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Results for “DNA sensing”

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At least 145 records · Page 8Linked to original sources
PubMed2006

Comparison of the signaling and stability of electrochemical DNA sensors fabricated from 6- or 11-carbon self-assembled monolayers.

We have characterized the solution-phase and dry storage stability of electrochemical E-DNA sensors fabricated using mixed self-assembled monolayers (SAMs) composed of 6- or 11-carbon (C6 and C11, respectively) alpha,omega-thiol alcohols and the analogous C6- or C11-thiol-terminated stem-loop DNA probe. We find that the solution-phase and dry storage stability of C6-based E-DNA sensors are limited and poorly reproducible. The use of stabilizing agents bovine serum albumin plus either glucose or trehalose significantly improves the dry storage shelf life of such sensors: when using these preservatives, we observe only 7-9% sensor degradation after 1 month of storage in air at room temperature. In comparison, the stability of C11-based E-DNA sensors is significantly greater than that of the C6-based sensors; we observe only minor (5-8%) loss of signal upon storing these sensors for a week under ambient solution conditions or for more than a month in air in the presence of preservatives. Moreover, whereas the electron-transfer rate through C11 SAMs is slower than that observed for C6 SAMs, it is rapid enough to support good sensor performance. It thus appears that C11 SAMs provide a reasonable compromise between electron-transfer efficiency and sensor stability and are well suited for use in electronic DNA-sensing applications.

Alcohols↗
PubMed1992

Inhibition of class II MHC gene expression by anti-sense RNA in transgenic mice.

We have established transgenic mice carrying the anti-sense DNA to the gene encoding beta chain of the class II major histocompatibility complex (I-A) molecule. The amount of I-A molecule on splenic B lymphocytes from the mice was reduced in the presence of a large amount of the exogenous anti-sense RNA. The amount of I-A beta chain RNA was selectively reduced and inversely correlated with the amount of anti-sense RNA in the spleens. These results suggest that the I-A beta chain RNA is rapidly degraded by duplex formation with the anti-sense RNA in splenic B cells from the transgenic mice.

Animals↗
PubMed2006

Biosensors and bioelectrochemistry.

This review describes recent developments in the field of biosensors and bioelectrochemistry. Nanoparticles have been used to improve sensor performance and to develop biosensors based on new detection principles. Their use has extended into all areas of biosensor and bioelectrochemistry research. Other active areas of biosensor development include DNA sensing, immunosensing, direct electron transfer between an electrode and a redox protein or enzyme, and in vivo sensors.

Biosensing Techniques↗
PubMed2005

In my end is my beginning: control of end resection and DSBR pathway 'choice' by cyclin-dependent kinases.

The genome is constantly subjected to chemical alterations that have the potential to cause genetic mutation, chromosomal rearrangements and, in the case of multicellular organisms, cancer. Particular vulnerability exists during DNA replication, when the two DNA strands of a chromosome separate to form templates for the synthesis of sister chromatids. Attempted replication across a damaged or nicked DNA template can result in the formation of a double-strand break (DSB), arguably the most dangerous of DNA lesions. DSBs can also arise directly at any cell cycle stage following exposure to ionizing radiation or radiomimetic agents. To combat these recurrent threats of genomic instability, numerous distinct enzyme systems have evolved that sense DNA damage and coordinate its repair. Part of this coordination involves the activation of signal transduction cascades that target repair proteins, trigger DNA damage-dependent cell cycle checkpoints and profoundly affect chromatin neighboring a DSB. Here, we discuss current models of how lesion processing itself helps to coordinate these signals in dividing cells. Recent evidence in yeast of a role for cyclin-dependent kinases in DNA end resection suggests a possible solution to the long-standing puzzle of how DSBR pathway 'choice' is regulated through the cell cycle.

Cyclin-Dependent Kinases↗
PubMed1999

Highly sensitive detection of target gene by electrochemical method.

A highly sensitive DNA sensing method was developed using electrochemically active ligand. This method is based on the detection of electric current generated by electrochemically active ligand concentrated on the electrode. Electrochemically active, intercalating ligand can bind to the double stranded DNA of target gene sequence on the electrode, where the complementary single strand is immobilized as a probe. We succeeded in the detection of 0.1 amol target gene. The technique was applicable to the detection of 0.1-10 amol gene.

DNA↗
PubMed2005

Evidence that DNA damage detection machinery participates in DNA repair.

The toroidal Rad9-Rad1-Hus1 checkpoint complex (9-1-1) is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication and repair. 9-1-1 has been characterized as a sensor of DNA damage that functions in concert with the checkpoint control proteins ATM and ATR. However, recent data suggest that the 9-1-1 complex and its individual Rad9 component serve different and multiple functions in cells by sensing DNA damage, stimulating apoptosis, and regulating gene transcription. Recently it was reported that 9-1-1 interacts with and/or stimulates components of the base excision repair (BER) pathway including the S. pombe MutY homolog (MYH), human polymerase beta (Polbeta), and flap endonuclease 1 (FEN1). Furthermore, preliminary results indicate a stimulation of DNA ligase I. In this review, the likely direct participation of 9-1-1 in DNA repair is discussed.

Animals↗
PubMed1993

Complementation and recombination between mutants of complementary sense genes of DNA A of Abutilon mosaic virus.

Mutations have been inserted into the complementary sense ORFs of DNA A of Abutilon mosaic virus (AbMV). Mutation in both ORF AC1 and ORF AC2/3 resulted in clones incapable of infecting Nicotiana benthamiana following agroinoculation. Mutation in ORF AC1 but not in ORF AC2/3 prevented DNA replication in leaf discs. Thus ORF AC1 is essential for AbMV DNA replication whereas ORF AC2/3 is probably involved in some aspect of the spread of the virus through the plant. Coagroinoculation of both mutants and wild-type DNA B resulted in complementation and recombination. Complementation precedes recombination and once recombination is established mutant DNA can no longer be detected. Plants in which complementation alone is occurring are symptomless and contain relatively low amounts of virus double-stranded DNA whereas following recombination a wild-type infection is established.

Blotting, Southern↗
PubMed1998

2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.

DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.

2-Aminopurine↗
PubMed2006

Carbon nanotube DNA sensor and sensing mechanism.

We report the fabrication of single-walled carbon nanotube (SWNT) DNA sensors and the sensing mechanism. The simple and generic protocol for label-free detection of DNA hybridization is demonstrated with random sequence 15mer and 30mer oligonucleotides. DNA hybridization on gold electrodes, instead of on SWNT sidewalls, is mainly responsible for the acute electrical conductance change due to the modulation of energy level alignment between SWNT and gold contact. This work provides concrete experimental evidence on the effect of SWNT-DNA binding on DNA functionality, which will help to pave the way for future designing of SWNT biocomplexes for applications in biotechnology in general and also DNA-assisted nanotube manipulation techniques.

Biosensing Techniques↗
PubMed2001

DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme?

The DNA damage signalling pathway is a core element of the cellular response to genotoxic insult, and its components play key roles in defending against neoplastic transformation. Recent work has indicated that the human ATM and ATR proteins, and their yeast homologues, are intimately involved in sensing DNA damage, suggesting parallels with the DNA double-strand break repair enzyme DNA-PK.

Animals↗
PubMed2005

DNA mismatch detection by resonance energy transfer between ruthenium(II) and osmium(II) tris(2,2'-bipyridyl) chromophores.

Octahedral tris-chelate complexes [M(II)(bpy)(3)](2+) (M = Ru or Os, bpy = 2,2'-bipyridyl), covalently attached to the 3'- and 5'-phosphates of two oligonucleotides, are juxtaposed when hybridized contiguously to a fully complementary DNA target. Visible metal-to-ligand charge-transfer (MLCT) excitation of the [Ru(II)(bpy)(3)](2+) unit leads to resonance energy transfer to the MLCT state of the [Os(II)(bpy)(3)](2+) moiety, with the energy transfer efficiency depending on the degree of hybridization. The extent of attenuation of the intense red luminescence from the Ru(II) chromophore hence allows highly sensitive structural probing of the assembly and constitutes a novel approach to DNA sensing which is capable of detecting mutations.

Base Sequence↗
PubMed1996

The role of poly(ADP-ribosyl)ation in the adaptive response.

An involvement of the poly(ADP-ribosyl)ation system in the expression of the adaptive response has been demonstrated with inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase. This enzyme is a key component of a reaction cycle in chromatin, involving dynamic synthesis and degradation of variably sized ADP-ribose polymers in response to DNA strand breaks. The present report reviews recent work focussing on the response of the poly(ADP-ribosyl)ation system in low dose adaptation. The results suggest that adaptation of human cells to minute concentrations of an alkylating agent involves a different activation mechanism for poly(ADP-ribose) polymerase than DNA break-mediated stimulation after high dose treatment. Moreover, adaptation induces the formation of branched polymers with a very high binding affinity for histone tails and selected other proteins. High dose challenge treatment of adapted cells further enhances formation of branched polymers. We propose that apart from sensing DNA nicks, poly(ADP-ribose) polymerase may be part of pathway protecting cells from downstream events of DNA damage.

Adaptation, Physiological↗
PubMed2001

In vitro investigations of tumor targeting with (99m)Tc-labeled antisense DNA.

UNLABELLED: One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. METHODS: An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. RESULTS: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled antisense versus sense DNA, an increased accumulation of (99m)Tc-antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 10(5) antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. CONCLUSION: This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.

Autoradiography↗
PubMedDate not supplied

The role of STAT1 in activation of IL-3- and IL-5-induced eosinophils by interferon gamma.

Tyrosine phosphorylation of STAT1alpha in eosinophils after IFN-gamma stimulation has been shown, but the biological significance of eosinophil STAT1alpha activation in transmitting the signals through the IFN-gamma receptor remains unknown. The purpose of this study is to determine whether STAT1 is involved in the regulation of eosinophils by IFN-gamma-IFN-gamma receptor interaction. rhIL-3- and rhIL-5-induced eosinophils from CD34+ cells of cord blood on day 28 of culture were used. The cells were washed and further incubated in IL-3- and IL-5-free medium for 48 h. The induced eosinophils constitutively expressed CD69 and lost this expression after a further 48-hour incubation without the cytokines. IFN-gamma significantly upregulated CD69 expression on the 48-hour incubated cells. In inhibitory experiments on STAT1, a phosphorothioate oligo antisense DNA against STAT1alpha was added to IL-3- and IL-5-containing medium from day 15 to day 28 of culture. The oligo DNAs altered neither the expressions of myeloid cell marker CD9 and 13 nor the expression of IFN-gamma receptor on the cells. The added STAT1alpha antisense, but not sense, DNA significantly reduced STAT1alpha mRNA expression in the cells. The STAT1 antisense also significantly inhibited IFN-gamma-induced CD69 expression on the 48-hour incubated eosinophils. In conclusion, these results indicate that IFN-gamma induces CD69 expression in the induced eosinophils through STAT1alpha, suggesting that STAT1alpha may play a significant role in eosinophil regulation by IFN-gamma.

Antigens, CD↗
PubMed1999

Irradiation-induced rescue of thymocyte differentiation and V(D)J recombination in mice lacking the catalytic subunit of DNA-dependent protein kinase.

Scid mice express a truncated form of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and are unable to properly rearrange their Ig and TCR genes, resulting in a severe combined immunodeficiency that is characterized by arrested differentiation of B and T lymphocytes. Treatment of scid mice with low doses of gamma irradiation rescues rearrangements at several TCR loci and promotes limited thymocyte differentiation. The machinery responsible for sensing DNA damage and the mechanism by which irradiation compensates for the scid defect in TCR recombination remain unknown. Because DNA-PKcs is present in scid thymocytes, it may mediate some or all of the irradiation effects. To test this hypothesis, we examined the effects of irradiation on DNA-PKcs-deficient (slip) mice. Our data provide the first evidence that DNA-PKcs is not required for limited rescue of thymocyte differentiation or TCR rearrangements.

Animals↗
PubMed2005

New opportunities in chemosensitization and radiosensitization: modulating the DNA-damage response.

Many current cancer treatments, including certain classes of chemotherapeutics and radiation, induce cytotoxicity by damaging DNA. However, many cancers are resistant to these therapies, which represents a significant challenge in the clinic. Thus, modulating DNA-damage responses to selectively enhance the sensitivity of cancer cells to these therapies is highly desirable. When DNA damage is detected, DNA checkpoint mechanisms are activated to halt cells at various phases of the cell cycle. Simultaneously, DNA-damage sensors transduce signals to activate DNA-repair mechanisms via de novo expression or post-translational modification of enzymes required for DNA repair. p53 is the major player in a checkpoint that arrests cells at the G1/S boundary, while checkpoint kinase (Chk)1 is critical for the G2/M checkpoint and also the S checkpoint that prevents cell cycle progression after replication defects (intra-S-phase checkpoint) or S/M uncoupling (S/M checkpoint). Poly(ADP-ribose) polymerase is involved in sensing DNA single-strand breaks and inducing DNA repair via poly(ADP-ribosyl)ating various DNA-binding and DNA-repair proteins. In this review, strategies for implementing small-molecule inhibitors of poly(ADP-ribose) polymerase and Chk1, which are emerging as potential adjuncts to current therapies, are discussed.

Antineoplastic Agents↗
PubMed2003

Structural flexibility and the thermodynamics of helix exchange constrain attenuation and allosteric activation of hammerhead ribozyme TRAPs.

Perturbations of precleavage equilibria in RNA-cleaving ribozymes can be exploited to control cleavage kinetics. In the targeted ribozyme-attenuated probes (TRAP) design, antisense and attenuator sequences are appended onto the catalytic core of a ribozyme or deoxyribozyme. The attenuator anneals to conserved bases in the catalytic core to form an inactive conformation, which is activated upon binding of a sense strand oligonucleotide to the antisense module. In this work, the apparent Michaelis-Menton constant (K'm) for the binding of the RNA substrate to the ribozyme is shown to be within a factor of 2 for a number of constructs whose observed cleavage rates varied by several 100-fold. These observations rule out models of allosteric regulation based on modulation of substrate binding affinity, instead favoring a model in which regulation arises from equilibration between the active and inactive conformations of the TRAP. Free energies of formation for isolated helices that are exchanged during this reequilibration were determined from the concentration dependence of optical melt data. These values established that the thermodynamic stabilities of sense-antisense duplexes and of the attenuator-core duplexes correlate with observed rates of cleavage. Notably reduced cleavage rates are observed for TRAP ribozymes with extended antisense sequences, suggesting that tight binding of attenuator to the core is assisted by a long antisense portion. A construct with a 25-nucleotide antisense showed greater than 730-fold activation upon annealing with a 20-nucleotide DNA sense strand oligo, representing the greatest activation observed to date for the TRAP design.

Allosteric Regulation↗