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At least 19 recordsLinked to original sources
PubMed2000

DNA sensing on a DNA probe-modified electrode using ferrocenylnaphthalene diimide as the electrochemically active ligand.

Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA. On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode. Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s. The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode. Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level. The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol.

Base Sequence↗
PubMed2000

A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage.

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA-CAF-1 interaction in the context of DNA damage processing and checkpoint control.

Animals↗
PubMed2004

Development of a novel DNA sensing system using DNA aptamer inhibited enzymatic activity 1.

A novel DNA detection system has been developed using the DNA aptamer inhibiting thrombin activity. The complementary strand to the target DNA was inserted into the loop region of the G-quartet structure of this adapter aiming at the disruption of it by the hybridization of the target DNA with its complementary strand in the aptamer. Using this modified aptamer, we were able to detect the target DNA by measuring thrombin activity.

Aptamers, Nucleotide↗
PubMed2004

Development of a novel DNA sensing system using DNA aptamer that inhibits enzymatic activity 2.

We have developed novel DNA sensor system using the DNA aptamers for thrombin. We modified original thrombin aptamer by addition of the target DNA complementary strands to the 5' end or 3' end of aptamer. Inhibitory activity of this modified aptamer increased in the presence of target DNA. We were able to detect the target DNA by measuring thrombin activity using this aptamer without the separation of bound and free separation.

Aptamers, Nucleotide↗
PubMed2001

Electronic transduction of DNA sensing processes on surfaces: amplification of DNA detection and analysis of single-base mismatches by tagged liposomes.

Tagged, negatively charged, liposomes are used to amplify DNA sensing processes. The analyses of the target DNA are transduced electrochemically by using Faradaic impedance spectroscopy, or by microgravimetric measurements with Au-quartz crystals. By one method, a probe oligonucleotide (1) is assembled on Au-electrodes or Au-quartz crystals. The formation of the double-stranded assembly with the analyte DNA (2) is amplified by the association of the 3-oligonucleotide-functionalized liposomes to the sensing interface. The target DNA is analyzed by this method with a sensitivity limit that corresponds to 1 x 10(-12) M. A second method to amplify the sensing of the analyte involves the interaction of the 1-functionalized electrode or Au-quartz crystal with the target DNA sample (2) that is pretreated with the biotinylated oligonucleotide (4). The formation of the three-component double-stranded assembly between 1/2/4 is amplified by the association of avidin and biotin-labeled liposomes to the sensing interfaces. By the secondary association of avidin and biotin-tagged liposomes, a dendritic-type amplification of the analysis of the DNA is accomplished. The analyte DNA (2) is sensed by this method with a sensitivity limit corresponding to 1 x 10(-13) M. The biotin-tagged liposomes are also used to probe and amplify single-base mismatches in an analyte DNA. The 6-oligonucleotide-functionalized Au-electrode or Au-quartz crystal was used to differentiate the single-base mismatch (G) in the mutant (5) from the normal A-containing gene (5a). Polymerase-induced coupling of the biotinylated-C-base to the double-stranded assembly generated between 6 and 5 followed by the association of avidin and biotin-tagged liposomes is used to probe the single base mismatch. The functionalized liposomes provide a particulate building unit for the dendritic amplification of DNA sensing.

Base Pair Mismatch↗
PubMed2003

Binding discrimination of MutS to a set of lesions and compound lesions (base damage and mismatch) reveals its potential role as a cisplatin-damaged DNA sensing protein.

The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate. The rate of association, kon, for binding to the 1,2-d(GpG) adduct was 3.1 x 104 m-1 s-1 and the specificity of binding was essentially dependent on koff. DNA duplexes containing a single 1,2-d(ApG), 1,3-d(GpCpG) adduct, and an interstrand cross-link of cisplatin were not preferentially recognized. Among 12 DNA substrates, each containing a different cisplatin compound lesion derived from replicative misincorporation of one base opposite either of the 1,2-intrastrand adducts, 10 were specifically recognized including those that are more likely formed in vivo based on cisplatin mutation spectra. Moreover, among these lesions, two compound lesions formed when an adenine was misincorporated opposite a 1,2-d(GpG) adduct were not substrates for the MutY-dependent mismatch repair pathway. The ability of MutS to sense differentially various platinated DNA substrates suggests that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are more plausible critical lesions for MMR-mediated cisplatin cytotoxicity.

Adenosine Triphosphatases↗
PubMed1992

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression.

DNA, Viral↗
PubMed2006

Homogeneous DNA sensing using enzyme-inhibiting DNA aptamers.

A novel aptameric enzyme subunit (AES) which can detect target DNAs has been developed. AES is an enzyme-inhibiting aptamer bearing a target-molecule binding site which can allosterically control enzymatic activity. The thrombin-inhibiting aptamer bearing a G-quartet structure was chosen as the enzyme-inhibiting aptamer. The stem-and-loop structure, which contains a strand complementary to the target DNA, was inserted into the G-quartet structure of the thrombin-inhibiting aptamer to disrupt the G-quartet structure through the hybridization of the target DNA with the complementary strand in the AES. The disruption of the G-quartet structure led to a decrease of the inhibitory activity of the whole aptameric complex. Using this designed aptamer, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution without bound/free separation, and the lower detection limit was 20 nM.

Aptamers, Peptide↗
PubMed2005

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 (DeltaIp) is linearly dependent 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.

Base Sequence↗
PubMed2006

Hydrothermally shrunk alumina nanopores and their application to DNA sensing.

Hydrothermal treatment of anodized alumina membranes has been known for years and is believed to seal the pores by transforming aluminium oxide into lower density hydroxides. We demonstrate that, at least for 60 nm diameter pores grown from anodization in oxalic acid at 40 V, the hydrothermal treatment significantly shrinks but does not fully seal the nanopores. The pores shrink to a neck of less than 10 nm in diameter and 2-4 microm in length, in which the diffusion coefficient of ions is five orders of magnitude smaller than in the bulk. Because of a high electrolyte resistance through hydrothermally treated shrunken nanopores, they can be used for electrical sensing applications, as demonstrated using the example of DNA sensing. Hybridization of target DNA with a complementary ssDNA covalently immobilized inside the nanopores causes an increase in impedance by more than 50% while a noncomplementary ssDNA has no measurable effect.

Aluminum Oxide↗
PubMed2000

Ferroceneacetyl naphthalene diimide as a new electrochemical ligand for DNA sensing.

New electrochemically active DNA ligand 1 was synthesized by the connection of ferroceneacetic acid at the terminal amino moieties of two imide substituents of naphthalene diimide. Electrochemical experiments in aqueous solution containing DMSO showed that the selectivity for double stranded DNA of 1 has increased from that of ligand 2 previously reported. The peak current of 1 shifted toward the negative side from that of 2, thereby shortening the time required for gene detection.

DNA↗
PubMed2005

Electrochemical DNA sensing using osmium complexes as hybridization indicators.

A surface-based method for the study of the interactions of DNA with redox-active osmium complexes is described. The study was carried out using gold electrodes modified with DNA by adsorption and [Os(bpy)3]3+/2+ (bpy=2,2'-bipyridyl) or [Os(phen)3]3+/2+ (phen=1,10-phenantroline) as electrochemical indicators. The method, which is simple and reagent saving, allows the accumulation of osmium complexes on the DNA layer. The amount of osmium complex bound by the layer of double-stranded (dsDNA) or single-stranded DNA (ssDNA) adsorbed at gold electrodes was estimated from the cyclic voltammetric (CV) peak charge of osmium complex reduction. The dissociation constants (K) for the oxidized and reduced forms of a bound species are also estimated. [Os(phen)3]3+/2+ was applied to a probe for electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 70 pmol/cm2 on a gold electrode. Following hybridization with the complementary DNA, the osmium complex was electrochemically accumulated on the double-stranded DNA layer and the differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of [Os(phen)3]3+/2+ that was bound to the double-stranded DNA on the electrode.

Adsorption↗
PubMed1992

Inhibition of human immunodeficiency virus-1 proliferation by liposome-encapsulated sense DNA to the 5' tat splice acceptor site.

A liposome formulation containing a distearoylphosphatidylethanolamine analog was developed that was endocytosed by both lymphocytes and monocytes. This formulation was used to encapsulate sense and antisense 20-mer oligodeoxynucleotides to the 5' tat splice acceptor site of human immunodeficiency virus type 1. At a DNA concentration of 140 nM, the liposome-encapsulated sense DNA inhibited p24 production by as much as 84% in human peripheral blood leukocytes infected with "wild-type" virus. This treatment also reduced the number of peripheral blood leukocytes producing intracellular viral antigen by 71%. Of interest, no reduction in either parameter was observed for the antisense-containing liposomes. The results demonstrate the promise of a new liposomal delivery vehicle to inhibit human immunodeficiency virus replication by an entrapped oligodeoxynucleotide.

Antiviral Agents↗
PubMed2004

High-fidelity DNA sensing by protein binding fluctuations.

One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response. We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of-equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.

Adenosine Triphosphate↗
PubMed2004

ATM and ATR: sensing DNA damage.

Cellular response to genotoxic stress is a very complex process, and it usually starts with the "sensing" or "detection" of the DNA damage, followed by a series of events that include signal transduction and activation of transcription factors. The activated transcription factors induce expressions of many genes which are involved in cellular functions such as DNA repair, cell cycle arrest, and cell death. There have been extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process, including the mitogen-activated protein kinases (MAPKs) cascade. Although the initial activation of protein kinase cascade is not fully understood, human protein kinases ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) are emerging as potential sensors of DNA damage. Current progresses in ATM/ATR research and related signaling pathways are discussed in this review, in an effort to facilitate a better understanding of genotoxic stress response.

Animals↗
PubMed2001

Liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplification of antigen--antibody or oligonucleotide--DNA sensing processes by the precipitation of an insoluble product on electrodes.

Liposomes labeled with biotin and the enzyme horseradish peroxidase (HRP) are used as a probe to amplify the sensing of antigen-antibody interactions or oligonucleotide-DNA binding. The HRP-biocatalyzed oxidation of 4-chloro-1-naphthol (1) in the presence of H2O2, and the precipitation of the insoluble product 2 on electrode supports, are used as an amplification route for the sensing processes. The anti-dinitrophenyl antibody (DNP-Ab) is sensed by a dinitrophenyl-L-cysteine antigen monolayer associated with an Au electrode. A biotinylated anti-IgG-antibody (Fc-specific) is linked to the antigen-DNP-Ab complex, and the biotin-labeled HRP-liposomes associate with the assembly through an avidin bridge. The biocatalyzed precipitation of 2 on the electrode increases the electron-transfer resistances at the electrode-solution interface or the electrode resistance itself. The binding events of the different proteins on the electrode and the biocatalyzed precipitation of 2 on the conductive support are followed by Faradaic impedance spectroscopy or constant-current chronopotentiometry. DNP-Ab concentrations as low as 1 x 10(-11) g x mL(-1) can be detected by this method. The labeled liposomes were also used for the amplified detection of DNA 3. The oligonucleotide 4, complementary to a part of the target DNA 3 that is a model nucleic acid sequence for the Tay-Sachs genetic disorder, is assembled on an Au electrode. Hybridization of the analyte 3 followed by the association of the biotin-tagged oligonucleotide 5 yields a three-component double-stranded assembly. Sensing of the analyte 3 is amplified by the association of avidin, the labeled liposomes, and the subsequent biocatalyzed precipitation of 2 on the electrodes. The DNA 3 is detected with a sensitivity that corresponds to 6.5 x 10(-13) M. Faradaic impedance spectroscopy and chronopotentiometry were employed to follow the stepwise assembly of the systems and the electronic transduction of the detection of the analyte DNA 3.

Antigen-Antibody Reactions↗