been investigated by UV absorption and fluorescence spectroscopies; the mode of ct-DNA binding for 1 has been proposed. DNA cleavage activities by 1 were performed in the absence of external agents. The influences of different complex concentrations or reaction times on DNA cleavage were studied.     

Electrochemical DNA Probes

Abstract

A report on the status of the electrochemical DNA probes for the detection of a DNA sequence for environmental and clinical studies is presented. The literature on the electrochemistry of DNA in the last ten years is reviewed. Results obtained in this laboratory using an electroactive hybridization indicator for constructing an electrochemical DNA probe are reported.     

Synthesis,characterization, DNA binding and cleavage activity of homoleptic zinc(II) β-oxodithioester chelate complexes

New homoleptic zinc(II) complexes, [Zn(L)₂], where L = methyl-3-hydroxy-(3-pyridyl)-2-propenedithioate L1 1, and methyl-3-hydroxy-(4-pyridyl)-2-propenedithioate L2 2, have been synthesized and characterized by elemental (C, H, and N) analysis, ESI-MS, and (IR, UV–vis, NMR) spectroscopy; the structure of 1 has been deduced by X-ray crystallography. The DNA binding and cleavage activity of the complexes have been studied. The cleavage potential of pBR322 DNA by 1 and 2 has been checked. Complex 1, which contains nitrogen of the pyridine group in the 3-position enhances DNA cleavage potential in the presence of ascorbic acid; however, the complex is protective against DNA cleavage in the presence of DMSO or H₂O₂. Also, 1 causes cytotoxicity against the MCF-7 breast cancer cell line. The efficient cytotoxic activity and DNA cleavage ability of 1 in the presence of ascorbic acid shows its potential anticancer properties and the need for further investigations of its potential as an anticancer drug.     

A copper(II) complex of 1,4,7-triazacyclononane featuring acetate pendant: an efficient DNA cleavage agent

A new water-soluble copper(II) complex, Cu(TACNA)Br?·?0.375H₂O (1) [TACNA?=?1,4,7-triazacyclononane-N-acetate], has been synthesized to serve as artificial nucleases. The X-ray crystal structure of 1 indicates that one bromide and an oxygen from acetate pendant coordinate to copper(II) in addition to the nitrogen atoms in the TACN macrocycle, resulting in a five-coordinate complex with square-pyramidal geometry. The interaction of 1 with calf thymus DNA (ct-DNA) has been investigated by UV absorption and fluorescence spectroscopies, and the mode of ct-DNA binding for 1 has been proposed. In the absence of external agents, supercoiled plasmid DNA cleavage by 1 was performed under aerobic condition; the influences on DNA cleavage of different complex concentrations and reaction times were also studied. The cleavage of plasmid DNA likely involves oxidative mechanism.     

Oxidative DNA cleavage by Cu(II) complexes of 1,10-phenanthroline-5,6-dione

A dimeric dichloro-bridged copper(II) complex [Cu₂(pdon)₂Cl₄] · 2DMF (1) and two mononuclear copper(II) complexes [Cu(pdon)(DMSO)Cl₂] · DMSO · H₂O (2) and [Cu(pdon)₃] · (ClO₄)₂ · 2.25CH₃CN · 6H₂O (3) (pdon = 1,10-phenanthroline-5,6-dione) have been synthesized and characterized. Variable-temperature magnetic susceptibility studies indicate the existence of weak anti-ferromagnetic coupling in the binuclear complex. The interaction of these complexes with CT-DNA (calf thymus DNA) has been studied using absorption and emission spectral methods. The apparent binding constants (K _app) for 1, 2 and 3 are 5.20 × 10⁵, 2.68 × 10⁵ and 7.05 × 10⁵ M^?1, respectively, showing moderate intercalative binding modes. All of these complexes cleave plasmid DNA to nicked DNA in a sequential manner as the concentration or reaction time is increased. The cleavage mechanism between the complex and plasmid DNA is likely to involve singlet oxygen ¹O₂ and ?OH as reactive oxygen species.     

Synthesis,structure, DNA binding and cleavage ability of a new copper ciprofloxacin complex

The structure of 1 consists of [Cu(HCp)(phen)(H₂O)]²⁺ (HCp is ciprofloxacin and phen is 1,10-phenanthroline), two acetates, and four free water molecules. In each cation, copper displays a distorted square pyramid, coordinated to ring 3-carboxylate and 4-oxo oxygen from HCp, two nitrogens from phen, and one water molecule. There are five water molecules in each discrete complex with one coordinated to Cu center, and the other four linked to each other by intermolecular hydrogen bonds. Two uncoordinated acetates make the compound neutral. The complex exhibits higher DNA binding compared to HCp at the same conditions by fluorescence and viscosity measurements. Combining its structure with the DNA-binding result, the binding mechanism may be explained by intercalation. Moreover, 1 shows significant cleavage of DNA in the presence of a reducing agent, such as ascorbate by gel electrophoresis using supercoiled pBR322 DNA in Tris-HCl buffer (pH 7.4). The complex also has a higher activity against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Klebsiella pneumoniae than HCp.     

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.     

异核双金属探针在活细胞细胞核和线粒体中选择性定位DNA

用于实时成像活细胞DNA的细胞核探针是非常罕见的。通过Ru(N^N)₃、Ir(C^N)₂(N^N)、Re(CO)₃Cl(N^N)和Pt(C^N)(N^N)的结合,生成了4种异核双金属配合物：2种不同的功能金属位点被锚定在线性双齿螯和配体(N^N)-(N^N)的尾端,形成不对称哑铃状的双金属分子M₁-M₂,即Ru-Re、Ru-Pt、Ir-Re和Ir-Pt。Ru-Re和Ru-Pt的红色发射探针具有较大的斯托克斯位移、优异的核膜穿透能力、良好光稳定性的DNA结合能力。此外,Ru-Re和Ru-Pt探针的DNA成像可与专门用于活细胞细胞核DNA染色的Hoechst 33342商用染料相媲美,而Ir-Re和Ir-Pt探针直接靶向线粒体。不同探针的发光特性和选择性胞内定位高度依赖于在异核双金属配合物中的金属物种。     

Microarray Detection of Fetal DNA Levels in Maternal Plasma

Abstract

The discovery of fetal DNA in maternal plasma has made non‐invasive prenatal diagnosis possible. Microarrays are promising tools for detecting fetal DNA for such purposes. We report the development of a microarray based quantitative detection method and the investigation of fetal DNA levels at different gestation ages and in abnormal pregnancies. Samples from 66 male carriers at different gestation stages and 6 male carriers from abnormal pregnancies were collected and DNA microarrays were used to measure the level of fetal DNA in maternal plasma in these samples. The male‐specific DYS gene was used as the male fetus marker. Results showed that the fetal DNA levels in maternal plasma increased with the gestation age. The level of fetal DNA in Down's syndrome pregnancy samples was higher than in control samples, while no differences were found between gestational hypertension samples and the control.     

Synthesis,crystal structure and DNA interaction studies of three coordination polymers with mixed ligand

Three coordination polymers [Mn(Nip)(Pbim)] _n (1), [Co(Nip)(Pbim)] _n (2) and [Zn(Nip)(Pbim)] _n (3) [5-nitroisophthalate (Nip) and 2-(2-pyridyl)benzimidazole (Pbim)] were synthesised and characterised by elemental analysis, IR and single-crystal X-ray diffraction. Single-crystal X-ray diffraction analysis reveals that 1, 2 and 3 have 1-D ladder chain structures constructed from μ₃-bridge Nip ligands and metal atoms. All of these chain-like structures are finally packed into supramolecular networks through hydrogen bonds and π–π stacking interactions. Fluorescence spectral method has been used for the study on the interaction of film sperm DNA with complexes. The results show that the corresponding fluorescence spectrum appeared and the intensity was enhanced with the growth of the concentration of DNA. All of the results indicate that there exists strong interaction of the complexes with DNA.     

Synthesis,characterization and DNA nuclease activity of oxo-peroxomolybdenum(VI) complexes

The synthesis and structural characterization of two oxo-peroxo molybdenum(VI) complexes, [Mo(O)(O)₂(PAA)]^? (1) and [Mo(O)(O)₂(PAH)]^? (2), with phenylacetic acid (PAA) and 2-phenylacetylhydroxamic acid (PAHH) ligands have been accomplished. The coordination geometry of the oxo-peroxo molybdenum(VI) complexes is found to be pentagonal bipyramidal where, in both cases, the ligands are coordinated in bidentate fashion through oxygen atoms. The binding affinities of 1 and 2 with calf-thymus DNA (CT DNA) are determined using absorption spectroscopic measurements. The spectroscopic as well as cyclic voltammetric (CV) studies and viscosity measurements indicate that both complexes interact with CT DNA in the groove. The intrinsic binding constants are 5.2 × 10⁴ M^?1 and 7.3 × 10⁴ M^?1 for 1 and 2, respectively, from UV–vis studies. Complexes 1 and 2 show nuclease activity with plasmid DNA in the presence of H₂O₂. Concentration-dependent nuclease study suggests that 2 possesses higher ability to cleave plasmid DNA compared to 1. The experimental results of the binding of 1 and 2 with DNA are further supported by molecular docking studies.     

Hydrolysis of DNA by N-Phosphoryl Branched Peptide

A N-phosphoryl branched peptide (DIPP-Leu) ₂ -Lys-OCH ₃ was found to efficiently cleave double-strand DNA such as PUC19 and Lambda DNA at pH 8.5 in 100 mM Tris-HCl buffer. The T4 ligase experiment implied that the DNA cleavage occurs via a hydrolytic path.     

Label-Free DNA Biosensor for Electrochemical Detection of Short DNA Sequences Related to Human Papilloma Virus

Abstract

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of an electrochemical hybridization biosensor for the detection of short DNA fragments specific to the human papilloma virus (HPV). Unlabeled DNA probes have been immobilized by spontaneous coadsorption of thiolated single-stranded oligonucleotides (HS-ssDNA) onto the sensing surface of a screen-printed gold electrode (SPGE). The covalently immobilized single-stranded DNA probe (HS-ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. DNA is treated with acid (e.g., 0.5 M chloridric acid), and the acid-released purine bases are directly determined by square wave voltammetry (SWV).

Variables of the probe-immobilization and hybridization steps are optimized to offer convenient quantitation of HPV DNA target, in connection with a short hybridization time. Peak currents were found to increase in the following order: hybrid-modified SPGE, 11-base mismatched modified SPGE, 18-base mismatched SPGE, and the probe modified SPGE. Control experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. The effect of the target DNA concentration on the hybridization signal was also studied. Under optimal conditions, this sensor has a good calibration range with HPV DNA sequence detection limit of 2 pg · ml^?1 (S/N = 3).     

Trends and perspectives in DNA biosensors as diagnostic devices

Despite the civilization and technological development, taking care of health based on early diagnostics is still challenging. Currently, cancer accounts for more than 20% of all deaths. Cancer mortality dramatically rises every year because of poor diagnosis at the late stage and inefficiency of conventional methods for early-stage cancer detection. That is why there is a demand for automated, inexpensive, miniaturized, and portable testing devices with real-time response, high sensitivity, and selectivity for early medical diagnostics but also for screening air and water. DNA biosensors have excellent predispositions and are a significant promise to become a powerful tool used in prevention and monitoring of diseases, rationalization of the way of medical treatment, and improving the patient quality of life.     

Electrochemical DNA sensing based on gold nanoparticle amplification

A hybridization signal-amplified method based on a gold nanoparticle-supported DNA sequence for electrochemical DNA sensing has been investigated by cyclic voltammetry, differential-pulse voltammetry, and atomic-force microscopy (AFM). Quantitative analysis showed that the peak current increment (Ip) is linearly dependant on the concentration of the gold nanoparticle-supported DNA sequence Au2 over the range 0.51–8.58 pmol L^–1. AFM results indicated that the extent of surface hybridization was dependent on the concentration of the gold-nanoparticle-supported DNA sequence. Moreover, a new pair of peaks, which might arise from the special configuration of the gold-nanoparticle-supported DNA sequence, appeared in the cyclic voltammogram after hybridization. Although quite sensitive, this DNA sensing surface was not easily regenerated, so this kind of amplified method was suitable for disposable DNA sensors and chip-based gene diagnosis sensors.     

DNA cleavage activities of two dinuclear copper coordination polymers

The DNA cleavage activities of two coordination polymers of Robson-type macrocycles, {[Cu₄L¹(4,4′-bipy)₂](ClO₄)₄·H₂O}_∞ (1) and {[Cu₄L²(4,4′-bipy)₄](ClO₄)₄·2CH₃CN·2H₂O}_∞ (2) (where H₂L¹ and H₂L² are the [2 + 2] condensation products of 1,3-diaminopropane with 2,6-diformyl-4-methylphenol and 2,6-diformyl-4-fluorophenol, respectively), have been studied. The interactions of the complexes with calf thymus-DNA were investigated by UV–vis spectroscopy, CD spectroscopy, and gel electrophoresis. The binding constants of 1 and 2 are 7.2 × 10⁴ and 2.1 × 10⁵ M^?1, respectively. The complexes exhibit DNA cleavage activity, with the cleavage process involving oxidative cleavage of DNA.     

Ternary complexes of cobalt cysteinylglycine with histidylserine and histidylphenylalanine-stabilities and DNA cleavage properties

Interaction of cobalt cysteinylglycine with histidylserine and histidylphenylalanine was investigated in a 1 : 1 : 1 ratio at 35°C and 0·10 mol dm⁻³ ionic strength. Their stabilities and geometries were determined. Their DNA binding and cleavage properties were investigated. The intrinsic binding constants (K _b ) for DNA bound 1 and 2 (3·03 × 10³ M⁻¹ for 1 and 3·87 × 10³ M⁻¹ for 2) were determined. Even though the negative charge on the complexes reduced their affinity for DNA, there was an enhancement of binding through specificity. The degradation of plasmid DNA was achieved by cobalt dipeptide complexes [Co^II(CysGly)(HisSer)] (1) and [Co^II(CysGly)(HisPhe)] (2). Cleavage experiments revealed that 1 and 2 cleave supercoiled DNA (form I) to nicked circular (form II) through hydrolytic pathway at physiological pH. The DNA hydrolytic cleavage rate constants for complexes 1 and 2 were determined to be 0·62 h⁻¹, for 1 and 0·38 h⁻¹ for 2 respectively.     

Electrochemical DNA biosensor for the detection of short DNA species of Chronic Myelogenous Leukemia by using methylene blue

A novel assay for the voltammetric detection of 18-bases DNA sequences relating to Chronic Myelogenous Leukemia (CML, Type b3a2) using methylene blue (MB) as the hybridization indicator was reported. DNA was covalently attached onto a glassy carbon electrode (GCE) through amines of the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N′-ethyl carbodiimidehydrochloride (EDC). The covalently immobilized single-stranded DNA (ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. A significant increase of the peak current for methylene blue upon the hybridization of immobilized ssDNA with cDNA in the solution was observed. This peak current change was used to monitor the recognition of CML DNA sequence. This electrochemical approach is sequence specific as indicated by the control experiments in which no peak current change was observed if a non-complementary DNA sequence was used. Factors, such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated. Under optimal conditions, this sensor has a good calibration range between 1.25 × 10⁻⁷ and 6.75 × 10⁻⁷ M, with CML DNA sequence detection limit of 5.9 × 10⁻⁸ M.