Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “quadruplex”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources
PubMed2006

Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay.

For rapid identification of methicillin-resistant Staphylococcus aureus, molecular methods are generally targeting mecA and species-specific genes. Sa442 DNA fragment is a popular species-specific target. However, recently, there have been few reports on S. aureus isolates that are negative for Sa442 fragment; therefore, use of single gene or DNA-fragment-specific polymerase chain reaction (PCR) for identification of microbial isolate may result in misidentification. This study includes CoA gene in parallel with Sa442 marker for identification of S. aureus. This further improves the specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and can prevent misidentification of S. aureus isolates lacking Sa442 DNA fragment. In this study, the newly developed triplex real-time PCR assay was compared with a quadruplex conventional gel-based PCR assay using the same primer sets in both assays. The dual-labeled TaqMan probes (ProOligo, France) for these primers were specifically designed and used in a real-time PCR assay. The clinical isolates (n = 152) were subjected to both PCR assays. The results obtained from both assays proved that the primer and probe sets were 100% sensitive and 100% specific for identification of S. aureus and detection of methicillin resistance. This triplex real-time PCR assay represents a rapid and powerful method for S. aureus identification and detection of methicillin resistance.

Coenzyme A↗
PubMed2005

Overcoming the immortality of tumour cells by telomere and telomerase based cancer therapeutics--current status and future prospects.

A key property of malignant tumours is their immortality or limitless replicative potential. Cell replication is associated with the maintenance of telomeres and in the great majority of cases, through the reactivation of the reverse transcriptase telomerase. Targeting the telomere/telomerase machinery offers a novel and potentially broad-spectrum anticancer therapeutic strategy since telomerase is constitutively overexpressed in the vast majority of human cancers. Telomeres are also critically short in most tumours compared to normal tissues. Strategies that exploit these differences include the direct targeting of components of telomerase: the protein component hTERT or RNA component hTR. Examples of such agents include the small molecule hTERT inhibitor BIBR1532 and GRN163L, a thio-phosphoramidate oligonucleotide targeting the template region of hTR as a "template antagonist". Anti-tumour effects have been observed in both cell lines and, especially for GRN163L, in xenografted human tumours in mice. Effects, however, are largely dependent upon initial telomere length, which can result in a substantial lag before antitumour activity is observed in tumours possessing relatively long telomeres. An alternative approach is to target the telomere itself (Telomere Targeting Agents, TTAs). Several classes of small molecules have been described that induce the G-rich single-stranded overhang of telomeric DNA to fold into 4-stranded G-quadruplex structures. Such folding is incompatible with telomerase function and may induce rapid telomere uncapping. These molecules have shown potent telomerase inhibition in nanomolar concentrations in vitro and the rapid induction of senescence in cancer cells. The trisubstituted acridine based TTA, BRACO19, has demonstrated single agent activity against human tumour xenografts with anti-tumour effects apparent from only 7 days of treatment. In the near future, it is expected that lead examples from both the direct telomerase targeted agents (e.g., GRN163L) and from the distinct class of those targeting telomeres (e.g., AS1410 based on BRACO19) will enter Phase I clinical trial where clinical benefit from this class of novel drugs will be determined.

Cell Division↗
PubMed2006

TRF2 inhibition triggers apoptosis and reduces tumourigenicity of human melanoma cells.

The inhibition of the telomere-binding protein TRF2, by expressing the dominant negative form TRF2(DeltaBDeltaC), has been used as a model of anti-telomere strategy to induce a reversion of the malignant phenotype of M14 and JR5 human melanoma lines. Over-expression of TRF2(DeltaBDeltaC) induced apoptosis and reduced tumourigenicity exclusively in JR5 cells. p53 and Rb status and apoptotic response to DNA damage did not seem to account for the different response of the two lines to TRF2 inhibition. Interestingly, JR5 cells possess shorter and more dysfunctional telomeres compared to M14 line. Moreover, the treatment with the G-quadruplex-interacting agent (G4-ligand) RHPS4 sensitises M14 cells to TRF2 inhibition. These results demonstrate that TRF2 can impair tumuorigenicity of human cancer cells. They further suggest that a basal level of telomere instability favours an efficient response to TRF2 inhibition and that a combined anti-TRF2 and G4-ligand therapy would have synergistic inhibitory effects on tumour cell growth.

Apoptosis↗
PubMed2005

Polymorphism of four X-chromosomal STRs in a Polish population sample.

Allele frequencies for four X-chromosomal STR were determined in a population sample of 240 unrelated males and females from the northeastern Poland by multiplex PCR and subsequent automated fluorescent detection (ABI 310) using a commercially available multiplex PCR kit (Mentype Argus X-UL). Kinship tests revealed a typical X-linked inheritance with no mutation. The analysed quadruplex is a potential extension to a battery of autosomal systems in forensic applications, especially in the investigation of kinship analysis and deficiency cases.

Chromosomes, Human, X↗
PubMed2004

Impact of intrinsic DNA structure on processing of plasmids for gene therapy and DNA vaccines.

Several non-Watson Crick DNA structures have been discovered to date, which may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. In this study, intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rate, total plasmid yield, and topology (i.e. the relative proportions of supercoiled plasmid, open circular and linear forms), were determined. The stability of the inserted sequences were assessed using gel electrophoresis. Z-DNA was shown to be unstable in a batch Escherichia coli DH1 production system grown in complex medium. Encouragingly other sequences studied (triplex, bend and quadruplex) did not cause spontaneous deletions, and no detrimental effect was found on growth rate or on total plasmid yield; indicating that such sequences could be included in future DNA products without any detrimental effect on plasmid yields; although the intra molecular triplex studied significantly decreased the proportion of supercoiled species.

Base Sequence↗
PubMed2004

The human protein translin specifically binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n: a study of the binding parameters.

We have previously identified in human fibroblasts a multisubunit protein (designated PGB) that specifically bound single-stranded G-rich microsatellite DNA sequences. PGB was later found to be identical, or closely related to translin, an octameric protein that bound single-stranded DNA consisting of sequences flanking chromosomal translocations. Here, we report that recombinant translin binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n, with higher affinities (Kdis approximately = 2 nM and Kdis approximately = 12.5 nM, respectively, in 100 mM NaCl and 25 degrees C) than the affinity with which it binds a prototypical sequence flanking translocation sites (Kdis approximately = 23 nM). Translin also binds d(GT)n and d(TTAGGG)n overhangs linked to double-stranded DNA with equilibrium constants in the nanomolar range. Formation of DNA quadruplexes by the d(TTAGGG)n repeats inhibits their binding to translin. A further study of the binding parameters revealed that the minimal length of d(GT)n and d(TTAGGG)n oligonucleotides that a translin octamer can bind is 11 nucleotides, but that such oligonucleotides containing up to 30 nucleotides can bind only a single translin octamer. However, the oligonucleotides d(GT)27 and d(TTAGGG)9 bind two octamers with negative cooperativity. Translin does not detectably bind single-stranded d(GT)n sequences embedded within double-stranded DNA. Based on our data, we propose that translin might be involved in the control of recombination at d(GT)n.d(AC)n microsatellites and in telomere maintenance.

DNA-Binding Proteins↗
PubMed2006

Multiplex measurement of cytokine/receptor gene polymorphisms and interaction between interleukin-10 (-1082) genotype and chorioamnionitis in extreme preterm delivery.

OBJECTIVES: To establish a multiplex amplification refractory mutation system (ARMS) in fluid and dried whole blood, and to perform a pilot study to examine the role for single-nucleotide polymorphisms (SNPs) of inflammation-associated genes (interleukin [IL]-1 and -10, tumor necrosis factor-alpha [TNFA], and toll-like receptor-4 [TLR4]) and their interaction with clinical chorioamnionitis (CAM) in prematurity. METHODS: We established a quadruplex ARMS to detect the four above SNPs. Fifty-four women delivered at gestational age less than 32 weeks and 83 healthy female volunteers were genotyped. We compared (1) mothers of preterm infants with volunteers, and (2) women delivered before 29 weeks' gestation (n = 29) with those delivered at 29 to 31 completed weeks (n = 25). RESULTS: Multiplex ARMS is feasible using both fluid and dried whole blood. We found no overall differences in genotype and allele frequencies between mothers of preterm infants and volunteers. Among women who had a preterm delivery, those with both CAM and IL10(-1082)*G allele, the risk for delivery before 29 weeks was markedly increased (odds ratio [OR] 22, 95% confidence interval [CI] 2.5 - 191). CONCLUSION: The presence of both CAM and IL10(-1082)*G might play a role in extreme preterm delivery less than 29 weeks.

Chorioamnionitis↗
PubMed2006

Molecular mechanisms for maintenance of G-rich short tandem repeats capable of adopting G4 DNA structures.

Mammalian genomes contain several types of repetitive sequences. Some of these sequences are implicated in various specific cellular events, including meiotic recombination, chromosomal breaks and transcriptional regulation, and also in several human disorders. In this review, we document the formation of DNA secondary structures by the G-rich repetitive sequences that have been found in several minisatellites, telomeres and in various triplet repeats, and report their effects on in vitro DNA synthesis. d(GGCAG) repeats in the mouse minisatellite Pc-1 were demonstrated to form an intra-molecular folded-back quadruplex structure (also called a G4' structure) by NMR and CD spectrum analyses. d(TTAGGG) telomere repeats and d(CGG) triplet repeats were also shown to form G4' and other unspecified higher order structures, respectively. In vitro DNA synthesis was substantially arrested within the repeats, and this could be responsible for the preferential mutability of the G-rich repetitive sequences. Electrophoretic mobility shift assays using NIH3T3 cell extracts revealed heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A3, which were tightly and specifically bound to d(GGCAG) and d(TTAGGG) repeats with K(d) values in the order of nM. HnRNP A1 unfolded the G4' structure formed in the d(GGCAG)(n) and d(TTAGGG)(n) repeat regions, and also resolved the higher order structure formed by d(CGG) triplet repeats. Furthermore, DNA synthesis arrest at the secondary structures of d(GGCAG) repeats, telomeres and d(CGG) triplet repeats was efficiently repressed by the addition of hnRNP A1. High expression of hnRNPs may contribute to the maintenance of G-rich repetitive sequences, including telomere repeats, and may also participate in ensuring the stability of the genome in cells with enhanced proliferation. Transcriptional regulation of genes, such as c-myc and insulin, by G4 sequences found in the promoter regions could be an intriguing field of research and help further elucidate the biological functions of the hnRNP family of proteins in human diseases.

3T3 Cells↗
PubMed2006

DNA architecture: from G to Z.

G-quadruplexes and Z-DNA are two important non-B forms of DNA architecture. Results on novel structural elements, folding and unfolding kinetics, and interactions with small molecules and proteins have been reported recently for these forms. These results will enhance our understanding of the biology of these structures and provide a platform for drug design.

DNA↗
PubMed2001

Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways.

Calorimetry, Differential Scanning↗
PubMed2003

Increasing sensitivity to arsenic trioxide-induced apoptosis by altered telomere state.

In this work, we investigated the synergic effects between low-dose arsenic trioxide and diethyloxadicarbocyanine (DODC), a telomerase inhibitor, on cell apoptosis. Results revealed that low-dose arsenic could block cell cycle arrest at the G2/M phase and induce apoptosis, whereas DODC could block cell cycle arrest at the G0/G1 phase but not induce apoptosis. However, cells pretreated with DODC showed greater sensitivity to arsenic than untreated cells. The percentage of apoptosis produced by combination treatment with the two agents increased and that was similar to the effect of high-dose arsenic treatment alone. Further studies showed that DODC alone could induce hairpin G-quadruplex formation and inhibit telomerase activity in a dose-dependent manner. Compared with HT1080 cells, 293 cells were more sensitive to cell growth inhibition and apoptosis and were less sensitivity to telomerase activity. These results indicate that DODC can synergistically enhance the apoptosis induced by arsenic, suggesting the increased cell senescence in response to arsenic is induced by an altered telomere state rather than by a loss of telomerase. Thus clinical application of combination treatment with arsenic and telomerase inhibitor may have potential in cancer therapy.

Apoptosis↗
PubMed1995

NMR studies of drug-DNA complexes.

The application of high-resolution, multidimensional NMR techniques to the problem of determining the structure of drug-DNA complexes in solution has led to substantial progress in understanding the effect of drugs on DNA at the molecular level. With the development of isotopic labeling methods applied in three- and four-dimensional experiments, we anticipate that more complex drug-DNA systems will become amenable to structural analysis. In addition to implementing these newer techniques, progress will also be made in terms of investigating the structure of drug complexes with more unusual forms of DNA, such as triplexes, quadruplexes, multistranded junctions, and so forth.

Algorithms↗
PubMed2001

The intrinsically unstable life of DNA triplet repeats associated with human hereditary disorders.

Expansions of specific DNA triplet repeats are the cause of an increasing number of hereditary neurological disorders in humans. In some diseases, such as Huntington's and several spinocerebellar ataxias, the repetitive DNA sequences are translated into long tracts of the same amino acid (usually glutamine), which alters interactions with cellular constituents and leads to the development of disease. For other disorders, including common genetic disorders such as myotonic dystrophy and fragile X syndrome, the DNA repeat is located in noncoding regions of transcribed sequences and disease is probably caused by altered gene expression. In studies in lower organisms, mammalian cells, and transgenic mice, high frequencies of length changes (increases and decreases) occur in long DNA triplet repeats. These observations are similar to other types of repetitive DNA sequences, which also undergo frequent length changes at genomic loci. A variety of processes acting on DNA influence the genetic stability of DNA triplet repeats, including replication, recombination, repair, and transcription. It is not yet known how these different multienzyme systems interact to produce the genetic mutation of expanded repeats. In vitro studies have identified that DNA triplet repeats can adopt several unusual DNA structures, including hairpins, triplexes, quadruplexes, slipped structures, and highly flexible and writhed helices. The formation of stable unusual structures within the cell is likely to disturb DNA metabolism and be a critical intermediate in the molecular mechanism(s) leading to genetic instabilities of DNA repeats and, hence, to disease pathogenesis.

DNA Repair↗
PubMed2001

The use of the LightCycler for the detection of Y chromosome SNPs.

A novel methodology based on PCR monitoring on-line with fluorescent formats using the LightCycler for Y chromosome SNP typing is proposed. The main advantages of the system are the time necessary for the analysis (which is around 20 min), the robustness and the accuracy of the method and especially its sensitivity, which permits the detection of the male component in male-female mixtures up to 1:300 for some of the SNPs. Singleplexes of four different SNPs (M9, sY81, SRY-1532 and SRY-2627) as well as two duplexes (M9 and sY81 on the one hand and SRY-1532 and SRY-2627 on the other) were efficiently implemented. A simultaneous amplification and analysis of the four SNPs is also possible. It seems difficult with the current methodology to implement more than a quadruplex.

Alleles↗
PubMed2002

Detection of activity of telomerase in tumor cells using fiber optical biosensors.

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.

Biomarkers, Tumor↗
PubMed2003

The structure of telomeric DNA.

The telomere is a nucleoprotein complex located at the ends of eukaryotic chromosomes. It is essential for maintaining the integrity of the genome. It is not a linear structure and, for much of the cell cycle, telomeric DNA is maintained in a loop structure, which serves to protect the vulnerable ends of chromosomes. Many of the key proteins in the telomere have been identified, although their interplay is still imperfectly understood and structural data are only available on a few. Telomeric DNA itself comprises simple guanine-rich repeats for most of its length, culminating in a short overhang of single-stranded sequence at the extreme 3' ends. This can, at least in vitro, fold into a wide variety of four-stranded quadruplex structures, many of whose arrangements are being revealed by crystallographic and NMR studies.

DNA↗
PubMed1997

Unusual DNA conformations.

DNA is on the move across conformational space. Duplexes diversity and, joined by triplexes, quadruplexes, loops, bulges and multiarmed junctions, open the route to a bewildering array of increasingly complex conformations. In addition to this structural growth, DNA has come under increasing scrutiny thanks to the development of chemical and physical techniques for deforming its conformation and probing its properties. These investigations help us to learn more about the mechanics and the activity of this remarkably versatile macromolecule.

DNA↗
PubMed1998

Trinucleotide repeat DNA structures: dynamic mutations from dynamic DNA.

Models for the disease-associated expansion of (CTG)n.(CAG)n, (CGG)n.(CCG)n, and (GAA)n.(TTC)n trinucleotide repeats involve alternative DNA structures formed during DNA replication, repair and recombination. These repeat sequences are inherently flexible and can form a variety of hairpins, intramolecular triplexes, quadruplexes, and slipped-strand structures that may be important intermediates and result in their genetic instability.

Base Composition↗