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At least 37 records · Page 2Linked to original sources
PubMed2003

Combination of amplification and post-amplification strategies to improve optical DNA sensing.

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.

Biosensing Techniques↗
PubMed2000

Study on colloidal Au-enhanced DNA sensing by quartz crystal microbalance.

Colloidal Au is reported for enhancement the immobilization capacity and ultimately detection limit of DNA using quartz crystal microbalance (QCM). Immobilization of approximately 12 nm-diameter colloidal Au on to an Au-coated QCM resulted in an easier attachment of oligonucleotide, with a mercaptohexyl group at the 5'-phosphate end and an increased capacity for nucleic acid detection. DNA immobilization and hybridization was monitored from QCM frequency changes. Hybridization was induced by exposure of the DNA-containing films to complementary DNA in solution. A much higher sensitivity was obtained for the analyte. The Au nanoparticle films on the Au plate provide a novel means for the fabrication of DNA sensor.

Animals↗
PubMed2002

DNA sensing on glassy carbon electrodes by using hemin as the electrochemical hybridization label.

The electrochemical behavior of hemin, an iron complex of porphyrin, on binding to DNA at a glassy carbon electrode (GCE) and in solution, is described. Hemin, which interacts with covalently immobilized calf thymus DNA, was detected by use of a bare GCE, a double-stranded DNA-modified GCE (dsDNA-modified GCE), and a single-stranded DNA-modified GCE (ssDNA-modified GCE), in combination with differential pulse voltammetry (DPV). The structural conformation of DNA was determined from changes in the voltammetric signals acquired on reduction of hemin. As a result of its large steric structure and anionic substitution on its porphyrin plane, hemin intercalates between the base pairs of dsDNA. A scan-rate study for hemin and the dsDNA-hemin complex were also performed to determine the electrochemical behavior of the complex. The partition coefficient was obtained from the peak currents measured when different concentrations of hemin were in the presence of dsDNA. By observing the oxidation signals of guanine, damage to DNA after reaction with hemin at the GCE surface was also detected. The electrochemical detection of hybridization between the covalently immobilized probe and its target sequence was detected by use of hemin. These results demonstrate the use of DNA biosensors in conjunction with hemin for electrochemical detection of hybridization and damage to DNA.

Base Sequence↗
PubMed2004

Copper(II)-quenched oligonucleotide probes for fluorescent DNA sensing.

A copper(II)-quenched molecular beacon was prepared by attaching fluorescein to the 3'-end and a copper(II) complex to the 5'-end of DNA. In the presence of complementary DNA, copper(II) and dye are spatially separated in the duplex and fluorescence increases up to 15-fold, with excellent discrimination of single base mismatches.

Base Pair Mismatch↗
PubMed1999

Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Manganese water-soluble porphyrin senses DNA conformation.

We have determined the sites that are preferentially cleaved by Mn(T4MPyP) (where T4MPyP is the dianion of 5, 10, 15, 20, tetrakis (4-N-methylpyridine)porphyrin) on synthetic DNAs and on both intrinsically curved and average-shaped natural DNA sequences. On the basis of cleavage selectivity and of DNase I footprinting we show that the recognition specificity by this compound is based on steric properties: the preferred conformation is a DNA minor groove narrower than average and dimensionally defined. This conclusion is reached on the basis of: (i) the localization of the preferential cleavage sites at the 3' extremity of short A-tracts, known to undergo minor groove directional narrowing; (ii) the effects of temperature on cleavage specificity on curved sequences; (iii) the localization of cleavage sites in synthetic constructs whose crystal and solution structure was previously defined, and in programmed sequence variants thereoff; (iv) the effects of base substitutions on cleavage efficiency; (v) DNase I footprinting analysis. Several of these evidences argue against the possibility that Mn(T4MPyP)/DNA site selection occurs on the basis of electrostatic potential effects.Mn(T4MPyP) provides a tool for the analysis of DNA conformation whose selectivity is complementary to that of DNase I and hydroxyl radicals.

Animals↗
PubMed2005

Sensing DNA hybridization via ionic conductance through a nanoporous electrode.

We show that nanoporous alumina modified with covalently linked DNA can be used to detect target DNA by monitoring the increase in impedance at the electrode upon DNA hybridization, which resulted from blocking the pores to ionic flow. Using cyclic voltammetry, direct current conductance, and impedance spectroscopy we confirm the importance of pore size: the effect is observed with 20-nm-diameter pores and is absent for 200-nm pores.

Aluminum↗
PubMed2004

Transcription - guarding the genome by sensing DNA damage.

Cells induce the expression of DNA-repair enzymes, activate cell-cycle checkpoints and, under some circumstances, undergo apoptosis in response to DNA-damaging agents. The mechanisms by which these cellular responses are triggered are not well understood, but there is recent evidence that the transcription machinery might be used in DNA-damage surveillance and in triggering DNA-damage responses to suppress mutagenesis. Transcription might also act as a DNA-damage dosimeter where the severity of blockage determines whether or not to induce cell death. Could transcription therefore be a potential therapeutic target for anticancer strategies?

Aging↗
PubMed2002

A nick-sensing DNA 3'-repair enzyme from Arabidopsis.

DNA single-strand breaks, a major cause of genome instability, often produce unconventional end groups that must be processed to restore terminal moieties suitable for reparative DNA gap filling or ligation. Here, we describe a bifunctional repair enzyme from Arabidopsis (named AtZDP) that recognizes DNA strand breaks and catalyzes the removal of 3'-end-blocking lesions. The isolated C-terminal domain of AtZDP is by itself competent for 3'-end processing, but not for strand break recognition. The N-terminal domain instead contains three Cys(3)-His zinc fingers and recognizes various kinds of damaged double-stranded DNA. Gapped DNA molecules are preferential targets of AtZDP, which bends them by approximately 73 degrees upon binding, as measured by atomic force microscopy. Potential partners of AtZDP were identified in the Arabidopsis genome using the human single-strand break repairosome as a reference. These data identify a novel pathway for single-strand break repair in which a DNA-binding 3'-phosphoesterase acts as a "nick sensor" for damage recognition, as the catalyst of one repair step, and possibly as a nucleation center for the assembly of a fully competent repair complex.

Amino Acid Sequence↗
PubMed2003

Sensing DNA damage by PARP-like fingers.

PARP-like zinc fingers are protein modules, initially described as nick-sensors of poly(ADP-ribosyl)-polymerases (PARPs), which are found at the N-terminus of different DNA repair enzymes. I chose to study the role of PARP-like fingers in AtZDP, a 3' DNA phosphoesterase, which is the only known enzyme provided with three such finger domains. Here I show that PARP-like fingers can maintain AtZDP onto damaged DNA sites without interfering with its DNA end repair functions. Damage recognition by AtZDP fingers, in fact, relies on the presence of flexible joints within double-strand DNA and does not entail DNA ends. A single AtZDP finger is already capable of specific recognition. Two fingers strengthen the binding and extend the contacts on the bound DNA. A third finger further enhances the specific binding to damaged DNA sites. Unexpectedly, gaps but not nicks are bound by AtZDP fingers, suggesting that nicks on a naked DNA template do not provide enough flexibility for the recognition. Altogether these results indicate that AtZDP PARP-like fingers, might have a role in positioning the enzyme at sites of enhanced helical flexibility, where single-strand DNA breaks are present or are prone to occur.

Amino Acid Sequence↗
PubMed2006

Sensing DNA methylation in the protozoan parasite Entamoeba histolytica.

In the protozoan parasite Entamoeba histolytica, 5-methylcytosine (m5C) was found predominantly in repetitive elements. Its formation is catalysed by Ehmeth, a DNA methyltransferase that belongs to the Dnmt2 subfamily. Here we describe a 32 kDa nuclear protein that binds in vitro with higher affinity to the methylated form of a DNA encoding a reverse transcriptase of an autonomous non-long-terminal repeat retrotransposon (RT LINE) compared with the non-methylated RT LINE. This protein, named E. histolytica-methylated LINE binding protein (EhMLBP), was purified from E. histolytica nuclear lysate, identified by mass spectrometry, and its corresponding gene was cloned. EhMLBP corresponds to a gene of unknown function that shares strong homology with putative proteins present in Entamoeba dispar and Entamoeba invadens. In contrast, the homology dropped dramatically when non-Entamoebidae sequences were considered and only a weak sequence identity was found with Trypanosoma and several prokaryotic histone H1. Recombinant EhMLBP showed the same binding preference for methylated RT LINE as the endogenous EhMLBP. Deletion mapping analysis localized the DNA binding region at the C-terminal part of the protein. This region is sufficient to assure the binding to methylated RT LINE with high affinity. Western blot and immunofluorescence microscopy, using an antibody raised against EhMLBP, showed that it has a nuclear localization. Chromatin immunoprecipitation (ChIP) confirmed that EhMLBP interacts with RT LINE in vivo. Finally, we showed that EhMLBP can also bind rDNA episome, a DNA that is methylated in the parasite. This suggests that EhMLBP may serve as a sensor of methylated repetitive DNA. This is the first report of a DNA-methylated binding activity in protozoa.

Animals↗
PubMed2003

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Anti sense DNA down-regulates proteins kinase C-epsilon and enhances vasopressin-stimulated Na+ absorption in rabbit cortical collecting duct.

Hormonal activation of protein kinase C (PKC) is a major signaling mechanism regulating salt and water transport in the distal nephron. We used antisense DNA to down-regulate a PKC isoform in the rabbit cortical collecting duct (CCD) and examined its role in mediating arginine vasopressin's (AVP) effect on salt transport in the CCD. Immunoblots demonstrate that PKC-epsilon (diacylglycerol sensitive) and PKC-zeta (diacylglycerol insensitive) are the major PKC isoforms in both freshly isolated and primary cultures of rabbit CCDs. Rabbit CCDs grown on semi-permeable supports, displayed a positive baseline short circuit current (Isc), which was abolished by amiloride, demonstrating active Na+ absorption. Both AVP and 8-chloro-phenylthio-cAMP (8CPTcAMP) transiently increased Isc, however, within 40 min Isc fell below baseline. Down-regulation of PKC-epsilon, as confirmed by immunoblot, was achieved either by treatment with a PKC-epsilon-specific antisense oligonucleotide or 48 h of 1 microM PMA. In PKC-epsilon down-regulated cells, 8CPTcAMP produced a sustained, rather than transient, increase in Isc. We suggest cAMP stimulates Na+ transport, but secondary activation of PKC-epsilon results in the sustained inhibition of Na+ transport seen in response to vasopressin in the CCD.

Animals↗
PubMed2004

New acridone derivatives for the electrochemical DNA-hybridisation labelling.

In the field of DNA sensing, DNA hybridisation detection is generally performed by fluorescence microscopy. However, fluorescence instrumentation is difficult to miniaturise in order to produce fully integrated DNA chips. In this context, electrochemical detection of DNA hybridisation may avoid this limitation. Therefore, the use of DNA intercalators is particularly attractive due to their selectivity toward DNA double strand enabling DNA labelling without target chemical modification and, for most of them, to their electroactivity. We have synthesized a pyridoacridone derivative dedicated to DNA hybridisation electrochemical-sensing which presents good electrochemical reversibility, electroactivity at mild potentials and specificity toward DNA double strand. The electrochemical behaviour of this molecule has been assessed using cyclic voltammetry (CV). DNA/intercalator interactions were studied by differential pulse voltammetry (DPV) before application to hybridisation detection onto DNA sensors based on polypyrrole modified electrodes.

Acridines↗
PubMed2006

DNA counterion current and saturation examined by a MEMS-based solid state nanopore sensor.

Reports of DNA translocation measurements have been increasing rapidly in recent years due to advancements in pore fabrication and these measurements continue to provide insight into the physics of DNA translocations through MEMS based solid state nanopores. Specifically, it has recently been demonstrated that in addition to typically observed current blockages, enhancements in current can also be measured under certain conditions. Here, we further demonstrate the power of these nanopores for examining single DNA molecules by measuring these ionic currents as a function of the applied electric field and show that the direction of the resulting current pulse can provide fundamental insight into the physics of condensed counterions and the dipole saturation in single DNA molecules. Expanding on earlier work by Manning and others, we propose a model of DNA counterion ionic current and saturation of this current based on our experimental results. The work can have broad impact in understanding DNA sensing, DNA delivery into cells, DNA conductivity, and molecular electronics.

Biosensing Techniques↗