Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.     

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex.

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.     

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.     

Accumulation of M1dG DNA adducts after chronic exposure to PCBs, but not from acute exposure to polychlorinated aromatic hydrocarbons

Oxidative DNA damage is one of the key events thought to be involved in mutation and cancer. The present study examined the accumulation of M1dG, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, DNA adducts after single dose or 1-year exposure to polyhalogenated aromatic hydrocarbons (PHAH) in order to evaluate the potential role of oxidative DNA damage in PHAH toxicity and carcinogenicity. The effect of PHAH exposure on the number of M1dG adducts was explored initially in female mice exposed to a single dose of either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or a PHAH mixture. This study demonstrated that a single exposure to PHAH had no significant effect on the number of M1dG adducts compared to the corn oil control group. The role of M1dG adducts in polychlorinated biphenyl (PCB)-induced toxicity and carcinogenicity was further investigated in rats exposed for a year to PCB 153, PCB 126, or a mixture of the two. PCB 153, at doses up to 3000 microg/kg/day, had no significant effect on the number of M1dG adducts in liver and brain tissues from the exposed rats compared to controls. However, 1000 ng/kg/day of PCB 126 resulted in M1dG adduct accumulation in the liver. More importantly, coadministration of equal proportions of PCB 153 and PCB 126 resulted in dose-dependent increases in M1dG adduct accumulation in the liver from 300 to 1000 ng/kg/day of PCB 126 with 300-1000 microg/kg/day of PCB 153. Interestingly, the coadministration of different amounts of PCB 153 with fixed amounts of PCB 126 demonstrated more M1dG adduct accumulation with higher doses of PCB 153. These results are consistent with the results from cancer bioassays that demonstrated a synergistic effect between PCB 126 and PCB 153 on toxicity and tumor development. In summary, the results from the present study support the hypothesis that oxidative DNA damage plays a key role in toxicity and carcinogenicity following long-term PCB exposure.     

Optimizing the immobilization of single-stranded DNA onto glass beads

The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo(1), onto glass beads via a 5'-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo(1) concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89%. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 microg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo(2), was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.     

Attachment of gold nanoparticles to glassy carbon electrode and its application for the direct electrochemistry and electrocatalytic behavior of hemoglobin

Gold nanoparticles have been attached onto glassy carbon electrode surface through sulfhydryl-terminated monolayer and characterized by X-ray photoelectron spectroscopy, atomic force microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The gold nanoparticles-attached glassy carbon electrodes have been applied to the immobilization/adsorption of hemoglobin, with a monolayer surface coverage of about 2.1 x 10(-10) mol cm(-2), and consequently obtained the direct electrochemistry of hemoglobin. Gold nanoparticles, acting as a bridge of electron transfer, can greatly promote the direct electron transfer between hemoglobin and the modified glassy carbon electrode without the aid of any electron mediator. In phosphate buffer solution with pH 6.8, hemoglobin shows a pair of well-defined redox waves with formal potential (E0') of about -0.085 V (versus Ag/AgCl/saturated KCl). The immobilized hemoglobin maintained its biological activity, showing a surface controlled electrode process with the apparent heterogeneous electron transfer rate constant (ks) of 1.05 s(-1) and charge-transfer coefficient (a) of 0.46, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide. A potential application of the hemoglobin-immobilized gold nanoparticles modified glassy carbon electrode as a biosensor to monitor hydrogen peroxide has been investigated. The steady-state current response increases linearly with hydrogen peroxide concentration from 2.0 x 10(-6) to 2.4 x 10(-4) M. The detection limit (3sigma) for hydrogen peroxide is 9.1 x 10(-7) M.     

Studies on the inhibition of highly purified calf thymus 8S and 7.3S DNA polymerase alpha by aphidicolin.

On activated DNA aphidicolin competitively inhibits the incorporation of dCMP by both calf thymus DNA polymerase alpha A2 and C enzymes and inhibits the incorporation of the other three deoxynucleoside monophosphates apparently non-competitively. However, aphidicolin does not inhibit the incorporation of dAMP into poly(dT) . oligo(A)10 nor does it inhibit the incorporation of dGMP into poly(dC) . oligo(dG)10, but, it does competitively inhibit the incorporation of dTMP into poly(dA) . oligo(dT)10.     

Studies on the interaction mechanism between hexakis(imidazole) manganese(II) terephthalate and DNA and preparation of DNA electrochemical sensor

The interaction between hexakis(imidazole) manganese(II) terephthalate ([Mn(Im)(6)](teph).4H(2)O) and salmon sperm DNA in 0.2M pH 2.30 Britton-Robinson buffer solution was studied by fluorescence spectroscopy and cyclic voltammetry. Increasing fluorescence was observed for [Mn(Im)(6)](2+) with DNA addition, while quenching fluorescence phenomenon appeared for EB-DNA system when [Mn(Im)(6)](2+) was added. There were a couple quasi-reversible redox peaks of [Mn(Im)(6)](2+) from the cyclic voltammogram on the glassy carbon electrode. The peak current of [Mn(Im)(6)](2+) decreased with positive shift of the formal potential in the presence of DNA compared with that in the absence of DNA. All the experimental results indicate that [Mn(Im)(6)](2+) can bind to DNA mainly by intercalative binding mode. The binding ratio of the DNA-[Mn(Im)(6)](2+) association complex is calculated to be 1:1 and the binding constant is 4.44x10(3) M(-1). By using [Mn(Im)(6)](teph).4H(2)O as the electrochemical hybridization indicator, the DNA electrochemical sensor was prepared by covalent interaction and the selectivity of ssDNA modified electrode were described. The results demonstrate the use of electrochemical DNA biosensor in the determination of complementary ssDNA.     

Hybridization signal enhancement via long-stranded probe for detection of DNA using aquadichloro (benzimidazole)-copper(II) as hybridization indicator

A simple and sensitive method for electrochemical detection of DNA was designed. This DNA sensor was based on a "sandwich" detection strategy, which involved a long capture probe DNA immobilized on glassy carbon electrodes that flanked both the reference DNA and target DNA. Electrochemical signals were measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using aquadichloro(benzimidazole)-copper(II), Cu(bzim)(H(2)O)Cl(2), as an electroactive indicator. An improving amount of Cu(bzim)(H(2)O)Cl(2) was interacted with the hybrid DNA via the incorporation of a long-probe DNA and a reference DNA in this sensor. As a result of this effect, this sensor design significantly enhanced the sensitivity. With 48-mer probe DNA and 27-mer reference DNA, the proposed method could be used for detection of 21-mer ssDNA ranging from 1.32 x 10(-7) to 2.52 x 10(-6) M with a detection limit of 2.94 x 10(-8) M. Electrochemical DNA biosensors were also developed using the same long-probe sequence as the target sequence with the novel hybridization indicator, Cu(bzim) (H(2)O)Cl(2). The detection limits for the complementary 21-mer target and 27-mer target were 9.52 x 10(-8) M and 5.81 x 10(-8) M, respectively. The results showed that the sensor with long-probe DNA and reference DNA is far more sensitive than that with nonswitch assay.     

Rapid detection of ssDNA and RNA using multi-walled carbon nanotubes modified screen-printed carbon electrode

A method for rapid sensitive detection of DNA or RNA was designed using a composite screen-printed carbon electrode modified with multi-walled carbon nanotubes (MWNTs). MWNTs showed catalytic characteristics for the direct electrochemical oxidation of guanine or adenine residues of signal strand DNA (ssDNA) and adenine residues of RNA, leading to indicator-free detection of ssDNA and RNA concentrations. With an accumulation time of 5 min, the proposed method could be used for detection of calf thymus ssDNA ranging from 17.0 to 345 microg ml(-1) with a detection limit of 2.0 microg ml(-1) at 3 sigma and yeast tRNA ranging from 8.2 microg ml(-1) to 4.1 mg ml(-1). AC impedance was employed to characterize the surface of modified electrodes. The advantages of convenient fabrication, low-cost detection, short analysis time and combination with nanotechnology for increasing the sensitivity made the subject worthy of special emphasis in the research programs and sources of new commercial products.     

Carbon-nanotube-modified electrodes for amplified enzyme-based electrical detection of DNA hybridization

Carbon-nanotube-modified glassy carbon (CNT/GC) transducers have been developed for enhancing the sensitivity and stability of enzyme-based electrochemical bioassays of DNA hybridization. The amplified signal reflects the interfacial accumulation of phenolic products of the alkaline-phosphatase tracer onto the CNT layer. In particular, chronopotentiometric measurements (following short accumulation periods) offer a substantial enhancement of the response of enzymatically liberated alpha-naphthol. The CNT modifier also leads to a dramatic improvement in the stability of the amperometric response of alpha-naphthol. These advantages of CNT/GC electrodes are illustrated from comparison to unmodified glassy carbon electrodes. Factors influencing the adsorptive accumulation of alpha-naphthol, and the overall performance of the new DNA assay, are assessed and optimized. The attractive performance characteristics of the new multi-amplification electrochemical detection of DNA hybridization are reported in connection to the detection of nucleic acid sequences related to the breast cancer BRCA1 gene.     

Apparent stimulation of calf thymus DNA polymerase alpha by ATP.

The mechanism by which millimolar concentrations of ATP stimulate the activity and increase the processivity of calf thymus DNA polymerase alpha has been investigated with poly(dA)/oligo(dT) as template/primer to eliminate possible effects due to primer synthesis. The effect of ATP on the rate of DNA synthesis with this template/primer was found to be dependent upon whether or not the ATP was neutralized and the species of buffer used in the reaction. The present studies suggest that ATP stimulation of calf thymus DNA polymerase can be attributed to changes in the pH of the reaction mixture, a shift in the magnesium ion optimum, or both. Furthermore, effects of ATP on the processivity of DNA polymerase alpha could be mimicked by lowering the pH of the reaction mixture.     

A comparison of redox polymer and enzyme co-immobilization on carbon electrodes to provide membrane-less glucose/O2 enzymatic fuel cells with improved power output and stability

Glassy carbon and graphite electrodes modified with films of enzyme and osmium redox polymer, cross linked with poly (ethylene glycol) diglycidyl ether, were used for elaboration of a glucose/O(2) enzymatic fuel cell. The redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)(2)(polyvinylimidazole)(10)Cl](+) and [Os(4,4'-dichloro-2,2'-bipyridine)(2)(polyvinylimidazole)(10)Cl](+) were selected to facilitate transfer of electrons from the glucose oxidase (GOx) active site to the T1 Cu site of multicopper oxygenases of Trametes hirsuta laccase (ThLacc) and Myrothecium verrucaria bilirubin oxidase (MvBOD). Maximum power density at pH 5.5 of 3.5 μW cm(-2) at a cell voltage of 0.35 V was obtained for an assembled membrane-less fuel cell based on ThLacc on glassy carbon as cathode, in the presence of 0.1 M glucose, 37 °C, with lower power observed at pH 7.4 and 4.5. Replacement of the ThLacc cathode with that of MvBOD produced 10 μW cm(-2) at 0.25 V under pseudo-physiological conditions. Replacement of glassy carbon with graphite as base electrode material resulted in increased redox polymer loading, leading to an increase in power output to 43 μW cm(-2) at 0.25 V under similar conditions. Improved stabilization of biofilms was achieved through covalent anchoring of enzyme and redox polymer on graphite electrodes, derivatized via electrochemical reduction of the diazonium cation generated in situ from p-phenylenediamine. Enzymatic fuel cells using this approach retained 70% power at 24 h, whereas fuel cells prepared without chemical anchoring to graphite retained only 10% of power over the same interval.     

Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks.

DNA cleavage by eukaryotic type II DNA topoisomerase (EC 5.99.1.3) was strongly inhibited by an oligonucleotide containing 10 dGua residues. Catalytic activities of topoisomerase II, as measured by relaxation and decatenation reactions, were also inhibited by oligo(dG)10. Inhibition was specific to oligo(dG)10; other oligonucleotides, nucleotides, or single-stranded DNAs tested did not influence the activity of topoisomerase II. Oligo(dG)10 did not inhibit other activities such as restriction enzymes. Although the enzyme neither binds nor cleaves oligo(dG)10, inhibition can be explained by the finding that topoisomerase II binds tightly with aggregated oligo(dG) structures (estimated to contain between 20 and 30 molecules of monomeric oligo(dG)10) that form spontaneously prior to addition of enzyme. These aggregated oligo(dG)-topoisomerase complexes are large networks that can be pelleted by a 20-min centrifugation step in a Microfuge. Western blotting with a monoclonal antibody confirmed that topoisomerase II is trapped in these pellets. The ability of the enzyme to form large DNA-protein networks could be a biochemical mechanism by which topoisomerase II might promote or participate in chromosome condensation in vivo prior to mitosis.     

Studies on the processivity of highly purified calf thymus 8S and 7.3S DNA polymerase alpha

Template-challenge experiments indicate no gross difference in processivity of the calf thymus DNA polymerase α A and C enzymes. Both enzymes appear to be distributive. Results showing the apparent processive nature of both enzymes on poly (dC). oligo (dG)₁₀ when challenged with poly (dA). oligo (dT)₁₀ are explicable by the failure of both enzymes to bind to the challenging template rather than by the presence of an initiation factor which preferentially binds to certain templates.