Effects of trimethylamine N-oxide and urea on DNA duplex and G-quadruplex.

Science.gov (United States)

Ueda, Yu-Mi; Zouzumi, Yu-Ki; Maruyama, Atsushi; Nakano, Shu-Ichi; Sugimoto, Naoki; Miyoshi, Daisuke

2016-01-01

We systematically investigated effects of molecular crowding with trimethylamine N -oxide (TMAO) as a zwitterionic and protective osmolyte and urea as a nonionic denaturing osmolyte on conformation and thermodynamics of the canonical DNA duplex and the non-canonical DNA G-quadruplex. It was found that TMAO and urea stabilized and destabilized, respectively, the G-quadruplex. On the other hand, these osmolytes generally destabilize the duplex; however, it was observed that osmolytes having the trimethylamine group stabilized the duplex at the lower concentrations because of a direct binding to a groove of the duplex. These results are useful not only to predict DNA structures and their thermodynamics under physiological environments in living cells, but also design of polymers and materials to regulate structure and stability of DNA sequences.

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

Science.gov (United States)

Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

2015-01-01

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

Science.gov (United States)

Zhang, Bo; Wu, Wen-Qiang; Liu, Na-Nv; Duan, Xiao-Lei; Li, Ming; Dou, Shuo-Xing; Hou, Xi-Miao; Xi, Xu-Guang

2016-01-01

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells. PMID:27471032

Protein complexation with DNA phosphates as a cause for DNA duplex destabilization : a thermodynamic model

NARCIS (Netherlands)

Genderen, van M.H.P.; Buck, H.M.

1989-01-01

Complexation of positively charged sites in a protein with the negative DNA phosphate groups shields the phosphate charges. This diminishes interstrand electrostatic repulsions, which stabilizes the duplex. When phosphate shidlding is present in one DNA strand only, the conformation of this strand

Structural studies of the 5'-phenazinium-tethered matched and G-A-mismatched DNA duplexes by NMR spectroscopy.

Science.gov (United States)

Maltseva, T; Sandström, A; Ivanova, I M; Sergeyev, D S; Zarytova, V F; Chattopadhyaya, J

1993-05-01

The mechanism through which modified oligo-DNA analogues act as antisense repressors at the transcriptional and translational level of gene expression is based on the information content in the nucleotide sequence which is determined by the specific base pairing. The efficiency of such action is largely determined by the stability of the duplex formed between the oligonucleotide reagent and the target sequence and also by the mismatched base pairing, such as G-A, that occurs during replication or recombination. We herein report that the phenazinium (Pzn)-tethered matched duplex p(d(TGTTTGGC)):(Pzn)-p(d(CCAAACA)) (III) (Tm = 50 degrees C) has a much larger stability than the parent matched duplex p(d(TGTTTGGC)):p(d(CCAAACA)) (I) (Tm = 30 degrees C). On the other hand, the Pzn-tethered G-A-mismatched duplex p(d(TGTTTGGC)):(Pzn)-p(d(ACAAACA)) (IV) (Tm = 34 degrees C) is only slightly more stable than its parent mismatched duplex p(d(TGTTTGGC)):p(d(ACAAACA)) (Tm = 25 degrees C). A detailed 500 MHz NMR study and constrained MD refinements of NMR-derived structures have been undertaken for the DNA duplexes (I), (II), (III) and (IV) in order to understand the structural basis of stabilization of Pzn-tethered matched DNA duplex (delta Tm = 20 degrees C) compared to mismatched duplex (delta Tm = 9 degrees C). Assignment of the 1H-NMR (500 MHz) spectra of the duplexes has been carried out by 2D NOESY, HOHAHA and DQF-COSY experiments. The torsion angles have been extracted from the J-coupling constants obtained by simulation of most of the DQF-COSY cross-peaks using program SMART. The solution structure of the duplexes were assessed by an iterative hybride relaxation matrix method (MORASS) combined with NOESY distances and torsion angles restrained molecular dynamics (MD) using program Amber 4.0. The standard Amber 4.0 force-field parameters were used for the oligonucleotide in conjunction with the new parameters for Pzn residue which was obtained by full geometry

Triple helical DNA in a duplex context and base pair opening

Science.gov (United States)

Esguerra, Mauricio; Nilsson, Lennart; Villa, Alessandra

2014-01-01

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region. PMID:25228466

Enhancement of fluorescence quenching and exciplex formation in DNA major groove by double incorporation of modified fluorescent deoxyuridines.

Science.gov (United States)

Tanaka, Makiko; Oguma, Kazuhiro; Saito, Yoshio; Saito, Isao

2012-06-15

5-(1-Naphthalenylethynyl)-2'-deoxyuridine ((N)U) and 5-[(4-cyano-1-naphthalenyl)ethynyl]-2'-deoxyuridine ((CN)U) were synthesized and incorporated into oligodeoxynucleotides. Fluorescence emissions of modified duplexes containing double (N)U were efficiently quenched depending upon the sequence pattern of the naphthalenes in DNA major groove, as compared to the duplex possessing single (N)U. When one of the naphthalene moieties has a cyano substituent, the exciplex emission from the chromophores in DNA major groove was observed at longer wavelength. Copyright © 2012 Elsevier Ltd. All rights reserved.

Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by 1H and 15N NMR studies

International Nuclear Information System (INIS)

Lin, Chin Hsiung; Hurley, L.H.

1990-01-01

(+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. The [6- 15 N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1 H and 15 N NMR. One-dimensional NOE difference and two-dimensional NOESY 1 H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6- 15 N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the 15 N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. The authors conclude that the covalently modified adenine N6 of the (+)-CC-1065-12-mer duplex adduct is predominantly in the doubly protonated form, in which calculations predict that the C6-N6 bond is shortened and the positive charge is delocalized over the entire adenine molecule

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

Science.gov (United States)

Odell, M; Shuman, S

1999-05-14

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions

International Nuclear Information System (INIS)

Chou, Shanho; Flynn, P.; Wang, A.; Reid, B.

1991-01-01

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(AACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but these results differ somewhat from previous theoretical model studies

Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

Science.gov (United States)

Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

2013-01-01

Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

Science.gov (United States)

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-05-23

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

Crystal structure of metallo DNA duplex containing consecutive Watson-Crick-like T-Hg(II)-T base pairs.

Science.gov (United States)

Kondo, Jiro; Yamada, Tom; Hirose, Chika; Okamoto, Itaru; Tanaka, Yoshiyuki; Ono, Akira

2014-02-24

The metallo DNA duplex containing mercury-mediated T-T base pairs is an attractive biomacromolecular nanomaterial which can be applied to nanodevices such as ion sensors. Reported herein is the first crystal structure of a B-form DNA duplex containing two consecutive T-Hg(II)-T base pairs. The Hg(II) ion occupies the center between two T residues. The N3-Hg(II) bond distance is 2.0 Å. The relatively short Hg(II)-Hg(II) distance (3.3 Å) observed in consecutive T-Hg(II)-T base pairs suggests that the metallophilic attraction could exist between them and may stabilize the B-form double helix. To support this, the DNA duplex is largely distorted and adopts an unusual nonhelical conformation in the absence of Hg(II). The structure of the metallo DNA duplex itself and the Hg(II)-induced structural switching from the nonhelical form to the B-form provide the basis for structure-based design of metal-conjugated nucleic acid nanomaterials. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

6-Thioguanine alters the structure and stability of duplex DNA and inhibits quadruplex DNA formation.

Science.gov (United States)

Marathias, V M; Sawicki, M J; Bolton, P H

1999-07-15

The ability to chemically synthesize biomolecules has opened up the opportunity to observe changes in structure and activity that occur upon single atom substitution. In favorable cases this can provide information about the roles of individual atoms. The substitution of 6-thioguanine (6SG) for guanine is a potentially very useful single atom substitution as 6SG has optical, photocrosslinking, metal ion binding and other properties of potential utility. In addition, 6-mercaptopurine is a clinically important pro-drug that is activated by conversion into 6SG by cells. The results presented here indicate that the presence of 6SG blocks the formation of quadruplex DNA. The presence of 6SG alters the structure and lowers the thermal stability of duplex DNA, but duplex DNA can be formed in the presence of 6SG. These results indicate that some of the cytotoxic activity of 6SG may be due to disruption of the quadruplex structures formed by telomere and other DNAs. This additional mode of action is consistent with the delayed onset of cytotoxicity.

Role of the heat capacity change in understanding and modeling melting thermodynamics of complementary duplexes containing standard and nucleobase-modified LNA.

Science.gov (United States)

Hughesman, Curtis B; Turner, Robin F B; Haynes, Charles A

2011-06-14

Melting thermodynamic data obtained by differential scanning calorimetry (DSC) are reported for 43 duplexed oligonucleotides containing one or more locked nucleic acid (LNA) substitutions. The measured heat capacity change (ΔC(p)) for the helix-to-coil transition is used to compute the changes in enthalpy and entropy for melting of an LNA-bearing duplex at the T(m) of its corresponding isosequential unmodified DNA duplex to allow rigorous thermodynamic analysis of the stability enhancements provided by LNA substitutions. Contrary to previous studies, our analysis shows that the origin of the improved stability is almost exclusively a net reduction (ΔΔS° thermodynamics and the increased melting temperature (ΔT(m)) of heteroduplexes formed between an unmodified DNA strand and a complementary strand containing any number and configuration of standard LNA nucleotides A, T, C, and G. This single-base thermodynamic (SBT) model requires only four entropy-related parameters in addition to ΔC(p). Finally, DSC data for 20 duplexes containing the nucleobase-modified LNAs 2-aminoadenine (D) and 2-thiothymine (H) are reported and used to determine SBT model parameters for D and H. The data and model suggest that along with the greater stability enhancement provided by D and H bases relative to their corresponding A and T analogues, the unique pseudocomplementary properties of D-H base pairs may make their use appealing for in vitro and in vivo applications.

Effect of structure variation of the aptamer-DNA duplex probe on the performance of displacement-based electrochemical aptamer sensors.

Science.gov (United States)

Pang, Jie; Zhang, Ziping; Jin, Haizhu

2016-03-15

Electrochemical aptamer-based (E-AB) sensors employing electrode-immobilized, redox-tagged aptamer probes have emerged as a promising platform for the sensitive and quick detection of target analytes ranging from small molecules to proteins. Signal generation in this class of sensor is linked to change in electron transfer efficiency upon binding-induced change in flexibility/conformation of the aptamer probe. Because of this signaling mechanism, signal gains of these sensors can be improved by employing a displacement-based recognition system, which links target binding with a large-scale flexibility/conformation shift from the aptamer-DNA duplex to the single-stranded DNA or the native aptamer. Despite the relatively large number of displacement-based E-AB sensor samples, little attention has been paid to the structure variation of the aptamer-DNA duplex probe. Here we detail the effects of complementary length and position of the aptamer-DNA duplex probe on the performance of a model displacement-based E-AB sensor for ATP. We find that, greater background suppression and signal gain are observed with longer complementary length of the aptamer-DNA duplex probe. However, sensor equilibration time slows monotonically with increasing complementary length; and with too many target binding sites in aptamer sequence being occupied by the complementary DNA, the aptamer-target binding does not occur and no signal gain observed. We also demonstrate that signal gain of the displacement-based E-AB sensor is strongly dependent on the complementary position of the aptamer-DNA duplex probe, with complementary position located at the electrode-attached or redox-tagged end of the duplex probe, larger background suppression and signal increase than that of the middle position are observed. These results highlight the importance of rational structure design of the aptamer-DNA duplex probe and provide new insights into the optimization of displacement-based E-AB sensors. Copyright

An exploration of sequence specific DNA-duplex/pyrene interactions for intercalated and surface-associated pyrene species. Final report, May 1, 1993--December 31, 1996

Energy Technology Data Exchange (ETDEWEB)

Netzel, T.L.

1997-03-01

The broad objective of this DOE sponsored work on photoinduced electron transfer (ET) within covalently modified DNA was to learn about the rates of Et among various DNA bases and commonly used organic electron donor (D) and acceptor (A) molecules. This hypothesis driven, multidisciplinary project combined skills in modified nucleic acid synthesis and in continuous and time-resolved optical spectroscopies. Covalently modified DNA chemistry as investigated in this program had two specific long term goals. The first was to use experimental and theoretical insights into the mechanisms of electron transfer (ET) reactions to design supramolecular assemblies of redox-active chromophores that function as efficient vectorial ET engines. The second was to construct oligonucleotide probes for real-time monitoring of intracellular processes involving DNA and RNA such as m-RNA expression and translocation. This research project laid the groundwork for studying ET reactions within DNA duplexes by examining the photophysics of uridine nucleosides which are covalently labeled at the 5-position with 1-pyrenyl chromophores.

Pulsed IR Heating Studies of Single-Molecule DNA Duplex Dissociation Kinetics and Thermodynamics

Science.gov (United States)

Holmstrom, Erik D.; Dupuis, Nicholas F.; Nesbitt, David J.

2014-01-01

Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10−11 liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20–100°C); 2), substantially superior spatial (≤20 μm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation. PMID:24411254

Duplex Alu Screening for Degraded DNA of Skeletal Human Remains

Directory of Open Access Journals (Sweden)

Fabian Haß

2017-10-01

Full Text Available The human-specific Alu elements, belonging to the class of Short INterspersed Elements (SINEs, have been shown to be a powerful tool for population genetic studies. An earlier study in this department showed that it was possible to analyze Alu presence/absence in 3000-year-old skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. We developed duplex Alu screening PCRs with flanking primers for two Alu elements, each combined with a single internal Alu primer. By adding an internal primer, the approximately 400–500 bp presence signals of Alu elements can be detected within a range of less than 200 bp. Thus, our PCR approach is suited for highly fragmented ancient DNA samples, whereas NGS analyses frequently are unable to handle repetitive elements. With this analysis system, we examined remains of 12 individuals from the Lichtenstein cave with different degrees of DNA degradation. The duplex PCRs showed fully informative amplification results for all of the chosen Alu loci in eight of the 12 samples. Our analysis system showed that Alu presence/absence analysis is possible in samples with different degrees of DNA degradation and it reduces the amount of valuable skeletal material needed by a factor of four, as compared with a singleplex approach.

Bis-pyrene-modified unlocked nucleic acids: synthesis, hybridization studies, and fluorescent properties

DEFF Research Database (Denmark)

Perlíková, Pavla; Ejlersen, Maria; Langkjaer, Niels

2014-01-01

Efficient synthesis of a building block for the incorporation of a bis-pyrene-modified unlocked nucleic acid (UNA) into oligonucleotides (DNA*) was developed. The presence of bis-pyrene-modified UNA within a duplex leads to duplex destabilization that is more profound in DNA*/RNA and less distinc......)uracil:pyrene exciplex emission in the single-stranded form. Such fluorescent properties enable the application of bis-pyrene-modified UNA in the development of fluorescence probes for DNA/RNA detection and for detection of deletions at specific positions....

A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.

Science.gov (United States)

Rampazzo, Rita de Cássia Pontello; Solcà, Manuela da Silva; Santos, Liliane Celestino Sales; Pereira, Lais de Novaes; Guedes, José Carlos Oliveira; Veras, Patrícia Sampaio Tavares; Fraga, Deborah Bittencourt Mothé; Krieger, Marco Aurélio; Costa, Alexandre Dias Tavares

2017-11-15

Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format

Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

Science.gov (United States)

Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

2017-04-01

Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone

OpenAIRE

Bergeron, François; Houde, Daniel; Hunting, Darel J.; Wagner, J. Richard

2004-01-01

2-Methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ–DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences ...

Duplex Healing of Selectively Thiolated Guanosine Mismatches through a Cd2+ Chemical Stimulus.

Science.gov (United States)

Lunn, Samantha M L; Hribesh, Samira; Whitfield, Colette J; Hall, Michael J; Houlton, Andrew; Bronowska, Agnieszka K; Tuite, Eimer M; Pike, Andrew R

2018-03-25

The on-column selective conversion of guanosine to thioguanosine (tG) yields modified oligomers that exhibit destabilisation over the fully complementary duplex. Restoration to a stabilised duplex is induced through thio-directed Cd 2+ coordination; a route for healing DNA damage. Short oligomers are G-specifically thiolated through a modified on-column protocol without the need for costly thioguanosine phosphoramidites. Addition of Cd 2+ ions to a duplex containing a highly disrupted tG central mismatch sequence, 3'-A 6 tG 4 T 6 -5', suggests a (tG) 8 Cd 2 central coordination regime, resulting in increased base stacking and duplex stability. Equilibrium molecular dynamic calculations support the hypothesis of metal-induced healing of the thiolated duplex. The 2 nm displacement of the central tG mismatched region is dramatically reduced after the addition of a chemical stimuli, Cd 2+ ions, returning to a minimized fluctuational state comparable to the unmodified fully complementary oligomer. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Long-Range Vibrational Dynamics Are Directed by Watson-Crick Base Pairing in Duplex DNA.

Science.gov (United States)

Hithell, Gordon; Shaw, Daniel J; Donaldson, Paul M; Greetham, Gregory M; Towrie, Michael; Burley, Glenn A; Parker, Anthony W; Hunt, Neil T

2016-05-05

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar-phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson-Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA.

Combined Triplex/Duplex Invasion of Double-Stranded DNA by "Tail-Clamp" Peptide Nucleic Acid

DEFF Research Database (Denmark)

Bentin, Thomas; Larsen, H. J.; Nielsen, Peter E.

2003-01-01

as determined by T-m measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C-50 measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed...

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

Science.gov (United States)

Yang, Zhiyu; Price, Nathan E.; Johnson, Kevin M.

2017-01-01

Abstract Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3’ddR5p) at the 3’-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3’ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3’ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. PMID:28531327

Unstable Hoogsteen base pairs adjacent to echinomycin binding sites within a DNA duplex

International Nuclear Information System (INIS)

Gilbert, D.E.; van der Marel, G.A.; van Boom, J.H.; Feigon, J.

1989-01-01

The bisintercalation complex present between the DNA octamer [d(ACGTACGT)] 2 and the cyclic octadepsipeptide antibiotic echinomycin has been studied by one- and two-dimensional proton NMR, and the results obtained have been compared with the crystal structures of related DNA-echinomycin complexes. Two echinomycins are found to bind cooperatively to each DNA duplex at the CpG steps, with the two quinoxaline rings of each echinomycin bisintercalating between the C·G and A·T base pairs. At low temperatures, the A·T base pairs on either side of the intercalation site adopt the Hoogsteen conformation, as observed in the crystal structures. However, as the temperature is raised, the Hoogsteen base pairs in the interior of the duplex are destabilized and are observed to be exchanging between the Hoogsteen base pair and either an open or a Watson-Crick base-paired state. The terminal A·T base pairs, which are not as constrained by the helix as the internal base pairs, remain stably Hoogsteen base-paired up to at least 45 degree C. The implications of these results for the biological role of Hoogsteen base pairs in echinomycin-DNA complexes in vivo are discussed

Binding modes and pathway of RHPS4 to human telomeric G-quadruplex and duplex DNA probed by all-atom molecular dynamics simulations with explicit solvent.

Science.gov (United States)

Mulholland, Kelly; Siddiquei, Farzana; Wu, Chun

2017-07-19

RHPS4, a potent binder to human telomeric DNA G-quadruplex, shows high efficacy in tumor cell growth inhibition. However, it's preferential binding to DNA G-quadruplex over DNA duplex (about 10 fold) remains to be improved toward its clinical application. A high resolution structure of the single-stranded telomeric DNA G-quadruplexes, or B-DNA duplex, in complex with RHPS4 is not available yet, and the binding nature of this ligand to these DNA forms remains to be elusive. In this study, we carried out 40 μs molecular dynamics binding simulations with a free ligand to decipher the binding pathway of RHPS4 to a DNA duplex and three G-quadruplex folders (parallel, antiparallel and hybrid) of the human telomeric DNA sequence. The most stable binding mode identified for the duplex, parallel, antiparallel and hybrid G-quadruplexes is an intercalation, bottom stacking, top intercalation and bottom intercalation mode, respectively. The intercalation mode with similar binding strength to both the duplex and the G-quadruplexes, explains the lack of binding selectivity of RHPS4 to the G-quadruplex form. Therefore, a ligand modification that destabilizes the duplex intercalation mode but stabilizes the G-quadruplex intercalation mode will improve the binding selectivity toward G-quadruplex. The intercalation mode of RHPS4 to both the duplex and the antiparallel and the hybrid G-quadruplex follows a base flipping-insertion mechanism rather than an open-insertion mechanism. The groove binding, the side binding and the intercalation with flipping out of base were observed to be intermediate states before the full intercalation state with paired bases.

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

Science.gov (United States)

Yang, Zhiyu; Price, Nathan E; Johnson, Kevin M; Wang, Yinsheng; Gates, Kent S

2017-06-20

Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components

Directory of Open Access Journals (Sweden)

Marian Filipiak

2008-03-01

Full Text Available An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5’-phosphate end to amino group of cysteamine self-assembled monolayer (SAM on gold electrode surface with the use of activating reagents – water soluble 1-ethyl-3(3’- dimethylaminopropyl-carbodiimide (EDC and N-hydroxy-sulfosuccinimide (NHS. The hybridization reaction on the electrode surface was detected via methylene blue (MB presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs give a broad perspectives for analytical application of the biosensor.

Chemically modified oligonucleotides with efficient RNase H response

DEFF Research Database (Denmark)

Vester, Birte; Boel, Anne Marie; Lobedanz, Sune

2008-01-01

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly...... in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage....

Duplex Interrogation by a Direct DNA Repair Protein in Search of Base Damage

Science.gov (United States)

Yi, Chengqi; Chen, Baoen; Qi, Bo; Zhang, Wen; Jia, Guifang; Zhang, Liang; Li, Charles J.; Dinner, Aaron R.; Yang, Cai-Guang; He, Chuan

2012-01-01

ALKBH2 is a direct DNA repair dioxygenase guarding mammalian genome against N1-methyladenine, N3-methylcytosine, and 1,N6-ethenoadenine damage. A prerequisite for repair is to identify these lesions in the genome. Here we present crystal structures of ALKBH2 bound to different duplex DNAs. Together with computational and biochemical analyses, our results suggest that DNA interrogation by ALKBH2 displays two novel features: i) ALKBH2 probes base-pair stability and detects base pairs with reduced stability; ii) ALKBH2 does not have nor need a “damage-checking site”, which is critical for preventing spurious base-cleavage for several glycosylases. The demethylation mechanism of ALKBH2 insures that only cognate lesions are oxidized and reversed to normal bases, and that a flipped, non-substrate base remains intact in the active site. Overall, the combination of duplex interrogation and oxidation chemistry allows ALKBH2 to detect and process diverse lesions efficiently and correctly. PMID:22659876

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

Science.gov (United States)

Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.

2005-09-01

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: predictive thermodynamic model.

Science.gov (United States)

Moreira, Bernardo G; You, Yong; Owczarzy, Richard

2015-03-01

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5' end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2 kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine-purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications. Copyright © 2015. Published by Elsevier B.V.

The first example of a Hoogsteen base-paired DNA duplex in dynamic equilibrium with a Watson-Crick base-paired duplex--a structural (NMR), kinetic and thermodynamic study.

Science.gov (United States)

Isaksson, J; Zamaratski, E; Maltseva, T V; Agback, P; Kumar, A; Chattopadhyaya, J

2001-06-01

A single-point substitution of the O4' oxygen by a CH2 group at the sugar residue of A6 (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C1G2C3G4A5A6T7T8C9G10C11G12)2(-3), has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)(1B): Keq = k1/k(-1) = 0.56+/-0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9 0.8 sec(-1); deltaHdegrees++ = 164+/-14 kJ/mol; -TdeltaS degrees++ (298K) = -92 kJ/mol giving a deltaG degrees++ 298 of 72 kJ/mol. Ea (k1) = 167 14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 0.6 sec-1, deltaH degrees++ = 153 13 kJ/mol; -TdeltaSdegrees++ (298K) = -82 kJ/mol giving a deltaGdegrees++(298) of 71 kJ/mol. Ea (k-1) = 155 13 kJ/mol]. Acomparison of deltaGdegrees++(298) of the forward (k1) and backward (k-1) conversions, (1A)(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition

DNA oligonucleotide duplexes containing intramolecular platinated cross-links: energetics, hydration, sequence, and ionic effects.

Science.gov (United States)

Kankia, Besik I; Soto, Ana Maria; Burns, Nicole; Shikiya, Ronald; Tung, Chang-Shung; Marky, Luis A

2002-11-05

The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross-links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross-links. The incorporation of these platinum adducts in a B-DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature-dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis-[Pt(NH(3))(2)[d(GpG

Molecular dynamics study of some non-hydrogen-bonding base pair DNA strands

Science.gov (United States)

Tiwari, Rakesh K.; Ojha, Rajendra P.; Tiwari, Gargi; Pandey, Vishnudatt; Mall, Vijaysree

2018-05-01

In order to elucidate the structural activity of hydrophobic modified DNA, the DMMO2-D5SICS, base pair is introduced as a constituent in different set of 12-mer and 14-mer DNA sequences for the molecular dynamics (MD) simulation in explicit water solvent. AMBER 14 force field was employed for each set of duplex during the 200ns production-dynamics simulation in orthogonal-box-water solvent by the Particle-Mesh-Ewald (PME) method in infinite periodic boundary conditions (PBC) to determine conformational parameters of the complex. The force-field parameters of modified base-pair were calculated by Gaussian-code using Hartree-Fock /ab-initio methodology. RMSD Results reveal that the conformation of the duplex is sequence dependent and the binding energy of the complex depends on the position of the modified base-pair in the nucleic acid strand. We found that non-bonding energy had a significant contribution to stabilising such type of duplex in comparison to electrostatic energy. The distortion produced within strands by such type of base-pair was local and destabilised the duplex integrity near to substitution, moreover the binding energy of duplex depends on the position of substitution of hydrophobic base-pair and the DNA sequence and strongly supports the corresponding experimental study.

Sequence distribution of acetaldehyde-derived N2-ethyl-dG adducts along duplex DNA.

Science.gov (United States)

Matter, Brock; Guza, Rebecca; Zhao, Jianwei; Li, Zhong-ze; Jones, Roger; Tretyakova, Natalia

2007-10-01

Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence

Correlation between the thermodynamic stability of DNA duplexes and the interaction and solvation energies of DNA building blocks

Czech Academy of Sciences Publication Activity Database

Řezáč, Jan; Hobza, Pavel

2008-01-01

Roč. 73, č. 2 (2008), s. 161-174 ISSN 0010-0765 R&D Projects: GA MŠk LC512; GA ČR GA203/05/0009; GA ČR(CZ) GD203/05/H001 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA duplex * interaction energy * stability Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.784, year: 2008

C-5 Propynyl Modifications Enhance the Mechanical Stability of DNA.

Science.gov (United States)

Aschenbrenner, Daniela; Baumann, Fabian; Milles, Lukas F; Pippig, Diana A; Gaub, Hermann E

2015-07-20

Increased thermal or mechanical stability of DNA duplexes is desired for many applications in nanotechnology or -medicine where DNA is used as a programmable building block. Modifications of pyrimidine bases are known to enhance thermal stability and have the advantage of standard base-pairing and easy integration during chemical DNA synthesis. Through single-molecule force spectroscopy experiments with atomic force microscopy and the molecular force assay we investigated the effect of pyrimidines harboring C-5 propynyl modifications on the mechanical stability of double-stranded DNA. Utilizing these complementary techniques, we show that propynyl bases significantly increase the mechanical stability if the DNA is annealed at high temperature. In contrast, modified DNA complexes formed at room temperature and short incubation times display the same stability as non-modified DNA duplexes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On the Formation and Properties of Interstrand DNA-DNA Cross-links Forged by Reaction of an Abasic Site With the Opposing Guanine Residue of 5′-CAp Sequences in Duplex DNA

Science.gov (United States)

Johnson, Kevin M.; Price, Nathan E.; Wang, Jin; Fekry, Mostafa I.; Dutta, Sanjay; Seiner, Derrick R.; Wang, Yinsheng; Gates, Kent S.

2014-01-01

We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5′-CAp sequence under conditions of reductive amination (NaCNBH3, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions and, once formed, the cross-link was stable to a variety of work-up conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LCMS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N2-amino group of the opposing guanine residue in 5′-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH3/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH3. PMID:23215239

3'-Pyrene-modified unlocked nucleic acids

DEFF Research Database (Denmark)

Ejlersen, Maria; Langkjær, Niels; Wengel, Jesper

2017-01-01

in the minor groove of DNA:DNA duplexes as confirmed by CD and UV/Vis absorption studies. Upon multiple incorporations of the monomer in single-stranded ONs, steady-state fluorescence emission studies revealed the formation of a pyrene excimer which in most cases was quenched upon duplex hybridization......, and fluorescence-based detection of mismatched hybridization was observed for some modified strand constitutions. Incorporation of the monomer in a triplex-forming oligonucleotide (TFO) strand lead to an increase of triplex melting temperature both at pH 6.0 and pH 7.0 for parallel triplexes - again an effect...

Exploring the Interactions of the Dietary Plant Flavonoids Fisetin and Naringenin with G-Quadruplex and Duplex DNA, Showing Contrasting Binding Behavior: Spectroscopic and Molecular Modeling Approaches.

Science.gov (United States)

Bhattacharjee, Snehasish; Chakraborty, Sandipan; Sengupta, Pradeep K; Bhowmik, Sudipta

2016-09-01

Guanine-rich sequences have the propensity to fold into a four-stranded DNA structure known as a G-quadruplex (G4). G4 forming sequences are abundant in the promoter region of several oncogenes and become a key target for anticancer drug binding. Here we have studied the interactions of two structurally similar dietary plant flavonoids fisetin and naringenin with G4 as well as double stranded (duplex) DNA by using different spectroscopic and modeling techniques. Our study demonstrates the differential binding ability of the two flavonoids with G4 and duplex DNA. Fisetin more strongly interacts with parallel G4 structure than duplex DNA, whereas naringenin shows stronger binding affinity to duplex rather than G4 DNA. Molecular docking results also corroborate our spectroscopic results, and it was found that both of the ligands are stacked externally in the G4 DNA structure. C-ring planarity of the flavonoid structure appears to be a crucial factor for preferential G4 DNA recognition of flavonoids. The goal of this study is to explore the critical effects of small differences in the structure of closely similar chemical classes of such small molecules (flavonoids) which lead to the contrasting binding properties with the two different forms of DNA. The resulting insights may be expected to facilitate the designing of the highly selective G4 DNA binders based on flavonoid scaffolds.

Free energy landscape and transition pathways from Watson–Crick to Hoogsteen base pairing in free duplex DNA

Science.gov (United States)

Yang, Changwon; Kim, Eunae; Pak, Youngshang

2015-01-01

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events. PMID:26250116

Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

DEFF Research Database (Denmark)

Izvolsky, K I; Demidov, V V; Nielsen, P E

2000-01-01

I restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate...... DNA duplexes in a virtually sequence-unrestricted manner....

Toehold-Mediated Displacement of an Adenosine-Binding Aptamer from a DNA Duplex by its Ligand.

Science.gov (United States)

Monserud, Jon H; Macri, Katherine M; Schwartz, Daniel K

2016-10-24

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand-displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high-throughput single-molecule FRET methods, we compared the kinetics of a putative aptamer-ligand and aptamer-complement strand-displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand-displacement concept can be extended to functional DNA-ligand systems. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

NMR studies of abasic sites in DNA duplexes: Deoxyadenosine stacks into the helix opposite acyclic lesions

International Nuclear Information System (INIS)

Kalnik, M.W.; Chang, Chienneng; Johnson, F.; Grollman, A.P.; Patel, D.J.

1989-01-01

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C)·d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated AP P 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C-)·d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated AP E 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the AP P 9-mer and AP E 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H 2 O and D 2 O solution between -5 and 5 degree C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and El14) and stacks between the flanking G4·C15 and G6·C13 Watson-Crick base pairs in both the AP P 9-mer and AP E 9-mer duplexes. Proton NMR parameters for the Ap P 9-mer and AP E 9-mer duplexes are similar to those reported previously. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the AP P 9-mer and AP E 9-mer duplexes (5 degree C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles

Free energy landscape and transition pathways from Watson-Crick to Hoogsteen base pairing in free duplex DNA.

Science.gov (United States)

Yang, Changwon; Kim, Eunae; Pak, Youngshang

2015-09-18

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson-Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine-thymine (A-T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry. Detection of Abasic Sites in Duplex DNA by EPR Spectroscopy

DEFF Research Database (Denmark)

Sigurdsson, Snorri; Vogel, Stefan; Shelke, Sandip

2010-01-01

and the nitroxide spin label. The spin label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy and give information about other structural deformations as well as local conformational changes in DNA. For example, reduced mobility of the spin label in a mismatched pair...... label out of the duplex and toward the solution. Thus, reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for EPR studies of nucleic acids...

Simultaneous Binding of Hybrid Molecules Constructed with Dual DNA-Binding Components to a G-Quadruplex and Its Proximal Duplex.

Science.gov (United States)

Asamitsu, Sefan; Obata, Shunsuke; Phan, Anh Tuân; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2018-03-20

A G-quadruplex (quadruplex) is a nucleic acid secondary structure adopted by guanine-rich sequences and is considered to be relevant to various pharmacological and biological contexts. Although a number of researchers have endeavored to discover and develop quadruplex-interactive molecules, poor ligand designability originating from topological similarity of the skeleton of diverse quadruplexes has remained a bottleneck for gaining specificity for individual quadruplexes. This work reports on hybrid molecules that were constructed with dual DNA-binding components, a cyclic imidazole/lysine polyamide (cIKP), and a hairpin pyrrole/imidazole polyamide (hPIP), with the aim toward specific quadruplex targeting by reading out the local duplex DNA sequence adjacent to designated quadruplexes in the genome. By means of circular dichroism (CD), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and NMR techniques, we showed the dual and simultaneous recognition of the respective segment via hybrid molecules, and the synergistic and mutual effect of each binding component that was appropriately linked on higher binding affinity and modest sequence specificity. Monitoring quadruplex and duplex imino protons of the quadruplex/duplex motif titrated with hybrid molecules clearly revealed distinct features of the binding of hybrid molecules to the respective segments upon their simultaneous recognition. A series of the systematic and detailed binding assays described here showed that the concept of simultaneous recognition of quadruplex and its proximal duplex by hybrid molecules constructed with the dual DNA-binding components may provide a new strategy for ligand design, enabling targeting of a large variety of designated quadruplexes at specific genome locations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

Science.gov (United States)

Soto, Ana Maria; Kankia, Besik I.; Dande, Prasad; Gold, Barry; Marky, Luis A.

2002-01-01

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔHITC–Δ(TΔS) compensation, negative ΔΔV and a net release of

Widespread Transient Hoogsteen Base-Pairs in Canonical Duplex DNA with Variable Energetics

Science.gov (United States)

Alvey, Heidi S.; Gottardo, Federico L.; Nikolova, Evgenia N.; Al-Hashimi, Hashim M.

2015-01-01

Hoogsteen base-pairing involves a 180 degree rotation of the purine base relative to Watson-Crick base-pairing within DNA duplexes, creating alternative DNA conformations that can play roles in recognition, damage induction, and replication. Here, using Nuclear Magnetic Resonance R1ρ relaxation dispersion, we show that transient Hoogsteen base-pairs occur across more diverse sequence and positional contexts than previously anticipated. We observe sequence-specific variations in Hoogsteen base-pair energetic stabilities that are comparable to variations in Watson-Crick base-pair stability, with Hoogsteen base-pairs being more abundant for energetically less favorable Watson-Crick base-pairs. Our results suggest that the variations in Hoogsteen stabilities and rates of formation are dominated by variations in Watson-Crick base pair stability, suggesting a late transition state for the Watson-Crick to Hoogsteen conformational switch. The occurrence of sequence and position-dependent Hoogsteen base-pairs provide a new potential mechanism for achieving sequence-dependent DNA transactions. PMID:25185517

Dual door entry to exciplex emission in a chimeric DNA duplex containing non-nucleoside-nucleoside pair.

Science.gov (United States)

Bag, Subhendu Sekhar; Talukdar, Sangita; Kundu, Rajen; Saito, Isao; Jana, Subhashis

2014-01-25

Dual door entry to exciplex formation was established in a chimeric DNA duplex wherein a fluorescent non-nucleosidic base surrogate () is paired against a fluorescent nucleosidic base surrogate (). Packing of the nucleobases via intercalative stacking interactions led to an exciplex emission either via FRET from the donor or direct excitation of the FRET acceptor .

Synthesis, DNA Binding, and Anticancer Properties of Bis-Naphthalimide Derivatives with Lysine-Modified Polyamine Linkers

Directory of Open Access Journals (Sweden)

Yu Huang

2018-01-01

Full Text Available A series of bis-naphthalimide derivatives with different diamine linkers were designed and synthesized. All of the synthesized bis-naphthalimide derivatives were characterized by NMR and HRMS spectra. The binding ability between the compounds and CT DNA was evaluated by using UV–Vis titration experiments. The bis-naphthalimide compound with an ethylenediamine linker showed the largest binding constant with CT DNA. Hence, it was used as the model compound to study the DNA binding selectivity by UV–Vis titration aiming at different DNA duplexes. As a result, this compound showed binding preference to AT-rich duplexes. The DNA binding modes of the compounds were also measured by viscosity titration. The cytotoxicity of the compounds was evaluated by MTT assay. Compounds with 1,6-diaminohexane or 1,4-phenylenedimethanamine linkers showed higher cytotoxicity compared with other bis-naphthalimide derivatives.

Structural Properties of G,T-Parallel Duplexes

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Anna Aviñó

2010-01-01

Full Text Available The structure of G,T-parallel-stranded duplexes of DNA carrying similar amounts of adenine and guanine residues is studied by means of molecular dynamics (MD simulations and UV- and CD spectroscopies. In addition the impact of the substitution of adenine by 8-aminoadenine and guanine by 8-aminoguanine is analyzed. The presence of 8-aminoadenine and 8-aminoguanine stabilizes the parallel duplex structure. Binding of these oligonucleotides to their target polypyrimidine sequences to form the corresponding G,T-parallel triplex was not observed. Instead, when unmodified parallel-stranded duplexes were mixed with their polypyrimidine target, an interstrand Watson-Crick duplex was formed. As predicted by theoretical calculations parallel-stranded duplexes carrying 8-aminopurines did not bind to their target. The preference for the parallel-duplex over the Watson-Crick antiparallel duplex is attributed to the strong stabilization of the parallel duplex produced by the 8-aminopurines. Theoretical studies show that the isomorphism of the triads is crucial for the stability of the parallel triplex.

LNA effects on DNA binding and conformation

DEFF Research Database (Denmark)

Pabon-Martinez, Y Vladimir; Xu, You; Villa, Alessandra

2017-01-01

-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.......The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined...... hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked...

[A Duplex PCR Method for Detection of Babesia caballi and Theileria equi].

Science.gov (United States)

Zhang, Yang; Zhang, Yu-ting; Wang, Zhen-bao; Bolati; Li, Hai; Bayinchahan

2015-04-01

To develop a duplex PCR assay for detection of Babesia caballi and Theileria equi. Two pairs of primers were designed according to the BC48 gene of B. caballi and 18 s rRNA gene of T. equi, and a duplex PCR assay was developed by the optimization of reaction conditions. The specificity, sensitivity and reliability of the method were tested. The horse blood samples of suspected cases were collected from Yili region, and detected by the duplex PCR, microspopy, conventional PCR, and fluorescence quantitative PCR, and the results were compared. Using the duplex PCR assay, the specific fragments of 155 bp and 280 bp were amplified from DNA samples of B. caballi and T. equi, respectively. No specific fragment was amplified from DNA samples of B. bigemina, Theilerdia annulata, Theilerdia sergenti, Toxoplasma gondii, Neospora caninum, and Trypanosoma evansi. The limit of detection was 4.85 x 10(5) copies/L for B. caballi DNA and 4.85 x 10(4) copies/µl for T. equi DNA, respectively. Among the 24 blood samples, 11 were found B. caballi-positive by the duplex PCR assay, and 18 were T. equi-positive. The coincidence rate of microscopy, conventional PCR, and fluorescence quantitative PCR with duplex PCR was 91.7% (22/24), 95.8% (23/24), and 95.8% (23/24), respectively. A duplex PCR assay for simultaneous detection of B. caballi and T. equi is established.

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

Science.gov (United States)

König, Sebastian L. B.; Huppert, Julian L.; Sigel, Roland K. O.; Evans, Amanda C.

2013-01-01

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions. PMID:23771141

Carotid Consensus Panel duplex criteria can replace modified University of Washington criteria without affecting accuracy.

Science.gov (United States)

Kim, Ann H; Augustin, Gener; Shevitz, Andrew; Kim, Hannah; Trivonovich, Michael R; Powell, Alexis R; Kumins, Norman; Tarr, Robert; Kashyap, Vikram S

2018-04-01

The decision to intervene for internal carotid stenosis often depends on the degree of stenosis seen on duplex ultrasound (US). The aim of this study is to compare the diagnostic accuracy of two criteria: modified University of Washington (UW) and 2003 Carotid Consensus Panel (CCP). All patients undergoing US in an accredited (IAC) vascular laboratory from January 2010 to June 2015 were reviewed ( n=18,772 US exams). Patients receiving a neck computed tomography angiography (CTA) within 6 months of the US were included in the study ( n=254). The degree of stenosis was determined by UW/CCP criteria and confirmed on CTA images using North American Symptomatic Carotid Endarterectomy Trial (NASCET)/European Carotid Surgery Trial (ECST) schema. Kappa analysis with 95% confidence intervals (CIs) were utilized to determine duplex-CTA agreement. A total of 417 carotid arteries from 221 patients were assessed in this study. The modified UW criteria accurately classified 266 (63.9%, kappa = 0.321, 95% CI 0.255 to 0.386) cases according to NASCET-derived measurements. The sensitivity, specificity, and accuracy at ≥ 60% stenosis were 65.7%, 81.3%, and 81.9%. The CCP criteria resulted in 296 (70.9%) accurate diagnoses (kappa = 0.359, 95% CI 0.280 to 0.437). At ≥ 70% stenosis, the sensitivity, specificity and accuracy were 38.8%, 91.6%, and 87.1% for NASCET. Comparison of the duplex results to ECST-derived CTA measurements revealed a similar trend (UW 53.1%, κ = 0.301 vs CCP 62.1%, κ = 0.315). The CCP criteria demonstrate a higher concordance rate with measurements taken from CTAs. The CCP criteria may be more sensitive in classifying clinically significant degrees of stenosis without a loss in diagnostic accuracy.

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite the cyclic analog of 2-deoxyribose

International Nuclear Information System (INIS)

Kalnik, M.W.; Chang, C.N.; Grollman, A.P.; Patel, D.J.

1988-01-01

Proton and phosphorus NMR studies are reported for the complementary d(C-A-T-G-A-G-T-A-C) x d(G-T-A-C-F-C-A-T-G) nonanucleotide duplex (designated AP/sub F/ 9-mer duplex) which contains a stable abasic site analog, F, in the center of the helix. This oligodeoxynucleotide contains a modified tetrahydrofuran moiety, isosteric with 2-deoxyribofuranose, which serves as a structural analog of a natural apurinic/apyrimidinic site. Exchangeable and nonexchangeable base and sugar protons, including those located at the abasic site, have been assigned in the complementary AP/sub F/ 9-mer duplex by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H 2 O and D 2 O solution at low temperature (0 0 C). These studies indicate that A5 inserts into the helix opposite the abasic site F14 and stacks with flanking G4 x C15 and G6 x C13 Watson-Crick base pairs. Base-sugar proton NOE connectivities were measured through G4-A5-G6 on the unmodified strand and between the base protons of C15 and the sugar protons of the 5'-flanking residue F14 on the modified strand. These studies establish that all glycosidic torsion angles are anti and that the helix is right-handed at and adjacent to the abasic site in the AP/sub F/ 9-mer duplex. Two of the 16 phosphodiester groups exhibit phosphorus resonances outside the normal spectral dispersion indicative of altered torsion angles at two of the phosphate groups in the backbone of the AP/sub F/ 9-mer duplex

Pyrene-modified unlocked nucleic acids: synthesis, thermodynamic studies, and fluorescent properties

DEFF Research Database (Denmark)

Karlsen, Kasper K; Pasternak, Anna; Jensen, Troels B

2012-01-01

-UNA modifications were studied. It was found that incorporation of pyrene-UNA monomers increased duplex stability relative to UNA monomers, and thermodynamic studies revealed significant mismatch discriminative capabilities of the pyrene-UNA modified oligonucleotides. Furthermore, the steady-state fluorescence...... emission intensities of pyrene-UNA modified oligonucleotides were increased upon hybridization to DNA, which to the best of our knowledge is unprecedented for an acyclic pyrene modification in DNA. Interestingly, pyrene excimer emission was observed for single-stranded oligonucleotides containing three...

Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

Science.gov (United States)

Ahour, F; Shamsi, A

2017-09-01

Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

Electroporation and microinjection successfully deliver single-stranded and duplex DNA into live cells as detected by FRET measurements.

Directory of Open Access Journals (Sweden)

Rosemary A Bamford

Full Text Available Förster resonance energy transfer (FRET technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5 complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Science.gov (United States)

Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

2016-01-01

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

Excited-State Dynamics of a DNA Duplex in a Deep Eutectic Solvent Probed by Femtosecond Time-Resolved IR Spectroscopy.

Science.gov (United States)

de La Harpe, Kimberly; Kohl, Forrest R; Zhang, Yuyuan; Kohler, Bern

2018-03-08

To better understand how the solvent influences excited-state deactivation in DNA strands, femtosecond time-resolved IR (fs-TRIR) pump-probe measurements were performed on a d(AT) 9 ·d(AT) 9 duplex dissolved in a deep eutectic solvent (DES) made from choline chloride and ethylene glycol in a 1:2 mol ratio. This solvent, known as ethaline, is a member of a class of ionic liquids capable of solubilizing DNA with minimal disruption to its secondary structure. UV melting analysis reveals that the duplex studied here melts at 18 °C in ethaline compared to 50 °C in aqueous solution. Ethaline has an excellent transparency window that facilitates TRIR measurements in the double-bond stretching region. Transient spectra recorded in deuterated ethaline at room temperature indicate that photoinduced intrastrand charge transfer occurs from A to T, yielding the same exciplex state previously detected in aqueous solution. This state decays via charge recombination with a lifetime of 380 ± 10 ps compared to the 300 ± 10 ps lifetime measured earlier in D 2 O solution. The TRIR data strongly suggest that the long-lived exciplex forms exclusively in the solvated duplex, and not in the denatured single strands, which appear to have little, if any, base stacking. The longer lifetime of the exciplex state in the DES compared to aqueous solution is suggested to arise from reduced stabilization of the charge transfer state, resulting in slower charge recombination on account of Marcus inverted behavior.

Increased Y-chromosome detection by SRY duplexing

DEFF Research Database (Denmark)

Hansen, Morten Høgh; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

2012-01-01

Determining fetal sex noninvasively is dependent of a robust assay. We designed a novel SRY assay and combined it with a SRY assay from literature forming a duplex assay with the same fluorescent dye to increase detection of Y-chromosome at low cell-free fetal DNA or chimeric DNA concentrations....

Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin. Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves As An Atypical DNA Intercalator

Science.gov (United States)

Fekry, Mostafa I.; Szekely, Jozsef; Dutta, Sanjay; Breydo, Leonid; Zang, Hong; Gates, Kent S.

2012-01-01

Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product leinamycin. Previous studies suggested that alkylation of duplex DNA by activated leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because leinamycin does not contain a classical noncovalent DNA-binding motif such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by leinamycin provided evidence that activated leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 103 M−1. Activated leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated leinamycin with palindromic duplexes containing 5'-CG and 5'-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of leinamycin with respect to target guanine residues. The data allows construction of a model for the leinamycin-DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA. PMID:21954957

An unusual mode of DNA duplex association: Watson-Crick interaction of all-purine deoxyribonucleic acids.

Science.gov (United States)

Battersby, Thomas R; Albalos, Maria; Friesenhahn, Michel J

2007-05-01

Nucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N(3)-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids.

Directed Formation of DNA Nanoarrays through Orthogonal Self-Assembly

Directory of Open Access Journals (Sweden)

Eugen Stulz

2011-06-01

Full Text Available We describe the synthesis of terpyridine modified DNA strands which selectively form DNA nanotubes through orthogonal hydrogen bonding and metal complexation interactions. The short DNA strands are designed to self-assemble into long duplexes through a sticky-end approach. Addition of weakly binding metals such as Zn(II and Ni(II induces the formation of tubular arrays consisting of DNA bundles which are 50-200 nm wide and 2-50 nm high. TEM shows additional long distance ordering of the terpy-DNA complexes into fibers.

Visualizing Transient Watson-Crick Like Mispairs in DNA and RNA Duplexes

Science.gov (United States)

Kimsey, Isaac J.; Petzold, Katja; Sathyamoorthy, Bharathwaj; Stein, Zachary W.; Al-Hashimi, Hashim M.

2015-01-01

Rare tautomeric and anionic nucleobases are believed to play fundamental biological roles but their prevalence and functional importance has remained elusive because they exist transiently, in low-abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10−3-10−5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases. PMID:25762137

Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes.

Science.gov (United States)

Kimsey, Isaac J; Petzold, Katja; Sathyamoorthy, Bharathwaj; Stein, Zachary W; Al-Hashimi, Hashim M

2015-03-19

Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10(-3) to 10(-5)) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.

Recognition of base J in duplex DNA by J-binding protein

NARCIS (Netherlands)

Sabatini, Robert; Meeuwenoord, Nico; van Boom, Jacques H.; Borst, Piet

2002-01-01

beta-d-Glucosylhydroxymethyluracil, also called base J, is an unusual modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously found a J-binding

Insights into Watson–Crick/Hoogsteen breathing dynamics and damage repair from the solution structure and dynamic ensemble of DNA duplexes containing m1A

Science.gov (United States)

Sathyamoorthy, Bharathwaj; Shi, Honglue; Zhou, Huiqing; Xue, Yi; Rangadurai, Atul; Merriman, Dawn K.

2017-01-01

Abstract In the canonical DNA double helix, Watson–Crick (WC) base pairs (bps) exist in dynamic equilibrium with sparsely populated (∼0.02–0.4%) and short-lived (lifetimes ∼0.2–2.5 ms) Hoogsteen (HG) bps. To gain insights into transient HG bps, we used solution-state nuclear magnetic resonance spectroscopy, including measurements of residual dipolar couplings and molecular dynamics simulations, to examine how a single HG bp trapped using the N1-methylated adenine (m1A) lesion affects the structural and dynamic properties of two duplexes. The solution structure and dynamic ensembles of the duplexes reveals that in both cases, m1A forms a m1A•T HG bp, which is accompanied by local and global structural and dynamic perturbations in the double helix. These include a bias toward the BI backbone conformation; sugar repuckering, major-groove directed kinking (∼9°); and local melting of neighboring WC bps. These results provide atomic insights into WC/HG breathing dynamics in unmodified DNA duplexes as well as identify structural and dynamic signatures that could play roles in m1A recognition and repair. PMID:28369571

Screening of Modified RNA duplexes

DEFF Research Database (Denmark)

Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form......Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...

Superimposed Code Theorectic Analysis of DNA Codes and DNA Computing

Science.gov (United States)

2010-03-01

that the hybridization that occurs between a DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research...ssDNA single stranded DNA WC Watson – Crick A Adenine C Cytosine G Guanine T Thymine ... Watson - Crick (WC) duplex, e.g., TCGCA TCGCA . Note that non-WC duplexes can form and such a formation is called a cross-hybridization. Cross

Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

Energy Technology Data Exchange (ETDEWEB)

Lucarelli, Fausto; Marrazza, Giovanna; Palchetti, Ilaria; Cesaretti, S.; Mascini, Marco

2002-09-26

This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence. The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised. The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.

Normalized cDNA libraries

Science.gov (United States)

Soares, Marcelo B.; Efstratiadis, Argiris

1997-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

Science.gov (United States)

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-09-18

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

Thermodynamics of sequence-specific binding of PNA to DNA

DEFF Research Database (Denmark)

Ratilainen, T; Holmén, A; Tuite, E

2000-01-01

For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

Insights into Watson-Crick/Hoogsteen breathing dynamics and damage repair from the solution structure and dynamic ensemble of DNA duplexes containing m1A.

Science.gov (United States)

Sathyamoorthy, Bharathwaj; Shi, Honglue; Zhou, Huiqing; Xue, Yi; Rangadurai, Atul; Merriman, Dawn K; Al-Hashimi, Hashim M

2017-05-19

In the canonical DNA double helix, Watson-Crick (WC) base pairs (bps) exist in dynamic equilibrium with sparsely populated (∼0.02-0.4%) and short-lived (lifetimes ∼0.2-2.5 ms) Hoogsteen (HG) bps. To gain insights into transient HG bps, we used solution-state nuclear magnetic resonance spectroscopy, including measurements of residual dipolar couplings and molecular dynamics simulations, to examine how a single HG bp trapped using the N1-methylated adenine (m1A) lesion affects the structural and dynamic properties of two duplexes. The solution structure and dynamic ensembles of the duplexes reveals that in both cases, m1A forms a m1A•T HG bp, which is accompanied by local and global structural and dynamic perturbations in the double helix. These include a bias toward the BI backbone conformation; sugar repuckering, major-groove directed kinking (∼9°); and local melting of neighboring WC bps. These results provide atomic insights into WC/HG breathing dynamics in unmodified DNA duplexes as well as identify structural and dynamic signatures that could play roles in m1A recognition and repair. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Thermodynamic and structural properties of the specific binding between Ag⁺ ion and C:C mismatched base pair in duplex DNA to form C-Ag-C metal-mediated base pair.

Science.gov (United States)

Torigoe, Hidetaka; Okamoto, Itaru; Dairaku, Takenori; Tanaka, Yoshiyuki; Ono, Akira; Kozasa, Tetsuo

2012-11-01

Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences. Copyright © 2012 Elsevier

A new general model for predicting melting thermodynamics of complementary and mismatched B-form duplexes containing locked nucleic acids: application to probe design for digital PCR detection of somatic mutations.

Science.gov (United States)

Hughesman, Curtis; Fakhfakh, Kareem; Bidshahri, Roza; Lund, H Louise; Haynes, Charles

2015-02-17

Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a

Positive cooperativity of the specific binding between Hg2+ ion and T:T mismatched base pairs in duplex DNA

International Nuclear Information System (INIS)

Torigoe, Hidetaka; Miyakawa, Yukako; Ono, Akira; Kozasa, Tetsuo

2012-01-01

Highlights: ► Hg 2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio. ► The binding constant between Hg 2+ and the T:T mismatched base pair was 10 6 M −1 . ► The binding constant was larger than those for nonspecific metal–DNA interactions. ► The binding constant for the second Hg 2+ was larger than that for the first Hg 2+ . ► The positive cooperative binding was observed between Hg 2+ and multiple T:T. - Abstract: Metal-mediated base pairs by the interaction between metal ions and artificial bases in oligonucleotides have been developed for their potential applications in nanotechnology. We recently found that a natural T:T mismatched base pair bound with Hg 2+ ion to form a novel T–Hg–T base pair. Here, we examined the thermodynamic properties of the binding between Hg 2+ and each of the single and double T:T mismatched base pair duplex DNAs by isothermal titration calorimetry. Hg 2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio with 10 6 M −1 binding constant, which was significantly larger than those for nonspecific metal ion–DNA interactions. In the Hg 2+ –double T:T mismatched base pair interaction, the affinity for the second Hg 2+ binding was significantly larger than that for the first Hg 2+ binding. The positively cooperative binding may be favorable to align multiple Hg 2+ in duplex DNA for the application of the metal-mediated base pairs in nanotechnology.

Molecular Modeling of the Major DNA Adduct Formed from Food Mutagen Ochratoxin A in NarI Two-Base Deletion Duplexes: Impact of Sequence Context and Adduct Ionization on Conformational Preference and Mutagenicity.

Science.gov (United States)

Kathuria, Preetleen; Sharma, Purshotam; Manderville, Richard A; Wetmore, Stacey D

2017-08-21

Exposure to ochratoxin A (OTA), a possible human carcinogen, leads to many different DNA mutations. As a first step toward understanding the structural basis of OTA-induced mutagenicity, the present work uses a robust computational approach and a slipped mutagenic intermediate model previously studied for C 8 -dG aromatic amine adducts to analyze the conformational features of postreplication two-base deletion DNA duplexes containing OT-dG, the major OTA lesion at the C 8 position of guanine. Specifically, a total of 960 ns of molecular dynamics simulations (excluding trial simulations) were carried out on four OT-dG ionization states in three sequence contexts within oligomers containing the NarI recognition sequence, a known hotspot for deletion mutations induced by related adducts formed from known carcinogens. Our results indicate that the structural properties and relative stability of the competing "major groove" and "stacked" conformations of OTA adducted two-base deletion duplexes depend on both the OTA ionization state and the sequence context, mainly due to conformation-dependent deviations in discrete local (hydrogen-bonding and stacking) interactions at the lesion site, as well as DNA bending. When the structural characteristics of the OT-dG adducted two-base deletion duplexes are compared to those associated with previously studied C 8 -dG adducts, a greater understanding of the effects of the nucleobase-carcinogen linkage, and size of the carcinogenic moiety on the conformational preferences of damaged DNA is obtained. Most importantly, our work predicts key structural features for OT-dG-adducted deletion DNA duplexes, which in turn allow us to develop hypotheses regarding OT-dG replication outcomes. Thus, our computational results are valuable for the design and interpretation of future biochemical studies on the potentially carcinogenic OT-dG lesion.

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

Science.gov (United States)

Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

2015-10-01

Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

Science.gov (United States)

Russo Krauss, Irene; Ramaswamy, Sneha; Neidle, Stephen; Haider, Shozeb; Parkinson, Gary N

2016-02-03

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

Science.gov (United States)

Su'etsugu, Masayuki; Takata, Makoto; Kubota, Toshio; Matsuda, Yusaku; Katayama, Tsutomu

2004-06-01

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. Copyright Blackwell Publishing Limited

Promotion of double-duplex invasion of peptide nucleic acids through conjugation with nuclear localization signal peptide.

Science.gov (United States)

Aiba, Yuichiro; Honda, Yuta; Komiyama, Makoto

2015-03-02

Pseudo-complementary peptide nucleic acid (pcPNA), as one of the most widely used synthetic DNA analogues, invades double-stranded DNA according to Watson-Crick rules to form invasion complexes. This unique mode of DNA recognition induces structural changes at the invasion site and can be used for a range of applications. In this paper, pcPNA is conjugated with a nuclear localization signal (NLS) peptide, and its invading activity is notably promoted both thermodynamically and kinetically. Thus, the double-duplex invasion complex is formed promptly at low pcPNA concentrations under high salt conditions, where the invasion otherwise never occurs. Furthermore, NLS-modified pcPNA is successfully employed for site-selective DNA scission, and the targeted DNA is selectively cleaved under conditions that are not conducive for DNA cutters using unmodified pcPNAs. This strategy of pcPNA modification is expected to be advantageous and promising for a range of in vitro and in vivo applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probabilistic Medium Access Control for Full-Duplex Networks with Half-Duplex Clients

OpenAIRE

Chen, Shih-Ying; Huang, Ting-Feng; Lin, Kate Ching-Ju; Hong, H. -W. Peter; Sabharwal, Ashutosh

2016-01-01

The feasibility of practical in-band full-duplex radios has recently been demonstrated experimentally. One way to leverage full-duplex in a network setting is to enable three-node full-duplex, where a full- duplex access point (AP) transmits data to one node yet simultaneously receives data from another node. Such three-node full-duplex communication however introduces inter-client interference, directly impacting the full-duplex gain. It hence may not always be beneficial to enable three-nod...

2'β-Fluoro-Tricyclo Nucleic Acids (2'F-tc-ANA): Thermal Duplex Stability, Structural Studies, and RNase H Activation.

Science.gov (United States)

Istrate, Alena; Katolik, Adam; Istrate, Andrei; Leumann, Christian J

2017-08-01

We describe the synthesis, thermal stability, structural and RNase H activation properties of 2'β-fluoro-tricyclo nucleic acids (2'F-tc-ANA). Three 2'F-tc-ANA nucleosides (T, 5Me C and A) were synthesized starting from a previously described fluorinated tricyclo sugar intermediate. NMR analysis and quantum mechanical calculations indicate that 2'F-tc-ANA nucleosides prefer sugar conformations in the East and South regions of the pseudorotational cycle. UV-melting experiments revealed that non-consecutive insertions of 2'F-tc-ANA units in DNA reduce the affinity to DNA and RNA complements. However, an oligonucleotide with five contiguous 2'F-tc-ANA-T insertions exhibits increased affinity to complementary RNA. Moreover, a fully modified 10-mer 2'F-tc-ANA oligonucleotide paired to both DNA (+1.6 °C/mod) and RNA (+2.5 °C/mod) with significantly higher affinity compared to corresponding unmodified DNA, and similar affinity compared to corresponding tc-DNA. In addition, CD spectroscopy and molecular dynamics simulations indicate that the conformation of the 2'F-tc-ANA/RNA duplex is similar to that of a DNA/RNA duplex. Moreover, in some sequence contexts, 2'F-tc-ANA promotes RNase H-mediated cleavage of a complementary RNA strand. Taken together, 2'F-tc-ANA represents a nucleic acid analogue that offers the advantage of high RNA affinity while maintaining the ability to activate RNase H, and can be considered a prospective candidate for gene silencing applications. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA immobilization on polymer-modified Si surface by controlling pH

International Nuclear Information System (INIS)

Demirel, Goekcen Birlik; Caykara, Tuncer

2009-01-01

A novel approach based on polymer-modified Si surface as DNA sensor platforms is presented. The polymer-modified Si surface was prepared by using 3-(methacryloxypropyl)trimethoxysilane [γ-MPS] and poly(acrylamide) [PAAm]. Firstly, a layer of γ-MPS was formed on the hydroxylated silicon surface as a monolayer and then modified with different molecular weight of PAAm to form polymer-modified surface. The polymer-modified Si surface was used for dsDNA immobilization. All steps about formation of layer structure were characterized by ellipsometry, atomic force microscopy (AFM), attenuated total reflectance Fourier transformed infrared (ATR-FTIR), and contact angle (CA) measurements. We found that in this case the amount of dsDNA immobilized onto the surface was dictated by the electrostatic interaction between the substrate surface and the DNA. Our results thus demonstrated that DNA molecules could be immobilized differently onto the polymer-modified support surface via electrostatic interactions.

A Dual-Specific Targeting Approach Based on the Simultaneous Recognition of Duplex and Quadruplex Motifs.

Science.gov (United States)

Nguyen, Thi Quynh Ngoc; Lim, Kah Wai; Phan, Anh Tuân

2017-09-20

Small-molecule ligands targeting nucleic acids have been explored as potential therapeutic agents. Duplex groove-binding ligands have been shown to recognize DNA in a sequence-specific manner. On the other hand, quadruplex-binding ligands exhibit high selectivity between quadruplex and duplex, but show limited discrimination between different quadruplex structures. Here we propose a dual-specific approach through the simultaneous application of duplex- and quadruplex-binders. We demonstrated that a quadruplex-specific ligand and a duplex-specific ligand can simultaneously interact at two separate binding sites of a quadruplex-duplex hybrid harbouring both quadruplex and duplex structural elements. Such a dual-specific targeting strategy would combine the sequence specificity of duplex-binders and the strong binding affinity of quadruplex-binders, potentially allowing the specific targeting of unique quadruplex structures. Future research can be directed towards the development of conjugated compounds targeting specific genomic quadruplex-duplex sites, for which the linker would be highly context-dependent in terms of length and flexibility, as well as the attachment points onto both ligands.

AUTENTIKASI DAGING AYAM SEGAR DARI KONTAMINASI DAGING BABI MENGGUNAKAN GEN CYT-B DENGAN ANALISIS DUPLEX- POLYMERASE CHAIN REACTION

Directory of Open Access Journals (Sweden)

Bayu Setya Hertanto

2017-05-01

Full Text Available Halal is one of important aspects in consumer protection. Meat and processed meat products are food that should be controlled strictly because those are prone to be adulterated by pork contamination. Therefore, it is necessary to provide detection technique which is accurate, fast and cheap. The objective of this research was to identify the presence of impurities of pork meat on raw chicken meat using gene Cyt-b with duplex-PCR analysis. This research used six samples of raw chicken meat and raw pork. Raw chicken meat was bought from supermarkets in the city of Surakarta and raw pork was obtained from pig slaughterhouse. The percentage of raw pork contamination on raw chicken meat was designed as much as 1, 5, 10, and 25%, respectively. The DNA genome was isolated according to DNA isolation protocol from Genomic DNA Mini Kit. In addition, duplex-PCR was performed based on protocol of KAPA2G Fast Multiplex PCR kit. The data was descriptively analyzed by directly looking the DNA bands on the gel documentation apparatus. The result showed that specific DNA bands for chicken and pig were completely appeared on 1.5% of agarose gels. Duplex-PCR detect contamination of pork on raw meat of chicken at all contamination levels. This research proved that the duplex-PCR detect the contamination of pork until the level of 1%.

Improved quantification accuracy for duplex real-time PCR detection of genetically modified soybean and maize in heat processed foods

Directory of Open Access Journals (Sweden)

CHENG Fang

2013-04-01

Full Text Available Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several real-time PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR was further validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.

Validation of a novel duplex ultrasound objective structured assessment of technical skills (DUOSATS) for arterial stenosis detection.

Science.gov (United States)

Jaffer, U; Singh, P; Pandey, V A; Aslam, M; Standfield, N J

2014-01-01

Duplex ultrasound facilitates bedside diagnosis and hence timely patient care. Its uptake has been hampered by training and accreditation issues. We have developed an assessment tool for Duplex arterial stenosis measurement for both simulator and patient based training. A novel assessment tool: duplex ultrasound assessment of technical skills was developed. A modified duplex ultrasound assessment of technical skills was used for simulator training. Novice, intermediate experience and expert users of duplex ultrasound were invited to participate. Participants viewed an instructional video and were allowed ample time to familiarize with the equipment. Participants' attempts were recorded and independently assessed by four experts using the modified duplex ultrasound assessment of technical skills. 'Global' assessment was also done on a four point Likert scale. Content, construct and concurrent validity as well as reliability were evaluated. Content and construct validity as well as reliability were demonstrated. The simulator had good satisfaction rating from participants: median 4; range 3-5. Receiver operator characteristic analysis has established a cut point of 22/ 34 and 25/ 40 were most appropriate for simulator and patient based assessment respectively. We have validated a novel assessment tool for duplex arterial stenosis detection. Further work is underway to establish transference validity of simulator training to improved skill in scanning patients. We have developed and validated duplex ultrasound assessment of technical skills for simulator training.

DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

Science.gov (United States)

Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

2010-06-01

A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

Method for construction of normalized cDNA libraries

Science.gov (United States)

Soares, Marcelo B.; Efstratiadis, Argiris

1998-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G x T and G x O4meT pairing in dodecanucleotide duplexes

International Nuclear Information System (INIS)

Kalnik, M.W.; Kouchakdjian, M.; Li, B.F.L.; Swann, P.F.; Patel, D.J.

1988-01-01

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G x T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O 4 meT-G-C-G) duplex (designated G x O 4 meT 12-mer) containing G x T and G x O 4 meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G x T 12-mer and G x O 4 meT 12-mer duplexes in H 2 O and D 2 O solution. The guanosine and thymidine imino protons in the G x T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G x T 12-mer duplex. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G x T mismatch or in G x C base pairs indicates that hydrogen bonding to O 4 meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH 3 group of O 4 meT across the pair and NOEs to the imino protons of flanking base pairs. Taken together with data from the NMR of nonexchangeable protons, this shows that both G and O 4 meT have anti-glycosidic torsion angles and are stacked into the duplex. Comparison of the intensity of the NOEs between the guanosine imino proton and the OCH 3 of O 4 meT as well as other protons in its vicinity demonstrates that the OCH 3 group of O 4 meT adopts the syn orientation with respect to N3 of the methylated thymidine. The authors propose an alternate base pairing mode stabilized by one short hydrogen bond between the 2-amino group of guanosine and the 2-carbonyl group of O 4 met

Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping

Science.gov (United States)

Hendershot, Jenna M.; O'Brien, Patrick J.

2014-01-01

Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping. PMID:25324304

Role of minor groove width and hydration pattern on amsacrine interaction with DNA.

Directory of Open Access Journals (Sweden)

Deepak K Jangir

Full Text Available Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.

Science.gov (United States)

Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

2016-03-22

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Two-dimensional 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex and a quinoxaline ([MeCys3, MeCys7]TANDEM) drug duplex complex

International Nuclear Information System (INIS)

Powers, R.; Olsen, R.K.; Gorenstein, D.G.

1989-01-01

Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCGGG), and its quinoxaline ([MeCys3, MeCys7]TANDEM) drug duplex complex has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The 31P chemical shifts of this 10 base pair oligonucleotide follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. While the 31P chemical shifts show sequence-specific variations, they also do not generally follow the Calladine rules previously demonstrated. 31P NMR also provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the drug to the duplex. Although the quinoxaline drug, [MeCys3, MeCys7]TANDEM, is generally expected to bind to duplex DNA by bis-intercalation, only small 31P chemical shift changes are observed upon binding the drug to duplex d(CCCGATCGGG). Additionally, only small perturbations in the 1H NMR and UV spectra are observed upon binding the drug to the decamer, although association of the drug stabilizes the duplex form relative to the other states. These results are consistent with a non-intercalative mode of association of the drug. Modeling and molecular mechanics energy minimization demonstrate that a novel structure in which the two quinoxaline rings of the drug binds in the minor groove of the duplex is possible

Modified DNA extraction for rapid PCR detection of methicillin-resistant staphylococci

International Nuclear Information System (INIS)

Japoni, A.; Alborzi, A.; Rasouli, M.; Pourabbas, B.

2004-01-01

Nosocomial infection caused by methicillin-resistant staphylococci poses a serious problem in many countries. The aim of this study was to rapidly and reliably detect methicillin-resistant-staphylococci in order to suggest appropriate therapy. The presence or absence of the methicillin-resistance gene in 115 clinical isolates of staphylococcus aureus and 50 isolates of coagulase negative staphylococci was examined by normal PCR. DNA extraction for PCR performance was then modified by omission of achromopeptadiase and proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. All isolates with Mic>8 μ g/ml showed positive PCR. No differences in PCR detection have been observed when normal and modified DNA extractions have been performed. Our modified DNA extraction can quickly detect methicillin-resistant staphylococci by PCR. The advantage of rapid DNA extraction extends to both reduction of time and cost of PCR performance. This modified DNA extraction is suitable for different PCR detection, when staphylococci are the subject of DNA analysis

Comparison of duplex PCR and phenotypic analysis in differentiating Candida dubliniensis from Candida albicans from oral samples.

Science.gov (United States)

Sampath, Asanga; Weerasekera, Manjula; Dilhari, Ayomi; Gunasekara, Chinthika; Bulugahapitiya, Uditha; Fernando, Neluka; Samaranayake, Lakshman

2017-12-01

Candida dubliniensis shares a wide range of phenotypic characteristics with Candida albicans including a common trait called germ tube positivity. Hence, laboratory differentiation of these two species is cumbersome. Duplex PCR analyses for C. albicans and C. dubliniensis was performed directly on DNA extracted from a total of 122 germ tube positive isolates derived from 100 concentrated oral rinse samples from a random cohort of diabetics attending a clinic in Sri Lanka. These results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA of the yeasts. Performance efficacy of duplex PCR was then compared with phenotypic identification using a standard battery of phenotypic tests. Of the 122 germ tube positive isolates three were identified by duplex PCR as C. dubliniensis and the remainder as C. albicans. On the contrary, when the standard phenotypic tests, sugar assimilation and chlamydospore formation, were used to differentiate the two species 13 germ tube positive isolates were erroneously identified as C. dubliniensis. Duplex PCR was found to be rapid, sensitive and more specific than phenotypic identification methods in discriminating C. dubliniensis from C. albicans. This is also the first report on the oral carriage of C. dubliniensis in a Sri Lankan population.

Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.

Science.gov (United States)

Tripathi, Prashant; Moinuddin; Dixit, Kiran; Mir, Abdul Rouf; Habib, Safia; Alam, Khursheed; Ali, Asif

2014-07-01

Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 μM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients. Copyright © 2014 Elsevier Inc. All rights reserved.

Development and evaluation of a single-step duplex PCR for simultaneous detection of Fasciola hepatica and Fasciola gigantica (family Fasciolidae, class Trematoda, phylum Platyhelminthes).

Science.gov (United States)

Le, Thanh Hoa; Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Le, Xuyen Thi Kim; Hoang, Chau Thi Minh; De, Nguyen Van

2012-08-01

A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

Science.gov (United States)

Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Le, Xuyen Thi Kim; Hoang, Chau Thi Minh; De, Nguyen Van

2012-01-01

A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap. PMID:22692744

Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

International Nuclear Information System (INIS)

Kouchakdjian, M.; Patel, D.J.; Eisenberg, M.; Johnson, F.; Grollman, A.P.

1991-01-01

Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N 2 -propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9)·(G10-T11-A12-C13-F14-C15-A16-T17-G18) X·F 9-mer duplex. Two-dimensional NMR experiments establish that the X·F 9-mer helix is right-handed with Watson-Crick A·T and G·C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4·C15 and G6·C13 which flank the lesion site, as well as to the H1' and H1 double-prime protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4·C15 and G6·C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N 2 -propanodeoxyguanosine adduct fits into the cavity generated by the abasic site

Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats.

Science.gov (United States)

Cui, Yanyan; Zhang, Yan; Jian, Fuchun; Zhang, Longxian; Wang, Rongjun; Cao, Shuxuan; Wang, Xiaoxing; Yan, Yaqun; Ning, Changshen

2017-05-01

Theileria spp. and Anaplasma spp., which are important tick-borne pathogens (TBPs), impact the health of humans and animals in tropical and subtropical areas. Theileria and Anaplasma co-infections are common in sheep and goats. Following alignment of the relevant DNA sequences, two primer sets were designed to specifically target the Theileria spp. 18S rRNA and Anaplasma spp. 16S rRNA gene sequences. Genomic DNA from the two genera was serially diluted tenfold for testing the sensitivities of detection of the primer sets. The specificities of the primer sets were confirmed when DNA from Anaplasma and Theileria (positive controls), other related hematoparasites (negative controls) and ddH 2 O were used as templates. Fifty field samples were also used to evaluate the utility of single PCR and duplex PCR assays, and the detection results were compared with those of the PCR methods previously published. An optimized duplex PCR assay was established from the two primer sets based on the relevant genes from the two TBPs, and this assay generated products of 298-bp (Theileria spp.) and 139-bp (Anaplasma spp.). The detection limit of the assay was 29.4 × 10 -3  ng per μl, and there was no cross-reaction with the DNA from other hematoparasites. The results showed that the newly developed duplex PCR assay had an efficiency of detection (P > 0.05) similar to other published PCR methods. In this study, a duplex PCR assay was developed that can simultaneously identify Theileria spp. and Anaplasma spp. in sheep and goats. This duplex PCR is a potentially valuable assay for epidemiological studies of TBPs in that it can detect cases of mixed infections of the pathogens. Copyright © 2017 Elsevier Inc. All rights reserved.

Rolling replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro

International Nuclear Information System (INIS)

Shavitt, O.; Livneh, Z.

1989-01-01

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers

Selective detection and quantification of modified DNA with solid-state nanopores.

Science.gov (United States)

Carlsen, Autumn T; Zahid, Osama K; Ruzicka, Jan A; Taylor, Ethan W; Hall, Adam R

2014-10-08

We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

Repair of Clustered Damage and DNA Polymerase Iota.

Science.gov (United States)

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Accurate and rapid modeling of iron–bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis

OpenAIRE

Harsch, Andreas; Marzilli, Lisa A.; Bunt, Richard C.; Stubbe, Joanne; Vouros, Paul

2000-01-01

Bleomycin B2 (BLM) in the presence of iron [Fe(II)] and O2 catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe–BLM was incorporated into each an...

Structure of DNA toroids and electrostatic attraction of DNA duplexes

International Nuclear Information System (INIS)

Cherstvy, A G

2005-01-01

DNA-DNA electrostatic attraction is considered as the driving force for the formation of DNA toroids in the presence of DNA condensing cations. This attraction comes from the DNA helical charge distribution and favours hexagonal toroidal cross-sections. The latter is in agreement with recent cryo-electron microscopy studies on DNA condensed with cobalt hexammine. We treat the DNA-DNA interactions within the modern theory of electrostatic interaction between helical macromolecules. The size and thickness of the toroids is calculated within a simple model; other models of stability of DNA toroids are discussed and compared

Approximation methods for the stability analysis of complete synchronization on duplex networks

Science.gov (United States)

Han, Wenchen; Yang, Junzhong

2018-01-01

Recently, the synchronization on multi-layer networks has drawn a lot of attention. In this work, we study the stability of the complete synchronization on duplex networks. We investigate effects of coupling function on the complete synchronization on duplex networks. We propose two approximation methods to deal with the stability of the complete synchronization on duplex networks. In the first method, we introduce a modified master stability function and, in the second method, we only take into consideration the contributions of a few most unstable transverse modes to the stability of the complete synchronization. We find that both methods work well for predicting the stability of the complete synchronization for small networks. For large networks, the second method still works pretty well.

Quantification of Chemical and Mechanical Effects on the Formation of the G-Quadruplex and i-Motif in Duplex DNA.

Science.gov (United States)

Selvam, Sangeetha; Mandal, Shankar; Mao, Hanbin

2017-09-05

The formation of biologically significant tetraplex DNA species, such as G-quadruplexes and i-motifs, is affected by chemical (ions and pH) and mechanical [superhelicity (σ) and molecular crowding] factors. Because of the extremely challenging experimental conditions, the relative importance of these factors on tetraplex folding is unknown. In this work, we quantitatively evaluated the chemical and mechanical effects on the population dynamics of DNA tetraplexes in the insulin-linked polymorphic region using magneto-optical tweezers. By mechanically unfolding individual tetraplexes, we found that ions and pH have the largest effects on the formation of the G-quadruplex and i-motif, respectively. Interestingly, superhelicity has the second largest effect followed by molecular crowding conditions. While chemical effects are specific to tetraplex species, mechanical factors have generic influences. The predominant effect of chemical factors can be attributed to the fact that they directly change the stability of a specific tetraplex, whereas the mechanical factors, superhelicity in particular, reduce the stability of the competing species by changing the kinetics of the melting and annealing of the duplex DNA template in a nonspecific manner. The substantial dependence of tetraplexes on superhelicity provides strong support that DNA tetraplexes can serve as topological sensors to modulate fundamental cellular processes such as transcription.

DNA glue

DEFF Research Database (Denmark)

Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

2008-01-01

Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

Energy Technology Data Exchange (ETDEWEB)

Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

2009-05-11

The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

DNA-modified electrodes (Ⅶ)——Preparation and characterization of DNA-bonded and DNA-adsorbed SAM/Au electrodes

Institute of Scientific and Technical Information of China (English)

陆琪; 庞代文; 胡深; 程介克; 蔡雄伟; 施财辉; 毛秉伟; 戴鸿平

1999-01-01

Two kinds of DNA-modified electrodes were prepared by covalent and adsorptive immobilization of DNA onto self-assembled monolayers of 2, 2’-dithiodiethanol on gold electrodes and characterized by cyclic voltammetry, Xray photoelectron spectroscopy and scanning tunneling microscopy. The results suggest that the methods are satisfactory for the immobilization of DNA on electrodes.

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus".

Science.gov (United States)

Selvaraj, Vijayanandraj; Maheshwari, Yogita; Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg; Yokomi, Raymond

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.

Validation of eDNA markers for New Zealand mudsnail surveillance and initial eDNA monitoring at Mississippi River Basin sites

Science.gov (United States)

Merkes, Christopher; Turnquist, Keith N.; Rees, Christopher B.; Amberg, Jon J.

2015-01-01

The performance of newly developed New Zealand mudsnail (Potamopyrgus antipodarum; NZMS) genetic markers for environmental (eDNA) analysis of water were compared across two laboratories. The genetic markers were tested in four quantitative polymerase chain reaction assays targeting two regions of the NZMS mitochondrial genome, specifically the cytochrome c oxidase subunit 1 (coi) and cytochrome b (cytb) genes. In a blind study, analysts tested each sample eight times with each assay. There were 10 expected-negative samples from the Black River in La Crosse, Wisconsin, 10 expected-positive samples from the Black Earth Creek in Black Earth, Wisconsin, and 10 known-positive samples from the Black River spiked with NZMS DNA. Previously extracted samples, kept at the Upper Midwest Environmental Sciences Center, were pooled by sample location and then equal quantities were distributed between the Upper Midwest Environmental Sciences Center and the Molecular Conservation Genetics Laboratory at the University of Wisconsin-Stevens Point for analysis. The assays tested were (1) the assay targeting cytb with a minor groove binder probe described by Goldberg and others (2013), (2) the cytb assay with a modified double-quenched probe, (3) an assay targeting coi with a double-quenched probe, and (4) a duplex reaction combining the modified cytb assay and the coi assay. Samples were considered positive for the presence of NZMS DNA when quantitative polymerase chain reaction amplification and probe signal was higher than the normalized threshold value above baseline fluorescence. For the duplex assay, samples were considered positive only when both probe signals were higher than the normalized threshold value above baseline fluorescence. Positive results were then confirmed by sequencing the products.

Initiation of the microgene polymerization reaction with non-repetitive homo-duplexes

International Nuclear Information System (INIS)

Itsko, Mark; Zaritsky, Arieh; Rabinovitch, Avinoam; Ben-Dov, Eitan

2008-01-01

Microgene Polymerization Reaction (MPR) is used as an experimental system to artificially simulate evolution of short, non-repetitive homo-duplex DNA into multiply-repetitive products that can code for functional proteins. Blunt-end ligation by DNA polymerase is crucial in expansion of homo-duplexes (HDs) into head-to-tail multiple repeats in MPR. The propagation mechanism is known, but formation of the initial doublet (ID) by juxtaposing two HDs and polymerization through the gap has been ambiguous. Initiation events with pairs of HDs using Real-Time PCR were more frequent at higher HD concentrations and slightly below the melting temperature. A process molecularity of about 3.1, calculated from the amplification efficiency and the difference in PCR cycles at which propagation was detected at varying HD concentrations, led to a simple mechanism for ID formation: the gap between two HDs is bridged by a third. Considering thermodynamic aspects of the presumed intermediate 'nucleation complex' can predict relative propensity for the process with other HDs

DNA Electrochemistry with Tethered Methylene Blue

Science.gov (United States)

Pheeney, Catrina G.

2012-01-01

Methylene blue (MB′), covalently attached to DNA through a flexible C12 alkyl linker, provides a sensitive redox reporter in DNA electrochemistry measurements. Tethered, intercalated MB′ is reduced through DNA-mediated charge transport; the incorporation of a single base mismatch at position 3, 10, or 14 of a 17-mer causes an attenuation of the signal to 62 ± 3% of the well-matched DNA, irrespective of position in the duplex. The redox signal intensity for MB′–DNA is found to be least 3-fold larger than that of Nile blue (NB)–DNA, indicating that MB′ is even more strongly coupled to the π-stack. The signal attenuation due to an intervening mismatch does, however, depend on DNA film density and the backfilling agent used to passivate the surface. These results highlight two mechanisms for reduction of MB′ on the DNA-modified electrode: reduction mediated by the DNA base pair stack and direct surface reduction of MB′ at the electrode. These two mechanisms are distinguished by their rates of electron transfer that differ by 20-fold. The extent of direct reduction at the surface can be controlled by assembly and buffer conditions. PMID:22512327

Molecular detection of infectious bronchitis and Newcastle disease viruses in broiler chickens with respiratory signs using Duplex RT-PCR.

Science.gov (United States)

Saba Shirvan, Aylar; Mardani, Karim

2014-01-01

Infectious bronchitis (IB) and Newcastle disease (ND) are highly contagious and the most economically important diseases of the poultry affecting respiratory tract and causing economic losses in poultry industry throughout the world. In the present study, the simultaneous detection and differentiation of causative agents of these diseases were investigated using duplex-RT-PCR. RNA was extracted from vaccinal and reference strains of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) and then cDNA was synthesized. Using two universal primer sets for detection of IBV and NDV, the duplex-RT-PCR was developed. In order to assess the efficiency of the developed duplex RT-PCR, a number of 12 broiler farms with the symptoms of respiratory tract infection was sampled (trachea, lung and kidney were sampled from affected birds suspicious for IBV and NDV infections). After RNA extraction from tissues and cDNA synthesis, the presence of IBV and NDV genome were investigated using duplex-PCR. The results showed that three of twelve examined broiler farms were positive for IBV and two farms were positive for NDV and IBV. The results revealed that the duplex-RT-PCR is a quick and sensitive procedure for simultaneously detecting IBV and NDV in birds with respiratory infections.

Solution structure of the luzopeptin-DNA complex

International Nuclear Information System (INIS)

Zhang, Xiaolu; Patel, D.J.

1991-01-01

The luzopeptin-d(C-A-T-G) complex (1 drug/duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. Once equivalent of luzopeptin binds to the self-complementary tetranucleotide duplex with the 2-fold symmetry of the antitumor agent and the DNA oligomer retained on complex formation. The authors have assigned the exchangeable and nonexchangeable proton resonances of luzopeptin and the d(C-A-T-G) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site in duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the luzopeptin and the DNA in the complex. The formation of cis peptide bonds for luzopeptin in the complex results in an increased separation of the long sides of the rectangular cyclic depsipeptide backbone and reorients in the glycine amide proton so that it can form an intermolecular hydrogen bond with the 2-carbonyl of T3 in the complex. This observation explains, in part, the requirement for Watson-Crick A·T pairs to be sandwiched between the quinolines at the bisintercalation site in the luzopeptin-DNA complex. The NMR studies on the luzopeptin-d(C-A-T-G) complex unequivocally establish that antitumor agents can undergo conformational transitions on complex formation with DNA, and it is the conformation of the drug in the complex that should serve as the starting point for drug design studies. The above structural details on the solution structure of the luzopeptin-DNA complex also explain the sequence selectivity of luzopeptin for bisintercalation at d(C-A)·d(T-G) steps in the d(C-A-T-G) duplex in solution

Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II)

International Nuclear Information System (INIS)

Sundquist, W.I.; Lippard, S.J.; Stollar, B.D.

1987-01-01

Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemothrapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)/sub b/, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations of cis-DDP bound to DNA. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds [Pt(en)Cl 2 ], [Pt(dach)Cl 2 ], and [Pt(NH 3 ) 2 (cbdca)] (carboplatin) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA. This antibody crossreacted with unmodified, denatured DNA. The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities

Development of a panel of seven duplex real-time PCR assays for detecting 13 streptococcal superantigens.

Science.gov (United States)

Yang, Peng; Peng, Xiaomin; Cui, Shujuan; Shao, Junbin; Zhu, Xuping; Zhang, Daitao; Liang, Huijie; Wang, Quanyi

2013-07-30

Streptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria. A panel consisting of seven duplex real-time PCR assays was developed to simultaneously detect 13 streptococcal SAgs and one internal control which may be important in the control of GAS-mediated diseases. Primer and probe sequences were selected based on the highly conserved region from an alignment of nucleotide sequences of the 13 streptococcal SAgs. The reaction conditions of the duplex real-time PCR were optimized and the specificity of the duplex assays was evaluated using SAg positive strains. The limit of detection of the duplex assays was determined by using 10-fold serial dilutions of the DNA of 13 streptococcal SAgs and compared to a conventional polymerase chain reaction (PCR) method for evaluating the duplex assays sensitivity. Using the duplex assays, we were able to differentiate between 13 SAgs from Streptococcus strains and other non-Streptococcus bacteria without cross-reaction. On the other hand, the limit of detection of the duplex assays was at least one or two log dilutions lower than that of the conventional PCR. The panel was highly specific (100%) and the limit of detection of these duplex groups was at least ten times lower than that obtained by using a conventional PCR method.

pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

Science.gov (United States)

Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

2009-11-23

Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

Influence of nucleotide modifications at the C2' position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA.

Science.gov (United States)

Copp, William; Denisov, Alexey Y; Xie, Jingwei; Noronha, Anne M; Liczner, Christopher; Safaee, Nozhat; Wilds, Christopher J; Gehring, Kalle

2017-09-29

Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2'-deoxyribose, 2'-O-methyl-ribose, 2'-deoxy-2'-fluoro-ribose, arabinose and 2'-deoxy-2'-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2' modifications gave a variety of effects. Arabinose and 2'-deoxy-2'-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2'-O-methyl and 2'-deoxy-2'-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

Science.gov (United States)

Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

2015-01-01

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

OpenAIRE

Parsons, C A; West, S C

1990-01-01

T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding comple...

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.

Science.gov (United States)

Chen, Dan; Pan, Shiyang; Xie, Erfu; Gao, Li; Xu, Huaguo; Xia, Wenying; Xu, Ting; Huang, Peijun

2017-01-01

Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, PDNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, PDNA, showing promising application in clinical diagnosis.

Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize.

Science.gov (United States)

Félix-Urquídez, Dalmira; Pérez-Urquiza, Melina; Valdez Torres, José-Benigno; León-Félix, Josefina; García-Estrada, Raymundo; Acatzi-Silva, Abraham

2016-01-05

Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%-100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio.

Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize

Science.gov (United States)

2015-01-01

Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%–100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio. PMID:26605751

NMR and molecular modeling evidence for a G·A mismatch base pair in a purine-rich DNA duplex

International Nuclear Information System (INIS)

Li, Ying; Wilson, W.D.; Zon, G.

1991-01-01

1 H NMR experiments indicate that the oligomer 5'-d(ATGAGCGAATA) forms an unusual 10-base-pair duplex with 4 G·A base pairs and a 3' unpaired adenosine. NMR results indicate that guanoxine imino protons of the F·A mismatches are not hydrogen bonded but are stacked in the helix. A G→ I substitution in either G·A base pair causes a dramatic decrtease in duplex stability and indicates that hydrogen bonding of the guanosine amino group is critical. Nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) results indicate that the overall duplex conformation is in the B-family. Cross-strand NOEs in two-dimensional NOESY spectra between a mismatched AH2 and an AH1' of the other mismatched base pair and between a mismatched GH8 and GNH1 of the other mismatch establish a purine-purine stacking pattern, adenosine over adenosine and guanosine over guanosine, which strongly stabilizes the duplex. A computer graphics molecular model of the ususual duplex was constructed with G·A base pairs containing A-NH 2 to GN3 and G-NH 2 to AN7 hydrogen bonds and B-form base pairs on both sides of the G·A pairs [5'-d(ATGAGC)]. The energy-minimized duplex satisfies all experimental constraints from NOESY and COSY results. A hydrogen bond from G-NH 2 of the mismatch to a phosphate oxygen is predicted

Vesicoureteral Reflux and Duplex Systems

OpenAIRE

Thomas, John C.

2008-01-01

Vesicoureteral reflux (VUR) is the most common anomaly associated with duplex systems. In addition to an uncomplicated duplex system, reflux can also be secondary in the presence of an ectopic ureterocele with duplex systems. Controversy exists in regard to the initial and most definitive management of these anomalies when they coexist. This paper will highlight what is currently known about duplex systems and VUR, and will attempt to provide evidence supporting the various surgical approa...

Modifying and adapting a plant-based DNA extraction protocol for ...

African Journals Online (AJOL)

... a 100 apparently healthy individuals residing in Calabar. The modified DNA procedure yielded good quality genomic DNA which was used in carrying out allele specific polymerase chain reaction which also yielded good quality amplicons. This method is simple and suitable for the extraction of DNA from human red cell.

Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

International Nuclear Information System (INIS)

Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

1991-01-01

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

Science.gov (United States)

Benacer, Douadi; Zain, Siti Nursheena Mohd; Lewis, John W; Khalid, Mohd Khairul Nizam Mohd; Thong, Kwai Lin

2017-01-01

This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

Base Pair Opening in a Deoxynucleotide Duplex Containing a cis-syn Thymine Cyclobutane Dimer Lesion

Science.gov (United States)

Wenke, Belinda B.; Huiting, Leah N.; Frankel, Elisa B.; Lane, Benjamin F.; Núñez, Megan E.

2014-01-01

The cis-syn thymine cyclobutane dimer is a DNA photoproduct implicated in skin cancer. We compared the stability of individual base pairs in thymine dimer-containing duplexes to undamaged parent 10-mer duplexes. UV melting thermodynamic measurements, CD spectroscopy, and 2D NOESY NMR spectroscopy confirm that the thymine dimer lesion is locally and moderately destabilizing within an overall B-form duplex conformation. We measured the rates of exchange of individual imino protons by NMR using magnetization transfer from water and determined the equilibrium constant for the opening of each base pair Kop. In the normal duplex Kop decreases from the frayed ends of the duplex toward the center, such that the central TA pair is the most stable with a Kop of 8×10−7. In contrast, base pair opening at the 5’T of the thymine dimer is facile. The 5’T of the dimer has the largest equilibrium constant (Kop =3×10−4) in its duplex, considerably larger than even the frayed penultimate base pairs. Notably, base pairing by the 3’T of the dimer is much more stable than by the 5’T, indicating that the predominant opening mechanism for the thymine dimer lesion is not likely to be flipping out into solution as a single unit. The dimer asymmetrically affects the stability of the duplex in its vicinity, destabilizing base pairing on its 5’ side more than on the 3’ side. The striking differences in base pair opening between parent and dimer duplexes occur independently of the duplex-single strand melting transitions. PMID:24328089

Structure of DNA damaged by UV and psoralen

International Nuclear Information System (INIS)

Sung-hou Kim; Tomic, M.T.; Wemmer, D.E.; Pearlman, D.; Holbrook, S.

1988-01-01

The authors have used NMR methods to determine a three-dimensional model of an 8 base-pair DNA fragment cross-linked with psoralen. The duplex form of the self-complementary deoxyribonucleotide d-GGGTACCC, contains a psoralen cross-linkable site at the center of the duplex. The cross-link was formed by UV irradiation of a mixture of the purified DNA octamer and 4'-(aminomethyl)-4,5',8-trimethylpsoralen (AMT). Structural information was obtained using one and two-dimensional NMR techniques. Two-dimensional NOE experiments were used to assign the spectrum and estimate distances for many pairs of protons in the cross-linked DNA. Structural parameters obtained are qualitatively consistent with a previously proposed model for kinked and unwound cross-linked B-form DNA derived from crystallography and molecular modeling. The NMR derived model has a 53 degree bend into the major groove occuring primarily at the site of drug addition, and a 56 degree unwinding spanning the 8 base pair duplex. (author)

Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids - TINA

DEFF Research Database (Denmark)

Schneider, Uffe V; Géci, Imrich; Jøhnk, Nina

2011-01-01

-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved......The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators....... Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para...

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta.

Science.gov (United States)

Sarkar, Sailendra Nath; Bakshi, Sankar; Mokkapati, Sanath K; Roy, Sujit; Sengupta, Dibyendu N

2004-07-16

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

Science.gov (United States)

Thakur, Manoj; Kumar, Mohan B J; Muniyappa, K

2016-10-18

Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

Validation of a novel venous duplex ultrasound objective structured assessment of technical skills for the assessment of venous reflux.

Science.gov (United States)

Jaffer, Usman; Normahani, Pasha; Lackenby, Kimberly; Aslam, Mohammed; Standfield, Nigel J

2015-01-01

Duplex ultrasound measurement of reflux time is central to the diagnosis of venous incompetence. We have developed an assessment tool for Duplex measurement of venous reflux for both simulator and patient-based training. A novel assessment tool, Venous Duplex Ultrasound Assessment of Technical Skills (V-DUOSATS), was developed. A modified DUOSATS was used for simulator training. Participants of varying skill level were invited to viewed an instructional video and were allowed ample time to familiarize with the Duplex equipment. Attempts made by the participants were recorded and independently assessed by 3 expert assessors and 5 novice assessors using the modified V-DUOSATS. "Global" assessment was also done by expert assessors on a 4-point Likert scale. Content, construct, and concurrent validities as well as reliability were evaluated. Content and construct validity as well as reliability were demonstrated. Receiver operator characteristic analysis-established cut points of 19/22 and 21/30 were most appropriate for simulator and patient-based assessment, respectively. We have validated a novel assessment tool for Duplex venous reflux measurement. Further work is required to establish transference validity of simulator training to improve skill in scanning patients. We have developed and validated V-DUOSATS for simulator training. Copyright © 2015 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.

Welding of duplex and super-duplex stainless steels

International Nuclear Information System (INIS)

Van Nassau, L.; Meelker, H.; Hilkes, J.

1994-01-01

After a recall of the commercial designation of duplex or super-duplex steels (22-27% Cr, 4-8% Ni, 0.1-0.3% N with or without Mo (1.5-4%)) and of some metallurgical properties (phase diagrams, microstructure, ferrite determination, heat treatment and aging), welding technologies are synthetically presented (advantages-disadvantages of each process, metals filler, parameters of the welding processes, heat treatments after welding, cleaning, passivation, properties (mechanical, corrosion resistance) of the welded pieces). (A.B.). 28 refs. 5 figs., 15 tabs., 1 annexe

Detection of chicken contamination in beef meatball using duplex-PCR Cyt b gene

Science.gov (United States)

Sari, E. P.; Kartikasari, L. R.; Cahyadi, M.

2017-04-01

Beef is one of expensive animal protein sources compared to other meats, on the other hand, chicken is cheap animal protein source. Mixing of chicken into beef meatball is possibly performed to decrease production cost. The aim of this study was to detect chicken contamination in beef meatball using Cytochrome b (Cyt b) gene by duplex-PCR. Sample was designed and prepared as follows, 100% of chicken meatball, 100% of beef meatball and serial level of chicken contaminations in beef meatball (1, 5, 10 and 25%, respectively). Isolation of DNA genome from meatball was according to the guideline of gSYNCTM DNA Extraction Kit for animal tissue. The PCR reaction was carried out using KAPA2G Fast Multiplex Mix. This study found that the DNA genome was succesfully extracted. Moreover, chicken contamination in beef meatball was indicated by the presence of 227 bp DNA band on 2% of agarose gels. Current study revealed that duplex-PCR using Cyt b gene as a genetic marker was able to detect chicken contamination in beef meatball until 1% of chicken meat in the sample. It can be effectively used to identify contamination and also authenticate species origin in animal products to protect consumer from undesirable contents in the food.

DNA-modified electrodes fabricated using copper-free click chemistry for enhanced protein detection.

Science.gov (United States)

Furst, Ariel L; Hill, Michael G; Barton, Jacqueline K

2013-12-31

A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development.

Law of mixture used to model the flow behavior of a duplex stainless steel at high temperatures

International Nuclear Information System (INIS)

Momeni, A.; Dehghani, K.; Poletti, M.C.

2013-01-01

In this investigation the flow curves of a duplex stainless steel were drawn by performing hot compression tests over a wide temperature range of 950–1200 °C and strain rates of 0.001–100 s −1 . The flow curves of ferrite and austenite phases in the duplex structure were depicted by conducting similar hot compression tests on two steels that were cast and prepared with the same chemical compositions. The flow curves of the austenitic steel were found typical of dynamic recrystallization. They were successfully modeled by using the experimental exponential equation proposed by Cingara and McQueen. The flow curves of the ferritic steel were typical of dynamic recovery. They were modeled by the dislocation density evolution function proposed by Estrin and Meckning. Comparing the flow curves of three studied steels, it was found that the flow curves of the duplex steel were very similar and close to those of the ferrite steel. It was understood that in a duplex structure of ferrite and austenite the flow behavior is mostly controlled by the softer phase, i.e. ferrite. The law of mixture was modified to consider the strain partitioning between ferrite and austenite. The distribution coefficients of ferrite and austenite were described and determined at different deformation conditions. The results of modeling satisfactorily predicted the experimental curves. It was shown that the influence of austenite on the flow behavior of the duplex structure is almost low. However, it increases as strain rate or temperature rises. - Highlights: ► Flow curves of austenite and ferrite in the duplex steel were modeled separately. ► The flow behavior of the duplex steel is mostly controlled by ferrite. ► The effect of austenite on flow curve increases with temperature and strain rate. ► The flow curve of the duplex steel is modeled by the modified law of mixture

Backbone conformation affects duplex initiation and duplex propagation in hybridisation of synthetic H-bonding oligomers.

Science.gov (United States)

Iadevaia, Giulia; Núñez-Villanueva, Diego; Stross, Alexander E; Hunter, Christopher A

2018-06-06

Synthetic oligomers equipped with complementary H-bond donor and acceptor side chains form multiply H-bonded duplexes in organic solvents. Comparison of the duplex forming properties of four families of oligomers with different backbones shows that formation of an extended duplex with three or four inter-strand H-bonds is more challenging than formation of complexes that make only two H-bonds. The stabilities of 1 : 1 complexes formed between length complementary homo-oligomers equipped with either phosphine oxide or phenol recognition modules were measured in toluene. When the backbone is very flexible (pentane-1,5-diyl thioether), the stability increases uniformly by an order of magnitude for each additional base-pair added to the duplex: the effective molarities for formation of the first intramolecular H-bond (duplex initiation) and subsequent intramolecular H-bonds (duplex propagation) are similar. This flexible system is compared with three more rigid backbones that are isomeric combinations of an aromatic ring and methylene groups. One of the rigid systems behaves in exactly the same way as the flexible backbone, but the other two do not. For these systems, the effective molarity for formation of the first intramolecular H-bond is the same as that found for the other two backbones, but additional H-bonds are not formed between the longer oligomers. The effective molarities are too low for duplex propagation in these systems, because the oligomer backbones cannot adopt conformations compatible with formation of an extended duplex.

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

Directory of Open Access Journals (Sweden)

Wilson Zoe A

2008-06-01

Full Text Available Abstract Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP and a complementary quenching probe (QP lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

Duplex Real-Time PCR Assay Distinguishes Aedes aegypti From Ae. albopictus (Diptera: Culicidae) Using DNA From Sonicated First-Instar Larvae.

Science.gov (United States)

Kothera, Linda; Byrd, Brian; Savage, Harry M

2017-11-07

Aedes aegypti (L.) and Ae. albopictus (Skuse) are important arbovirus vectors in the United States, and the recent emergence of Zika virus disease as a public health concern in the Americas has reinforced a need for tools to rapidly distinguish between these species in collections made by vector control agencies. We developed a duplex real-time PCR assay that detects both species and does not cross-amplify in any of the other seven Aedes species tested. The lower limit of detection for our assay is equivalent to ∼0.03 of a first-instar larva in a 60-µl sample (0.016 ng of DNA per real-time PCR reaction). The assay was sensitive and specific in mixtures of both species that reflected up to a 2,000-fold difference in DNA concentration. In addition, we developed a simple protocol to extract DNA from sonicated first-instar larvae, and used that DNA to test the assay. Because it uses real-time PCR, the assay saves time by not requiring a separate visualization step. This assay can reduce the time needed for vector control agencies to make species identifications, and thus inform decisions about surveillance and control. Published by Oxford University Press on behalf of Entomological Society of America 2017 This work is written by US Government employees and is in the public domain in the US.

Synthesis and Structural Characterization of 2'-Fluoro-α-L-RNA-Modified Oligonucleotides

DEFF Research Database (Denmark)

Bundgaard Jensen, Troels; Pasternak, Anna; Stahl Madsen, Andreas

2011-01-01

with the smallest destabilization towards RNA. Thermodynamic data show that the duplex formation with 2'-fluoro-α-L-RNA nucleotides is enthalpically disfavored but entropically favored. 2'-Fluoro-α-L-RNA nucleotides exhibit very good base pairing specificity following Watson-Crick rules. The 2'-fluoro......-α-L-RNA monomer was designed as a monocyclic mimic of the bicyclic α-L-LNA, and molecular modeling showed that this indeed is the case as the 2'-fluoro monomer adopts a C3'-endo/C2'-exo sugar pucker. Molecular modeling of modified duplexes show that the 2'-fluoro-α-L-RNA nucleotides partake in Watson-Crick base......We describe the synthesis and binding properties of oligonucleotides that contain one or more 2'-fluoro-α-L-RNA thymine monomer(s). Incorporation of 2'-fluoro-α-L-RNA thymine into oligodeoxynucleotides decreased thermal binding stability slightly upon hybridization with complementary DNA and RNA...

Genotoxic effect and antigen binding characteristics of SLE auto-antibodies to peroxynitrite-modified human DNA.

Science.gov (United States)

Khan, Md Asad; Alam, Khursheed; Mehdi, Syed Hassan; Rizvi, M Moshahid A

2017-12-01

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by auto-antibodies against native deoxyribonucleic acid after modification and is one of the reasons for the development of SLE. Here, we have evaluated the structural perturbations in human placental DNA by peroxynitrite using spectroscopy, thermal denaturation and high-performance liquid chromatography (HPLC). Peroxynitrite is a powerful potent bi-functional oxidative/nitrative agent that is produced both endogenously and exogenously. In experimental animals, the peroxynitrite-modified DNA was found to be highly immunogenic. The induced antibodies showed cross-reactions with different types of DNA and nitrogen bases that were modified with peroxynitrite by inhibition ELISA. The antibody activity was inhibited by approximately 89% with its immunogen as the inhibitor. The antigen-antibodies interaction between induced antibodies with peroxynitrite-modified DNA showed retarded mobility as compared to the native form. Furthermore, significantly increased binding was also observed in SLE autoantibodies with peroxynitrite-modified DNA than native form. Moreover, DNA isolated from lymphocyte of SLE patients revealed significant recognition of anti-peroxynitrite-modified DNA immunoglobulin G (IgG). Our data indicates that DNA modified with peroxynitrite presents unique antigenic determinants that may induce autoantibody response in SLE. Copyright © 2017 Elsevier Inc. All rights reserved.

Tricriticality in the q-neighbor Ising model on a partially duplex clique.

Science.gov (United States)

Chmiel, Anna; Sienkiewicz, Julian; Sznajd-Weron, Katarzyna

2017-12-01

We analyze a modified kinetic Ising model, a so-called q-neighbor Ising model, with Metropolis dynamics [Phys. Rev. E 92, 052105 (2015)PLEEE81539-375510.1103/PhysRevE.92.052105] on a duplex clique and a partially duplex clique. In the q-neighbor Ising model each spin interacts only with q spins randomly chosen from its whole neighborhood. In the case of a duplex clique the change of a spin is allowed only if both levels simultaneously induce this change. Due to the mean-field-like nature of the model we are able to derive the analytic form of transition probabilities and solve the corresponding master equation. The existence of the second level changes dramatically the character of the phase transition. In the case of the monoplex clique, the q-neighbor Ising model exhibits a continuous phase transition for q=3, discontinuous phase transition for q≥4, and for q=1 and q=2 the phase transition is not observed. On the other hand, in the case of the duplex clique continuous phase transitions are observed for all values of q, even for q=1 and q=2. Subsequently we introduce a partially duplex clique, parametrized by r∈[0,1], which allows us to tune the network from monoplex (r=0) to duplex (r=1). Such a generalized topology, in which a fraction r of all nodes appear on both levels, allows us to obtain the critical value of r=r^{*}(q) at which a tricriticality (switch from continuous to discontinuous phase transition) appears.

SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

DEFF Research Database (Denmark)

Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

2015-01-01

dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

[Analysis of Conformational Features of Watson-Crick Duplex Fragments by Molecular Mechanics and Quantum Mechanics Methods].

Science.gov (United States)

Poltev, V I; Anisimov, V M; Sanchez, C; Deriabina, A; Gonzalez, E; Garcia, D; Rivas, F; Polteva, N A

2016-01-01

It is generally accepted that the important characteristic features of the Watson-Crick duplex originate from the molecular structure of its subunits. However, it still remains to elucidate what properties of each subunit are responsible for the significant characteristic features of the DNA structure. The computations of desoxydinucleoside monophosphates complexes with Na-ions using density functional theory revealed a pivotal role of DNA conformational properties of single-chain minimal fragments in the development of unique features of the Watson-Crick duplex. We found that directionality of the sugar-phosphate backbone and the preferable ranges of its torsion angles, combined with the difference between purines and pyrimidines. in ring bases, define the dependence of three-dimensional structure of the Watson-Crick duplex on nucleotide base sequence. In this work, we extended these density functional theory computations to the minimal' fragments of DNA duplex, complementary desoxydinucleoside monophosphates complexes with Na-ions. Using several computational methods and various functionals, we performed a search for energy minima of BI-conformation for complementary desoxydinucleoside monophosphates complexes with different nucleoside sequences. Two sequences are optimized using ab initio method at the MP2/6-31++G** level of theory. The analysis of torsion angles, sugar ring puckering and mutual base positions of optimized structures demonstrates that the conformational characteristic features of complementary desoxydinucleoside monophosphates complexes with Na-ions remain within BI ranges and become closer to the corresponding characteristic features of the Watson-Crick duplex crystals. Qualitatively, the main characteristic features of each studied complementary desoxydinucleoside monophosphates complex remain invariant when different computational methods are used, although the quantitative values of some conformational parameters could vary lying within the

[Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].

Science.gov (United States)

Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

2012-01-01

Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.

Watson-Crick base pairing controls excited-state decay in natural DNA.

Science.gov (United States)

Bucher, Dominik B; Schlueter, Alexander; Carell, Thomas; Zinth, Wolfgang

2014-10-13

Excited-state dynamics are essential to understanding the formation of DNA lesions induced by UV light. By using femtosecond IR spectroscopy, it was possible to determine the lifetimes of the excited states of all four bases in the double-stranded environment of natural DNA. After UV excitation of the DNA duplex, we detected a concerted decay of base pairs connected by Watson-Crick hydrogen bonds. A comparison of single- and double-stranded DNA showed that the reactive charge-transfer states formed in the single strands are suppressed by base pairing in the duplex. The strong influence of the Watson-Crick hydrogen bonds indicates that proton transfer opens an efficient decay path in the duplex that prohibits the formation or reduces the lifetime of reactive charge-transfer states. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Full Duplex Emulation via Spatial Separation of Half Duplex Nodes in a Planar Cellular Network

DEFF Research Database (Denmark)

Thomsen, Henning; Kim, Dong Min; Popovski, Petar

2016-01-01

A Full Duplex Base Station (FD-BS) can be used to serve simultaneously two Half-Duplex (HD) Mobile Stations (MSs), one working in the uplink and one in the downlink, respectively. The same functionality can be realized by having two interconnected and spatially separated Half Duplex Base Stations...... (HD-BSs), which is a scheme termed CoMPflex (CoMP for In-Band Wireless Full Duplex). A FD-BS can be seen as a special case of CoMPflex with separation distance zero. In this paper we study the performance of CoMPflex in a two-dimensional cellular scenario using stochastic geometry and compare...

Nucleic acid nanomaterials: Silver-wired DNA

Science.gov (United States)

Auffinger, Pascal; Ennifar, Eric

2017-10-01

DNA double helical structures are supramolecular assemblies that are typically held together by classical Watson-Crick pairing. Now, nucleotide chelation of silver ions supports an extended silver-DNA hybrid duplex featuring an uninterrupted silver array.

Detection of DNA of genetically modified maize by a silicon nanowire field-effect transistor

International Nuclear Information System (INIS)

Pham, Van Binh; Tung Pham, Xuan Thanh; Duong Dang, Ngoc Thuy; Tuyen Le, Thi Thanh; Tran, Phu Duy; Nguyen, Thanh Chien; Nguyen, Van Quoc; Dang, Mau Chien; Tong, Duy Hien; Van Rijn, Cees J M

2011-01-01

A silicon nanowire field-effect transistor based sensor (SiNW-FET) has been proved to be the most sensitive and powerful device for bio-detection applications. In this paper, SiNWs were first fabricated by using our recently developed deposition and etching under angle technique (DEA), then used to build up the complete SiNW device based biosensor. The fabricated SiNW biosensor was used to detect DNA of genetically modified maize. As the DNA of the genetically modified maize has particular DNA sequences of 35S promoter, we therefore designed 21 mer DNA oligonucleotides, which are used as a receptor to capture the transferred DNA of maize. In our work, the SiNW biosensor could detect DNA of genetically modified maize with concentrations down to about 200 pM

Harvesting full-duplex rate gains in cellular networks with half-duplex user terminals

KAUST Repository

Alammouri, Ahmad; Elsawy, Hesham; Alouini, Mohamed-Slim

2016-01-01

Full-Duplex (FD) transceivers may be expensive in terms of complexity, power consumption, and price to be implemented in all user terminals. Therefore, techniques to exploit in-band full-duplex communication with FD base stations (BSs) and half

DNA translocation by human uracil DNA glycosylase: the case of single-stranded DNA and clustered uracils.

Science.gov (United States)

Schonhoft, Joseph D; Stivers, James T

2013-04-16

Human uracil DNA glycosylase (hUNG) plays a central role in DNA repair and programmed mutagenesis of Ig genes, requiring it to act on sparsely or densely spaced uracil bases located in a variety of contexts, including U/A and U/G base pairs, and potentially uracils within single-stranded DNA (ssDNA). An interesting question is whether the facilitated search mode of hUNG, which includes both DNA sliding and hopping, changes in these different contexts. Here we find that hUNG uses an enhanced local search mode when it acts on uracils in ssDNA, and also, in a context where uracils are densely clustered in duplex DNA. In the context of ssDNA, hUNG performs an enhanced local search by sliding with a mean sliding length larger than that of double-stranded DNA (dsDNA). In the context of duplex DNA, insertion of high-affinity abasic product sites between two uracil lesions serves to significantly extend the apparent sliding length on dsDNA from 4 to 20 bp and, in some cases, leads to directionally biased 3' → 5' sliding. The presence of intervening abasic product sites mimics the situation where hUNG acts iteratively on densely spaced uracils. The findings suggest that intervening product sites serve to increase the amount of time the enzyme remains associated with DNA as compared to nonspecific DNA, which in turn increases the likelihood of sliding as opposed to falling off the DNA. These findings illustrate how the search mechanism of hUNG is not predetermined but, instead, depends on the context in which the uracils are located.

Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

Science.gov (United States)

Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

2011-01-01

To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR.

Science.gov (United States)

Luo, Yakun; Liang, Lin; Zhou, Ling; Zhao, Kai; Cui, Shangjin

2015-07-01

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the simple, rapid, and specific amplification of DNA and has been used to detect viruses. A duplex nanoPCR molecular detection system was developed to detect pseudorabies virus (PRV) and porcine bocavirus (PBoV). Primers were selected to target conserved regions within the PRV gE gene and the PBoV NS1 gene. Under optimized nanoPCR reaction conditions, two specific fragments of 316 bp (PRV) and 996 bp (PBoV) were amplified by the duplex nanoPCR with a detection limit of 6 copies for PRV and 95 copies for PBoV; no fragments were amplified when other porcine viruses were used as template. When used to test 550 clinical samples, the duplex nanoPRC assay and a conventional duplex PCR assay provided very similar results (98.1% consistency); single PRV infections, single PBoV infections, and concurrent PRV and PBoV infections were detected in 37%, 15%, and 9% of the samples, respectively. The results indicate that the novel duplex nanoPCR assay is useful for the rapid detection of PRV and PBoV in pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

Achieving single channel, full duplex wireless communication

KAUST Repository

Choi, Jung Il; Jain, Mayank; Srinivasan, Kannan; Levis, Phil; Katti, Sachin

2010-01-01

This paper discusses the design of a single channel full-duplex wireless transceiver. The design uses a combination of RF and baseband techniques to achieve full-duplexing with minimal effect on link reliability. Experiments on real nodes show the full-duplex prototype achieves median performance that is within 8% of an ideal full-duplexing system. This paper presents Antenna Cancellation, a novel technique for self-interference cancellation. In conjunction with existing RF interference cancellation and digital baseband interference cancellation, antenna cancellation achieves the amount of self-interference cancellation required for full-duplex operation. The paper also discusses potential MAC and network gains with full-duplexing. It suggests ways in which a full-duplex system can solve some important problems with existing wireless systems including hidden terminals, loss of throughput due to congestion, and large end-to-end delays. Copyright 2010 ACM.

Modified beacon probe assisted dual signal amplification for visual detection of microRNA.

Science.gov (United States)

Sun, Xiuwei; Ying, Na; Ju, Chuanjing; Li, Zhongyi; Xu, Na; Qu, Guijuan; Liu, Wensen; Wan, Jiayu

2018-04-21

In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity. Copyright © 2018 Elsevier Inc. All rights reserved.

Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae

Directory of Open Access Journals (Sweden)

Hilary A. McManus

2017-05-01

Full Text Available Background Comparative studies of chloroplast genomes (plastomes across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae, one from Hydrodictyon reticulatum and the other from Pediastrum duplex. Methods Genomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed. Results The plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae. Discussion The complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique

Superimposed Code Theoretic Analysis of Deoxyribonucleic Acid (DNA) Codes and DNA Computing

Science.gov (United States)

2010-01-01

DNA strand and its Watson - Crick complement can be used to perform mathematical computation. This research addresses how the...Acid dsDNA double stranded DNA MOSAIC Mobile Stream Processing Cluster PCR Polymerase Chain Reaction RAM Random Access Memory ssDNA single stranded DNA WC Watson – Crick A Adenine C Cytosine G Guanine T Thymine ...are 5′→3′ and strands with strikethrough are 3′→5′. A dsDNA duplex formed between a strand and its reverse complement is called a

Silver-mediated base pairings: towards dynamic DNA nanostructures with enhanced chemical and thermal stability

International Nuclear Information System (INIS)

Swasey, Steven M; Gwinn, Elisabeth G

2016-01-01

The thermal and chemical fragility of DNA nanomaterials assembled by Watson–Crick (WC) pairing constrain the settings in which these materials can be used and how they can be functionalized. Here we investigate use of the silver cation, Ag + , as an agent for more robust, metal-mediated self-assembly, focusing on the simplest duplex building blocks that would be required for more elaborate Ag + –DNA nanostructures. Our studies of Ag + -induced assembly of non-complementary DNA oligomers employ strands of 2–24 bases, with varied base compositions, and use electrospray ionization mass spectrometry to determine product compositions. High yields of duplex products containing narrowly distributed numbers of Ag + can be achieved by optimizing solution conditions. These Ag + -mediated duplexes are stable to at least 60 mM Mg 2+ , higher than is necessary for WC nanotechnology schemes such as tile assemblies and DNA origami, indicating that sequential stages of Ag + -mediated and WC-mediated assembly may be feasible. Circular dichroism spectroscopy suggests simple helical structures for Ag + -mediated duplexes with lengths to at least 20 base pairs, and further indicates that the structure of cytosine-rich duplexes is preserved at high urea concentrations. We therefore propose an approach towards dynamic DNA nanomaterials with enhanced thermal and chemical stability through designs that combine sturdy silver-mediated ‘frames’ with WC paired ‘pictures’. (paper)

Duplex gall bladder: bystander or culprit.

Science.gov (United States)

Kumar, Jogender; Yadav, Arushi

2017-08-30

Gall bladder (GB) duplication is a rare anatomical malformation, which can be detected by preoperative imaging study. We present a case of duplex gall bladder in a 14-year-old boy who presented with abdominal pain. On ultrasound, he had right nephrolithiasis and duplex gall bladder. Duplex gall bladder was confirmed on MR cholangiopancreatography. There was a dilemma for surgical management of duplex gall bladder; however, he became asymptomatic after conservative treatment. Prophylactic surgery is not recommended for asymptomatic incidentally detected duplex gall bladder. Radiologists and paediatric surgeons should be sensitised about the exact anatomy of this entity. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Targeting human c-Myc promoter duplex DNA with actinomycin D by use of multi-way analysis of quantum-dot-mediated fluorescence resonance energy transfer

DEFF Research Database (Denmark)

Gholami, Somayeh; Kompany Zare, Mohsen

2013-01-01

Actinomycin D (Act D), an oncogenic c-Myc promoter binder, interferes with the action of RNA polymerase. There is great demand for high-throughput technology able to monitor the activity of DNA-binding drugs. To this end, binding of 7-aminoactinomycin D (7AAD) to the duplex c-Myc promoter...... pairs resulted in efficient energy transfer from drug to QD via fluorescence resonance energy transfer (FRET). Multi-way analysis of the three-way data array obtained from titration experiments was performed by use of restricted Tucker3 and hard trilinear decomposition (HTD). These techniques enable...... the important advantage over univariate classical methods of enabling us to investigate the source of variance in the fluorescence signal of the DNA-drug complex. It was established that hard trilinear decomposition analysis of FRET-measured data overcomes the problem of rank deficiency, enabling calculation...

O6-ethylguanine carcinogenic lesions in DNA: An NMR study of O6etG·T pairing in dodecanucloetide duplexes

International Nuclear Information System (INIS)

Kalnik, M.W.; Li, B.F.L.; Swann, P.F.; Patel, D.J.

1989-01-01

High-resolution two-dimensional NMR studies are reported on the self-complementary d-(C1-G2-C3-O 6 etG4-A5-G6-C7-T8-T9-G10-C11-G12) duplex (designated O 6 etG·T 12-mer) containing two symmetrically related O 6 etG·T lesion sites located four base pairs in from either end of the duplex. Parallel studies were undertaken on a related sequence containing O 6 meG·T lesion sites (designated O 6 meG·T 12-mer) in order to evaluate the influence of the size of the alkyl substituent on the structure of the duplex and were undertaken on a related sequence containing G·T mismatch sites (designated G·T 12-mer duplex), which served as the control duplex. The exchangeable and nonexchangeable proton and the phosphorus nuclei have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOE) and correlated spectra of the O 6 etG·T 12-mer, O 6 meG·T 12-mer, and G·T 12-mer duplexes in H 2 O and D 2 O solutions. The distance connectivities observed in the NOESY spectra of the O 6 alkG·T 12-mer duplexes establish that the helix is right-handed and all of the bases adopt an anti conformation of the glycosidic torsion angle including the O 6 alkG4 and T9 bases at the lesion site. These observations establish that the O 6 alkG4 and T9 residues are stacked into the duplex and that the O 6 CH 3 and O 6 CH 2 CH 3 groups of O 6 alkG4 adopt a syn orientation with respect to the N 1 of the alkylated guanine. Since the O 6 -alkyl group adopts a syn orientation, the separation between the O 6 of O 6 alkG4 and the O 4 of T9 in the major groove is increased, preventing the formation of a short hydrogen bond between the N 1 ring nitrogen of O 6 alkG4 and the imino proton of T9

Hairpin and duplex formation in DNA fragments CCAATTTTGG, CCAATTTTTTGG, and CCATTTTTGG: a proton NMR study

International Nuclear Information System (INIS)

Pramanik, P.; Kanhouwa, N.; Kan, L.

1988-01-01

Three DNA fragments, CCAATTTTGG (1), CCAATTTTTTGG (2), AND CCATTTTTGG (3), were studied by proton NMR spectroscopy in aqueous solution. All these oligodeoxyribonucleotides contain common sequences at the 5' and 3' ends (5'-CCA and TGG-3'). 2 as well as 3 forms only hairpin structures with four unpaired thymidylyl units, four and three base pair stems, respectively, in neutral solution under low and high NaCl concentrations. At high salt concentration the oligomer 1 forms a duplex structure with -TT- internal loop. On the other hand, the same oligomer forms a stable hairpin structure at low salt and low strand concentrations at pH 7. The hairpin structure of 1 has a stem containing only three base pairs (CCA x TGG) and a loop containing four nucleotides (-ATTT-) that includes a dissociated A x T base pair. The two secondary structures of 1 coexist in an aqueous solution containing 0.1 M NaCl, at pH 7. The equilibrium shifts to the hairpin side when the temperature is raised. The stabilities and base-stacking modes of all three oligonucleotides in tow different structures are reported

Base pairing and structural insights into the 5-formylcytosine in RNA duplex

Science.gov (United States)

Wang, Rui; Luo, Zhipu; He, Kaizhang; Delaney, Michael O.; Chen, Doris; Sheng, Jia

2016-01-01

Abstract 5-Formylcytidine (f5C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m5C) through 5-hydroxymethylcytidine (hm5C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f5C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5′-GUA(f5C)GUAC-3′]2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f5C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex. PMID:27079978

Overall performance of the duplex Stirling refrigerator

International Nuclear Information System (INIS)

Erbay, L. Berrin; Ozturk, M. Mete; Doğan, Bahadır

2017-01-01

Highlights: • Overall performance coefficient of duplex Stirling refrigerator was investigated. • A definite region for the coefficient of performance of the refrigerator in duplex Stirling is identified. • A definite region for the thermal efficiency of the heat engine in duplex Stirling is identified. • Benchmark values and design bounds of the duplex Stirling refrigerator were obtained. - Abstract: The duplex Stirling refrigerator is an integrated refrigerator consists of Stirling cycle engine and Stirling cycle refrigerator used for cooling. The equality of the work generation of the heat engine to the work consumption of the refrigerator is the primary constraint of the duplex Stirling. The duplex Stirling refrigerator is investigated thermodynamically by considering the effects of constructional and operational parameters which are namely the temperature ratios for heat engine and refrigerator, and the compression ratios for both sides. The primary concern is given to the parametric effects on the overall coefficient of performance of the duplex Stirling refrigerator. The given diagrams provide a design bounds and benchmark results that allows seeing the big picture about the cooling load and heat input relation. Moreover they ease to determine the corresponding work rate to the target cooling load. As regard to the obtained results, a definite region for coefficient of performance of the refrigerator and a definite region for the thermal efficiency of the heat engine of the duplex Stirling are identified.

Four highly pseudosymmetric and/or twinned structures of d(CGCGCG) ₂ extend the repertoire of crystal structures of Z-DNA

Energy Technology Data Exchange (ETDEWEB)

Luo, Zhipu; Dauter, Zbigniew; Gilski, Miroslaw

2017-10-30

DNA oligomer duplexes containing alternating cytosines and guanines in their sequences tend to form left-handed helices of the Z-DNA type, with the sugar and phosphate backbone in a zigzag conformation and a helical repeat of two successive nucleotides. Z-DNA duplexes usually crystallize as hexagonally arranged parallel helical tubes, with various relative orientations and translation of neighboring duplexes. Four novel high-resolution crystal structures of d(CGCGCG)₂duplexes are described here. They are characterized by a high degree of pseudosymmetry and/or twinning, with three or four independent duplexes differently oriented in a monoclinicP2₁lattice of hexagonal metric. The various twinning criteria give somewhat conflicting indications in these complicated cases of crystal pathology. The details of molecular packing in these crystal structures are compared with other known crystal forms of Z-DNA.

An improved method for detecting genetic variation in DNA using denaturing gradient gel electrophoresis

International Nuclear Information System (INIS)

Takahashi, Norio; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

1990-05-01

We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that use of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than use of DNA:DNA heteroduplexes, because preparation of RNA probes is easier than that of DNA probes. Three different 32 P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of a mismatch(es) was detected as a difference in the mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and three thalassemic human β-globin genes. The results from experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human β-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was undertaken in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for detecting heritable variation and should be a powerful tool for detecting fresh mutations in DNA, which occur outside the known restriction sites. (author)

Hydrophobically modified chitosan/gold nanoparticles for DNA delivery

International Nuclear Information System (INIS)

Bhattarai, Shanta Raj; Remant Bahadur, K.C.; Aryal, Santosh; Bhattarai, Narayan; Kim, Sun Young; Yi, Ho Keun; Hwang, Pyoung Han; Kim, Hak Yong

2008-01-01

Present study dealt an application of modified chitosan gold nanoparticles (Nac-6-Au) for the immobilization of necked plasmid DNA. Gold nanoparticles stabilized with N-acylated chitosan were prepared by graft-onto approach. The stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-vis, TEM, ELS and DLS. MTT assay was used for in vitro cytotoxicity of the nanoparticles into three different cell lines (NIH 3T3, CT-26 and MCF-7). The formulation of plasmid DNA with the nanoparticles corresponds to the complex forming capacity and in-vitro/in-vivo transfection efficiency was studied via gel electrophoresis and transfection methods, respectively. Results showed the modified chitosan gold nanoparticles were well-dispersed and spherical in shape with average size around 10∼12 nm in triple distilled water at pH 7.4, and showed relatively no cytotoxicity at low concentration. Addition of plasmid DNA on the aqueous solution of the nanoparticles markedly reduced surface potential (50.0∼66.6%) as well as resulted in a 13.33% increase in hydrodynamic diameters of the formulated nanoparticles. Transfection efficiency of Nac-6-Au/DNA was dependent on cell type, and higher β-galactosidase activity was observed on MCF-7 breast cancer cell. Typically, this activity was 5 times higher in 4.5 mg/ml nanoparticles concentration than that achieved by the nanoparticles of other concentrations (and/or control). However, this activity was lower in in-vitro and dramatically higher in in-vivo than that of commercially available transfection kit (Lipofectin (registered) ) and DNA. From these results, it can be expected to develop alternative new vectors for gene delivery

In situ detection of tandem DNA repeat length

Energy Technology Data Exchange (ETDEWEB)

Yaar, R.; Szafranski, P.; Cantor, C.R.; Smith, C.L. [Boston Univ., MA (United States)

1996-11-01

A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats of known lengths. Single-stranded loop structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase. 7 refs., 4 figs.

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

Science.gov (United States)

Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

2013-01-01

Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

Science.gov (United States)

Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

2017-09-01

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Buffer management in wireless full-duplex systems

KAUST Repository

Bouacida, Nader

2015-10-19

Wireless full-duplex radios can simultaneously transmit and receive using the same frequency. In theory, this can double the throughput. In fact, there is only little work addressing aspects other than throughput gains in full-duplex systems. Over-buffering in today\\'s networks or the so-called “bufferbloat” phenomenon creates excessive end-to-end delays resulting in network performance degradation. Our analysis shows that full-duplex systems may suffer from high latency caused by bloated buffers. In this paper, we address the problem of buffer management in full-duplex networks by using Wireless Queue Management (WQM), which is an active queue management technique for wireless networks. Our solution is based on Relay Full-Duplex MAC (RFD-MAC), an asynchronous media access control protocol designed for relay full-duplexing. We compare the performance of WQM in full-duplex environment to Drop Tail mechanism over various scenarios. Our solution reduces the end-to-end delay by two orders of magnitude while achieving similar throughput in most of the cases.

Thickness, morphology, and optoelectronic characteristics of pristine and surfactant-modified DNA thin films

International Nuclear Information System (INIS)

Arasu, Velu; Reddy Dugasani, Sreekantha; Son, Junyoung; Gnapareddy, Bramaramba; Ha Park, Sung; Jeon, Sohee; Jeong, Jun-Ho

2017-01-01

Although the preparation of DNA thin films with well-defined thicknesses controlled by simple physical parameters is crucial for constructing efficient, stable, and reliable DNA-based optoelectronic devices and sensors, it has not been comprehensively studied yet. Here, we construct DNA and surfactant-modified DNA thin films by drop-casting and spin-coating techniques. The DNA thin films formed with different control parameters, such as drop-volume and spin-speed at given DNA concentrations, exhibit characteristic thickness, surface roughness, surface potential, and absorbance, which are measured by a field emission scanning electron microscope, a surface profilometer, an ellipsometer, an atomic force microscope, a Kelvin probe force microscope, and an UV–visible spectroscope. From the observations, we realized that thickness significantly affects the physical properties of DNA thin films. This comprehensive study of thickness-dependent characteristics of DNA and surfactant-modified DNA thin films provides insight into the choice of fabrication techniques in order for the DNA thin films to have desired physical characteristics in further applications, such as optoelectronic devices and sensors. (paper)

Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

NARCIS (Netherlands)

Etson, Candice M.; Hamdan, Samir M.; Richardson, Charles C.; Oijen, Antoine M. van; Richardson, Charles C.

2010-01-01

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1:1 complex with

Practical, real-time, full duplex wireless

KAUST Repository

Jain, Mayank; Choi, Jung Il; Kim, Taemin; Bharadia, Dinesh; Seth, Siddharth; Srinivasan, Kannan; Levis, Philip; Katti, Sachin; Sinha, Prasun

2011-01-01

This paper presents a full duplex radio design using signal inversion and adaptive cancellation. Signal inversion uses a simple design based on a balanced/unbalanced (Balun) transformer. This new design, unlike prior work, supports wideband and high power systems. In theory, this new design has no limitation on bandwidth or power. In practice, we find that the signal inversion technique alone can cancel at least 45dB across a 40MHz bandwidth. Further, combining signal inversion cancellation with cancellation in the digital domain can reduce self-interference by up to 73dB for a 10MHz OFDM signal. This paper also presents a full duplex medium access control (MAC) design and evaluates it using a testbed of 5 prototype full duplex nodes. Full duplex reduces packet losses due to hidden terminals by up to 88%. Full duplex also mitigates unfair channel allocation in AP-based networks, increasing fairness from 0.85 to 0.98 while improving downlink throughput by 110% and uplink throughput by 15%. These experimental results show that a re- design of the wireless network stack to exploit full duplex capability can result in significant improvements in network performance. © 2011 ACM.

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

Science.gov (United States)

Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

2014-06-01

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

Rietveld and impedance analysis of cold and hot rolled duplex and lean duplex steels for application in paper and pulp industry

Energy Technology Data Exchange (ETDEWEB)

Esteves, Luiza; Lins, Vanessa de Freitas Cunha, E-mail: luizaeq@yahoo.com.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Departamento de Engenharia Quimica; Paiva, Paulo Renato Perdigao [Centro Federal de Educacao Tecnologica de Minas Gerais (CEFET), Belo Horizonte, MG (Brazil); Viana, Adolfo Kalergis do Nascimento [APERAM South America, Timoteo, MG (Brazil)

2017-01-15

In this study, X-Ray Diffraction (XRD) and Rietveld Refinement were performed to identify and quantify the ferrite and austenite phase of cold and hot rolled duplex stainless steels (UNS S31803) and lean duplex stainless steels (UNS S32304). Electrochemical impedance spectroscopy (EIS) was applied to evaluate the chemical behavior of duplex and lean duplex stainless steels in white, green, and black liquors of paper and pulp industry. Rietveld analysis results showed a higher austenite content than the standard limit for duplex steels in the hot rolled condition. The hot rolling condition plays a major role in improving corrosion resistance in white liquor mainly for the lean duplex steel. (author)

Rietveld and impedance analysis of cold and hot rolled duplex and lean duplex steels for application in paper and pulp industry

International Nuclear Information System (INIS)

Esteves, Luiza; Lins, Vanessa de Freitas Cunha; Viana, Adolfo Kalergis do Nascimento

2017-01-01

In this study, X-Ray Diffraction (XRD) and Rietveld Refinement were performed to identify and quantify the ferrite and austenite phase of cold and hot rolled duplex stainless steels (UNS S31803) and lean duplex stainless steels (UNS S32304). Electrochemical impedance spectroscopy (EIS) was applied to evaluate the chemical behavior of duplex and lean duplex stainless steels in white, green, and black liquors of paper and pulp industry. Rietveld analysis results showed a higher austenite content than the standard limit for duplex steels in the hot rolled condition. The hot rolling condition plays a major role in improving corrosion resistance in white liquor mainly for the lean duplex steel. (author)

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

Science.gov (United States)

Boiteux, Serge; Jinks-Robertson, Sue

2013-01-01

DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

Science.gov (United States)

Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

2000-05-01

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

Prolonged CT urography in duplex kidney.

Science.gov (United States)

Gong, Honghan; Gao, Lei; Dai, Xi-Jian; Zhou, Fuqing; Zhang, Ning; Zeng, Xianjun; Jiang, Jian; He, Laichang

2016-05-13

Duplex kidney is a common anomaly that is frequently associated with multiple complications. Typical computed tomography urography (CTU) includes four phases (unenhanced, arterial, parenchymal and excretory) and has been suggested to considerably aid in the duplex kidney diagnosi. Unfortunately, regarding duplex kidney with prolonged dilatation, the affected parenchyma and tortuous ureters demonstrate a lack of or delayed excretory opacification. We used prolonged-delay CTU, which consists of another prolonged-delay phase (1- to 72-h delay; mean delay: 24 h) to opacify the duplicated ureters and affected parenchyma. Seventeen patients (9 males and 8 females; age range: 2.5-56 y; mean age: 40.4 y) with duplex kidney were included in this study. Unenhanced scans did not find typical characteristics of duplex kidney, except for irregular perirenal morphology. Duplex kidney could not be confirmed on typical four-phase CTU, whereas it could be easily diagnosed in axial and CT-3D reconstruction using prolonged CTU (prolonged-delay phase). Between January 2005 and October 2010, in this review board-approved study (with waived informed consent), 17 patients (9 males and 8 females; age range: 2.5 ~ 56 y; mean age: 40.4 y) with suspicious duplex kidney underwent prolonged CTU to opacify the duplicated ureters and confirm the diagnosis. Our results suggest the validity of prolonged CTU to aid in the evaluation of the function of the affected parenchyma and in the demonstration of urinary tract malformations.

Zoster duplex: a clinical report and etiologic analysis.

Science.gov (United States)

Zhang, Feng; Zhou, Jin

2015-01-01

Herpes zoster (HZ) duplex is a rare disease presentation. The mechanisms of varicella zoster virus (VZV) reactivation in multiple dermal regions are unknown. To present a HZ duplex case occurring in an immunocompetent woman and to analyze the possible underlying causes of HZ duplex. We present a HZ duplex case in an immunocompetent woman and analyzed the possible contributing factors in 36 HZ duplex cases. Continuously distributed variables were categorized by numbers and percentages. In our study, 24 cases (66.7%) were from Asia, 16 cases (44.4%) were in individuals ≥ 50 years of age, and 17 cases (47.2%) occurred in immunocompromised patients. Of the 36 cases, 23 involved women (63.9%) and 13 involved men. Eighteen patients suffering from HZ duplex, 13 of which were women (72.2%), did not suffer from any chronic systemic disease or have a long history of taking drugs. HZ duplex is a rare event that can occur in both immunocompetent and immunosuppressed individuals. HZ duplex might be associated with the Asia region, advanced age, immunosuppression, and being female.

Preferential recognition of auto-antibodies against 4-hydroxynonenal modified DNA in the cancer patients.

Science.gov (United States)

Faisal, Mohammad; Shahab, Uzma; Alatar, Abdulrahman A; Ahmad, Saheem

2017-11-01

The structural perturbations in DNA molecule may be caused by a break in a strand, a missing base from the backbone, or a chemically changed base. These alterations in DNA that occurs naturally can result from metabolic or hydrolytic processes. DNA damage plays a major role in the mutagenesis, carcinogenesis, aging and various other patho-physiological conditions. DNA damage can be induced through hydrolysis, exposure to reactive oxygen species (ROS) and other reactive carbonyl metabolites including 4-hydroxynonenal (HNE). 4-HNE is an important lipid peroxidation product which has been implicated in the mutagenesis and carcinogenesis processes. The present study examines to probe the presence of auto-antibodies against 4-hydroxynonenal damaged DNA (HNE-DNA) in various cancer subjects. In this study, the purified calf thymus DNA was damaged by the action of 4-HNE. The DNA was incubated with 4-HNE for 24 h at 37°C temperature. The binding characteristics of cancer auto-antibodies were assessed by direct binding and competitive inhibition ELISA. DNA modifications produced hyperchromicity in UV spectrum and decreased fluorescence intensity. Cancer sera exhibited enhanced binding with the 4-HNE modified calf thymus DNA as compared to its native conformer. The 4-HNE modified DNA presents unique epitopes which may be one of the factors for the auto-antibody induction in cancer patients. The HNE modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients. © 2017 Wiley Periodicals, Inc.

Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

Science.gov (United States)

Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

2017-05-12

Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle.

Science.gov (United States)

Mazloum-Ardakani, Mohammad; Rajabzadeh, Nooshin; Benvidi, Ali; Heidari, Mohammad Mehdi

2013-12-15

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH-ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA. Copyright © 2013 Elsevier Inc. All rights reserved.

Maximizing synchronizability of duplex networks

Science.gov (United States)

Wei, Xiang; Emenheiser, Jeffrey; Wu, Xiaoqun; Lu, Jun-an; D'Souza, Raissa M.

2018-01-01

We study the synchronizability of duplex networks formed by two randomly generated network layers with different patterns of interlayer node connections. According to the master stability function, we use the smallest nonzero eigenvalue and the eigenratio between the largest and the second smallest eigenvalues of supra-Laplacian matrices to characterize synchronizability on various duplexes. We find that the interlayer linking weight and linking fraction have a profound impact on synchronizability of duplex networks. The increasingly large inter-layer coupling weight is found to cause either decreasing or constant synchronizability for different classes of network dynamics. In addition, negative node degree correlation across interlayer links outperforms positive degree correlation when most interlayer links are present. The reverse is true when a few interlayer links are present. The numerical results and understanding based on these representative duplex networks are illustrative and instructive for building insights into maximizing synchronizability of more realistic multiplex networks.

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (εdA) opposite thymidine in a DNA duplex. Nonplanar alignment of εdA(anti) and dT(anti) at the lesion site

International Nuclear Information System (INIS)

Kouchakdjian, M.; Patel, D.J.; Eisenberg, M.; Yarema, K.; Basu, A.; Essigmann, J.

1991-01-01

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C)·d(G-T-A-C-εA-C-A-T-G) nonanucleotide duplex (designated εdA·dT 9-mer duplex) containing 1,N 6 -ethenodeoxyadenosine (εdA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. The authors NMR studies have focused on the conformation of the εdA·dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5·εdA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and εdA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4·dC15 and dG6·dC13 pairs. Furthermore, the d(G4-T5-G6)·d(C13-εA14-C15) trinucleotide segment centered about the dT5·εdA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and εdA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the εdA·dT 9-mer duplex. The NMR data are consistent with a nonplanar alignment of εdA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4·dC15 base pair within the d(G4-T5-G6)·d(C13-εA14-C15) segment of the εdA·dT 9-mer duplex

AgI -Induced Switching of DNA Binding Modes via Formation of a Supramolecular Metallacycle.

Science.gov (United States)

Basak, Shibaji; Léon, J Christian; Ferranco, Annaleizle; Sharma, Renu; Hebenbrock, Marian; Lough, Alan; Müller, Jens; Kraatz, Heinz-Bernhard

2018-03-12

The histidine derivative L1 of the DNA intercalator naphthalenediimide (NDI) forms a triangular Ag I complex (C2). The interactions of L1 and of C2 with DNA were studied by circular dichroism (CD) and UV/Vis spectroscopy and by viscosity studies. Different binding modes were observed for L1 and for C2, as the Ag I complex C2 is too large in size to act as an intercalator. If Ag I is added to the NDI molecule that is already intercalated into a duplex, higher order complexes are formed within the DNA duplex and cause disruptions in the helical duplex structure, which leads to a significant decrease in the characteristic CD features of B-DNA. Thus, via addition of a metal we show how a classic and well-known organic intercalator unit can be turned into a partial metallo insertor. We also show how electrochemical impedance spectroscopy (EIS) can be used to probe DNA binding modes on DNA films that are immobilized on gold surfaces. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether-Modified Thymidine and o-Quinolinone Quinone Methide.

Science.gov (United States)

Gubu, Amu; Li, Long; Ning, Yan; Zhang, Xiaoyun; Lee, Seonghyun; Feng, Mengke; Li, Qiang; Lei, Xiaoguang; Jo, Kyubong; Tang, Xinjing

2018-04-17

Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst-free bioorthogonal ligation using vinyl thioether modified thymidine and o-quinolinone quinone methide. With the newly designed vinyl thioether-modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT-oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

Science.gov (United States)

Ordonez, Heather; Shuman, Stewart

2013-05-17

We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

Control of DNA hybridization by photoswitchable molecular glue.

Science.gov (United States)

Dohno, Chikara; Nakatani, Kazuhiko

2011-12-01

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.

On the Secrecy Degrees of Freedom with Full-Duplex Communication

DEFF Research Database (Denmark)

Mahmood, Nurul Huda; Mogensen, Preben Elgaard

2017-01-01

of such an application. The potential of full duplex nodes in improving the physical layer security of a communication link is investigated in this contribution. We specifically derive the information theoretic secrecy degrees of freedom measure for a pair of nodes communicating in full duplex mode. Moreover, closed...... and the delay reduction of full duplex communication are somewhat limited in realistic network settings, leading researchers to study other possible applications of full duplex communication which can provide significantly higher gains over half duplex communication. Physical layer security is an example...... form expressions for the instantaneous and ergodic throughput gain of full duplex communication over conventional half duplex is derived. The secrecy degrees of freedom with full duplex is shown to be two as opposed to that of zero in half duplex mode....

Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

Science.gov (United States)

Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

2013-10-30

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Silver(I)-Mediated Base Pairs in DNA Sequences Containing 7-Deazaguanine/Cytosine: towards DNA with Entirely Metallated Watson-Crick Base Pairs.

Science.gov (United States)

Méndez-Arriaga, José M; Maldonado, Carmen R; Dobado, José A; Galindo, Miguel A

2018-03-26

DNA sequences comprising noncanonical 7-deazaguanine ( 7C G) and canonical cytosine (C) are capable of forming Watson-Crick base pairs via hydrogen bonds as well as silver(I)-mediated base pairs by coordination to central silver(I) ions. Duplexes I and II containing 7C G and C have been synthesized and characterized. The incorporation of silver(I) ions into these duplexes has been studied by means of temperature-dependent UV spectroscopy, circular dichroism, and DFT calculations. The results suggest the formation of DNA molecules comprising contiguous metallated 7C G-Ag I -C Watson-Crick base pairs that preserve the original B-type conformation. Furthermore, additional studies performed on duplex III indicated that, in the presence of Ag I ions, 7C G-C and 7C A-T Watson-Crick base pairs ( 7C A, 7-deazadenine; T, thymine) can be converted to metallated 7C G-Ag I -C and 7C A-Ag I -T base pairs inside the same DNA molecule whilst maintaining its initial double helix conformation. These findings are very important for the development of customized silver-DNA nanostructures based on a Watson-Crick complementarity pattern. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Full-Duplex Relay Selection in Cognitive Underlay Networks

KAUST Repository

Khafagy, Mohammad Galal; Alouini, Mohamed-Slim; Aissa, Sonia

2017-01-01

In this work, we analyze the performance of full-duplex relay selection (FDRS) in spectrum-sharing networks. Contrary to half-duplex relaying, full-duplex relaying (FDR) enables simultaneous listening/forwarding at the secondary relay(s), thereby

Surface modification of 17-4PH stainless steel by DC plasma nitriding and titanium nitride film duplex treatment

International Nuclear Information System (INIS)

Qi, F.; Leng, Y.X.; Huang, N.; Bai, B.; Zhang, P.Ch.

2007-01-01

17-4PH stainless steel was modified by direct current (DC) plasma nitriding and titanium nitride film duplex treatment in this study. The microstructure, wear resistance and corrosion resistance were characterized by X-ray diffraction (XRD), pin-on-disk tribological test and polarization experiment. The results revealed that the DC plasma nitriding pretreatment was in favor of improving properties of titanium nitride film. The corrosion resistance and wear resistance of duplex treatment specimen was more superior to that of only coated titanium nitride film

Modified surface of titanium dioxide nanoparticles-based biosensor for DNA detection

Science.gov (United States)

Nadzirah, Sh.; Hashim, U.; Rusop, M.

2018-05-01

A new technique was used to develop a simple and selective picoammeter DNA biosensor for identification of E. coli O157:H7. This biosensor was fabricated from titanium dioxide nanoparticles that was synthesized by sol-gel method and spin-coated on silicon dioxide substrate via spinner. 3-Aminopropyl triethoxy silane (APTES) was used to modify the surface of TiO2. Simple surface modification approach has been applied; which is single dropping of APTES onto the TiO2 nanoparticles surface. Carboxyl modified probe DNA has been bind onto the surface of APTES/TiO2 without any amplifier element. Electrical signal has been used as the indicator to differentiate each step (surface modification of TiO2 and probe DNA immobilization). The I-V measurements indicate extremely low current (pico-ampere) flow through the device which is 2.8138E-10 A for pure TiO2 nanoparticles, 2.8124E-10 A after APTES modification and 3.5949E-10 A after probe DNA immobilization.

Glucose-nucleobase pairs within DNA: impact of hydrophobicity, alternative linking unit and DNA polymerase nucleotide insertion studies† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04850e

Science.gov (United States)

Vengut-Climent, Empar; Peñalver, Pablo; Lucas, Ricardo; Gómez-Pinto, Irene; Aviñó, Anna; Muro-Pastor, Alicia M.; Galbis, Elsa; de Paz, M. Violante; Fonseca Guerra, Célia; Bickelhaupt, F. Matthias; Eritja, Ramón; González, Carlos

2018-01-01

Recently, we studied glucose-nucleobase pairs, a binding motif found in aminoglycoside–RNA recognition. DNA duplexes with glucose as a nucleobase were able to hybridize and were selective for purines. They were less stable than natural DNA but still fit well on regular B-DNA. These results opened up the possible use of glucose as a non-aromatic DNA base mimic. Here, we have studied the incorporation and thermal stability of glucose with different types of anchoring units and alternative apolar sugar-nucleobase pairs. When we explored butanetriol instead of glycerol as a wider anchoring unit, we did not gain duplex thermal stability. This result confirmed the necessity of a more conformationally restricted linker to increase the overall duplex stability. Permethylated glucose-nucleobase pairs showed similar stability to glucoside-nucleobase pairs but no selectivity for a specific nucleobase, possibly due to the absence of hydrogen bonds between them. The three-dimensional structure of the duplex solved by NMR located both, the hydrophobic permethylated glucose and the nucleobase, inside the DNA helix as in the case of glucose-nucleobase pairs. Quantum chemical calculations on glucose-nucleobase pairs indicate that the attachment of the sugar to the DNA skeleton through the OH1 or OH4 positions yields the highest binding energies. Moreover, glucose was very selective for guanine when attached through OH1 or OH4 to the DNA. Finally, we examined DNA polymerase insertion of nucleotides in front of the saccharide unit. KF– polymerase from E. coli inserted A and G opposite glc and 6dglc with low efficiency but notable selectivity. It is even capable of extending the new pair although its efficiency depended on the DNA sequence. In contrast, Bst 2.0, SIII and BIOTAQ™ DNA polymerases seem to display a loop-out mechanism possibly due to the flexible glycerol linker used instead of deoxyribose. PMID:29780486

Harvesting full-duplex rate gains in cellular networks with half-duplex user terminals

KAUST Repository

AlAmmouri, Ahmad

2016-07-26

Full-Duplex (FD) transceivers may be expensive in terms of complexity, power consumption, and price to be implemented in all user terminals. Therefore, techniques to exploit in-band full-duplex communication with FD base stations (BSs) and half-duplex (HD) users\\' equipment (UEs) are required. In this context, 3-node topology (3NT) has been recently proposed for FD BSs to reuse the uplink (UL) and downlink (DL) channels with HD terminals within the same cell. In this paper, we present a tractable mathematical framework, based on stochastic geometry, for 3NT in cellular networks. To this end, we propose a design paradigm via pulse-shaping and partial overlap between UL and DL channels to maximize the harvested rate gains in 3NT. The results show that 3NT achieves a close performance to networks with FD BSs and FD UEs, denoted by 2-node topology (2NT) networks. A maximum of 5% rate loss is reported when 3NT is compared to 2NT with efficient self-interference cancellation (SIC). If the SIC in 2NT is not efficient, 3NT highly outperforms 2NT. Consequently, we conclude that, irrespective to the UE duplexing scheme, it is sufficient to have FD BSs to harvest FD rate gains.

Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates

Directory of Open Access Journals (Sweden)

Lavrik O. I.

2012-06-01

Full Text Available Aim. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ using 5-formyluridine (5-foU containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence of replication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.

Duplex kidney: not just a drooping lily.

Science.gov (United States)

Doery, Ashlea J; Ang, Eileen; Ditchfield, Michael R

2015-04-01

Duplex kidneys are common, mostly asymptomatic and of no clinical significance. However, they can be associated with significant pathology, often with long-term morbidity. There is minimal literature on the review of the duplex kidney, its associated anomalies and complications. The purpose of this paper is to review our experience of imaging the spectrum of abnormalities associated with duplex kidneys in the paediatric population and correlate this with contemporary literature. A retrospective review of the radiology database in a tertiary paediatric centre was performed. A word search of the Radiology Information System for 'duplex' of patients under the age of 16 was undertaken and limited to studies performed between 2006 and 2013. Two hundred seventy-four patients were identified (age range 0-16, median 3 years, gender 59.9% female) who had 836 studies: ultrasound 598/836 (71.6%), nuclear medicine 180/836 (21.5%), micturating cystourethrogram 52/836 (6.2%), MRI 5/836 (duplex and no complication (151/274 = 55.1%), upper moiety obstruction, lower moiety reflux/scarring, multicystic dysplastic kidney, abnormal ureteric insertion and other pathology. Duplex kidneys are common and often not clinically significant. However, this study demonstrates almost 50% of paediatric patients investigated for duplex kidneys had complications requiring treatment. The most common complications were upper moiety obstruction associated with a ureterocele and lower moiety vesicoureteric reflux. Ultrasound was the most common modality for early detection of these complications. © 2015 The Royal Australian and New Zealand College of Radiologists.

Full Duplex Communications in 5G Small Cells

DEFF Research Database (Denmark)

Mahmood, Nurul Huda; Gatnau, Marta; Berardinelli, Gilberto

2017-01-01

Full duplex communication promises system performance improvement over conventional half duplex communication by allowing simultaneous transmission and reception. However, such concurrent communication results in strong self interference and an increase in the overall network interference, and can...... only be exploited when traffic is available in both directions. The potential throughput gains of full duplex communication over conventional half duplex transmission in a small cell network with asymmetric traffic conditions is investigated in this contribution. The throughput performance gains...... are analysed using tools from stochastic geometry, and further confirmed through extensive system level simulations. Our findings explicitly quantify how the gains from full duplex communication depend on the traffic profile and the inter-cell interference coupling. The demonstrated throughput gains and delay...

Rapid detection of SNP (c.309T>G in the MDM2 gene by the Duplex SmartAmp method.

Directory of Open Access Journals (Sweden)

Yasuaki Enokida

Full Text Available BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

Timing, Coordination, and Rhythm : Acrobatics at the DNA Replication Fork

NARCIS (Netherlands)

Hamdan, Samir M.; Oijen, Antoine M. van

2010-01-01

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In

The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

Energy Technology Data Exchange (ETDEWEB)

Liu Lanxia; Bai Yuanyuan; Song Chunni; Zhu Dunwan; Song Liping; Zhang Hailing; Dong Xia; Leng Xigang, E-mail: lengxg@bme.org.c [Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Laboratory of Bioengineering (China)

2010-06-15

It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-10, IL-12, and TNF-{alpha}). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 {mu}g/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

Identification of a premature termination of DNA polymerization in ...

Indian Academy of Sciences (India)

2013-04-25

Apr 25, 2013 ... strands to the 5′ end of the template strands, forming double- stranded DNA with ... duplex stem is important for high processive polymerization. HIV-1 reverse ... KF has been used as a model to study DNA polymerase.

Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

Science.gov (United States)

Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

2013-01-01

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′on = 2.1(1) × 106 M−1 s−1) and 6 bp (k′on = 5.0(1) × 106 M−1 s−1) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s−1) to 6 bp (koff = 14 s−1) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s−1 to 4.0(4) s−1 between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s−1 to 0.17(7) s−1) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° duplex formation. PMID:23931323

Racemic DNA crystallography.

Science.gov (United States)

Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

2014-12-22

Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

DEFF Research Database (Denmark)

Kushon, S A; Jordan, J P; Seifert, J L

2001-01-01

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA...... structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic...

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

Science.gov (United States)

Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

2014-07-05

The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the

Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR.

Science.gov (United States)

Luo, Jun; Li, Junhua; Yang, Hang; Yu, Junping; Wei, Hongping

2017-10-01

Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from coexisting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylococci. In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swabs. The new molecular assay is based on detecting the cooccurrence of nuc and mecA markers in a single bacterial cell by utilizing droplet digital PCR (ddPCR) with the chimeric lysin ClyH for cell lysis. The method consists of (i) dispersion of an intact single bacterium into nanoliter droplets, (ii) temperature-controlled release of genomic DNA (gDNA) by ClyH at 37°C, and (iii) amplification and detection of the markers ( nuc and mecA ) using standard TaqMan chemistries with ddPCR. Results were analyzed based on MRSA index ratios used for indicating the presence of the duplex-positive markers in droplets. The method was able to achieve an absolute limit of detection (LOD) of 2,900 CFU/ml for MRSA in nasal swabs spiked with excess amounts of Escherichia coli , MSSA, and other mecA -positive bacteria within 4 h. Initial testing of 104 nasal swabs showed that the method had 100% agreement with the standard culture method, while the normal duplex qPCR method had only about 87.5% agreement. The single-bacterium duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens. Copyright © 2017 American Society for Microbiology.

Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

Science.gov (United States)

Hocek, Michal

2014-11-07

The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

Directory of Open Access Journals (Sweden)

Parvathi Chary

Full Text Available To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT, this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB

Science.gov (United States)

Yokoyama, Katsushi; Nogami, Hideki; Kabasawa, Mamiko; Ebihara, Sonomi; Shimowasa, Ai; Hashimoto, Keiko; Kawashima, Tsuyoshi; Ishijima, Sanae A.; Suzuki, Masashi

2009-01-01

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship. PMID:19468044

Conformation and dynamics of nucleotides in bulges and symmetric internal loops in duplex DNA studied by EPR and fluorescence spectroscopies

International Nuclear Information System (INIS)

Cekan, Pavol; Sigurdsson, Snorri Th.

2012-01-01

Highlights: ► Bulges and loops were studied by both EPR and fluorescence spectroscopies using the probe Ç/Ç f . ► One-base bulge was in a temperature-dependent equilibrium between looped-out and stacked states. ► Bases in two- and three-base bulges were stacked at all temperatures, resulting in DNA bending. ► Bases were stacked in symmetrical two- to five-base internal loops, according to EPR data. ► Unexpectedly high fluorescence for the smaller loops indicated local structural perturbations. -- Abstract: The dynamics and conformation of base bulges and internal loops in duplex DNA were studied using the bifunctional spectroscopic probe Ç, which becomes fluorescent (Ç f ) upon reduction of the nitroxide functional group, along with EPR and fluorescence spectroscopies. A one-base bulge was in a conformational equilibrium between looped-out and stacked states, the former favored at higher temperature and the latter at lower temperature. Stacking of bulge bases was favored in two- and three-base bulges, independent of temperature, resulting in DNA bending as evidenced by increased fluorescence of Ç f . EPR spectra of Ç-labeled three-, four- and five-base symmetrical interior DNA bulges at 20 °C showed low mobility, indicating that the spin-label was stacked within the loop. The spin-label mobility at 37 °C increased as the loops became larger. A considerable variation in fluorescence between different loops was observed, as well as a temperature-dependence within constructs. Fluorescence unexpectedly increased as the size of the loop decreased at 2 °C. Fluorescence of the smallest loops, where a single T·T mismatch was located between the stem region and the probe, was even larger than for the single strand, indicating a considerable local structural deformation of these loops from regular B-DNA. These results show the value of combining EPR and fluorescence spectroscopy to study non-helical regions of nucleic acids.

Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

DEFF Research Database (Denmark)

Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

2016-01-01

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs...

An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation.

Directory of Open Access Journals (Sweden)

Ke Shi

Full Text Available Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting-rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

Science.gov (United States)

Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

2016-01-01

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

Science.gov (United States)

Hellman, Lance M.; Spear, Tyler J.; Koontz, Colton J.; Melikishvili, Manana; Fried, Michael G.

2014-01-01

O6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs. PMID:25080506

Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2'-Amino-α-l-LNA Adenine Monomers

DEFF Research Database (Denmark)

Andersen, Nicolai K; Anderson, Brooke A; Wengel, Jesper

2013-01-01

The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-l-LNA are two interesting examples...... (ONs) modified with 2'-amino-α-l-LNA adenine monomers W-Z. The synthesis of the target phosphoramidites 1-4 is initiated from pentafuranose 5, which upon Vorbrüggen glycosylation, O2'-deacylation, O2'-activation and C2'-azide introduction yields nucleoside 8. A one-pot tandem Staudinger....... ONs modified with pyrene-functionalized 2'-amino-α-l-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation...

A Model of H-NS Mediated Compaction of Bacterial DNA

NARCIS (Netherlands)

Joyeux, M.; Vreede, J.

2013-01-01

The histone-like nucleoid structuring protein (H-NS) is a nucleoid-associated protein, which is involved in both gene regulation and DNA compaction. H-NS can bind to DNA in two different ways: in trans, by binding to two separate DNA duplexes, or in cis, by binding to different sites on the same

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

Science.gov (United States)

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Enzymatic recognition of DNA replication origins

International Nuclear Information System (INIS)

Stayton, M.M.; Bertsch, L.; Biswas, S.

1983-01-01

In this paper we discuss the process of recognition of the complementary-strand origin with emphasis on RNA polymerase action in priming M13 DNA replication, the role of primase in G4 DNA replication, and the function of protein n, a priming protein, during primosome assembly. These phage systems do not require several of the bacterial DNA replication enzymes, particularly those involved in the regulation of chromosome copy number of the initiatiion of replication of duplex DNA. 51 references, 13 figures, 1 table

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis.

Science.gov (United States)

Janwan, Penchom; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

2011-12-01

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

Science.gov (United States)

Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

2013-01-02

Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

Buffer management in wireless full-duplex systems

KAUST Repository

Bouacida, Nader; Showail, Ahmad; Shihada, Basem

2015-01-01

, we address the problem of buffer management in full-duplex networks by using Wireless Queue Management (WQM), which is an active queue management technique for wireless networks. Our solution is based on Relay Full-Duplex MAC (RFD

Radiation chemical and photochemical study of Z-DNA modified by 2-aminopurine and 8- bromodeoxyguanosine

International Nuclear Information System (INIS)

Kimura, T.; Kawai, K.; Majima, T.

2003-01-01

DNA is able to take a number of local conformations. (CG) n repeats have the highest potential to Z-DNA which has a left-handed zig-zag backbone and unusual syn-conformation purine base. Because of the polymorphic nature of dinucleotide repeats, it seems possible that Z-DNA forming sequences may provide a source of genetic variation if they occur in regions that are important for the regulation of gene activity. Here, we investigated structural properties of Z-DNA compared with those of B-DNA with respects to one-electron attachment reaction of 8-bromodeoxyguanosine (dBrG) and fluorescence properties of 2-aminopurine (Ap). To investigate one-electron attachment reaction of Z-DNA, we synthesized oligodeoxynucleotides modified by dBrG in which syn-conformation deoxyguanosine was stabilized by steric repulsion between 8-bromo group of dBrG and sugar moiety in Z-DNA. Debromination from the dBrG modified oligodeoxynucleotides occurred from the one-electron attachment during the gamma-ray irradiation. The structural dependence of B- and Z-DNA was observed in the one-electron attachment reaction. Interestingly, the higher conversion of dBrG were observed in the Z-DNA than in the B-DNA. Since the solvent accessibility to purine base in Z-DNA increases compared with that in B-DNA, it is suggested that the electron attachment is enhanced in Z-DNA than in B-DNA. Next we studied the fluorescence properties of Ap in left-handed Z-DNA and compared with those in B-DNA. Since photoexcited adenine analogue Ap can serve as a sensitive probe of DNA structural dynamics, we synthesized Ap- and dBrG-modified oligodeoxynucleotides. Higher intensity was observed in the steady-state fluorescence of Ap in Z-DNA than in B-DNA. A new peak at 275 nm was observed in the excitation spectrum measured at the Ap emission wavelength 370 nm in Z-DNA. This has been explained by the energy transfer from the excited nucleobases to Ap. It is found that Ap is a useful fluorescence probe of Z-DNA

Energy balance and deformation mechanisms of duplexes

Science.gov (United States)

Mitra, Gautam; Boyer, Steven E.

A duplex consists of a series of imbricate faults that are asymptotic to a roof thrust and a floor thrust. Depending on the final orientations of the imbricate faults and the final position of the branch lines, a duplex may be hinterland-dipping, foreland-dipping, or an antiformal stack. The exact geometry depends on various factors such as the initial dimensions of the individual slices (horses), their lithology, the amount of displacement (normalized to size of horse) on each fault, and the mechanics of movement along each fault. The energy required in duplex formation can be determined by calculating the total work involved in emplacing each horse: this is given by where W t=W p+W b+W g+W iWp is the work involved in initiating and propagating a fracture. Wb is the work involved in basal sliding, which may be frictional or some form of ductile flow, Wg is the work done against gravity during the emplacement of the horse, and Wi is the work involved in the internal deformation of the horse. By calculating and comparing these work terms it is possible to predict the conditions under which the different types of duplexes will form. Normally, the development of a hinterland-dipping duplex is most likely. However, if deformation conditions are favorable, displacements on individual imbricate faults may be very large compared to the size of the horses, leading to the formation of either antiformal stacks or foreland-dipping duplexes.

Duplex ultrasound surveillance after carotid artery endarterectomy.

Science.gov (United States)

Al Shakarchi, Julien; Lowry, Danielle; Nath, Jay; Khawaja, Aurangzaib Z; Inston, Nicholas; Tiwari, Alok

2016-06-01

After carotid endarterectomy (CEA), patients have been regularly followed up by duplex ultrasound imaging. However, the evidence for long-term follow-up is not clear, especially if the results from an early duplex scan are normal. This study assessed and systematically reviewed the evidence base for long-term surveillance after CEA and a normal early scan. Electronic databases were searched for studies assessing duplex surveillance after CEA in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The primary outcome for this study was the incidence of restenosis after a normal early scan. The secondary outcome was the number of reinterventions after a normal early scan. The review included seven studies that reported 2317 procedures. Of those patients with a normal early scan, 2.8% (95% confidence interval, 0.7%-6%) developed a restenosis, and 0.4% (95% confidence interval, 0%-0.9%) underwent a reintervention for their restenosis during the follow-up period. This review confirms that routine postoperative duplex ultrasound surveillance after CEA is not necessary if the early duplex scan is normal. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

Structure of a DNA glycosylase that unhooks interstrand cross-links

Energy Technology Data Exchange (ETDEWEB)

Mullins, Elwood A.; Warren, Garrett M.; Bradley, Noah P.; Eichman, Brandt F. (Vanderbilt)

2017-04-10

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

Sequence- and structure-dependent DNA base dynamics: Synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA

International Nuclear Information System (INIS)

Spaltenstein, A.; Robinson, B.H.; Hopkins, P.B.

1989-01-01

A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1 H NMR, CD, and thermal denaturation studies indicate that 1a (T) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules

Repeated DNA sequences in fungi

Energy Technology Data Exchange (ETDEWEB)

Dutta, S K

1974-11-01

Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10 to 20 percent repeated DNA sequences. There are approximately 100 to 110 copies of repeated DNA sequences of approximately 4 x 10/sup 7/ daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5/sup 0/C difference of T/sub e/50 (temperature at which 50 percent duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA. (auth)

Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

Science.gov (United States)

Dong, X Y; Li, W H; Zhu, J L; Liu, W J; Zhao, M Q; Luo, Y W; Chen, J D

2015-01-01

Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance.

Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus.

Science.gov (United States)

Bogomolni, Andrea L; Frasca, Salvatore; Matassa, Keith A; Nielsen, Ole; Rogers, Kara; De Guise, Sylvain

2015-04-01

Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10(0) to 10(9) copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10(0) to 10(9) copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.

On the Performance of In-Band Full-Duplex Cooperative Communications

KAUST Repository

Khafagy, Mohammad Galal

2016-01-01

-duplex potentials. Full-duplex relaying (FDR), where intermediate nodes may now support source-destination communication via simultaneous listening/forwarding, represents one of two full-duplex settings currently recommended for deployment in future fifth

Detecting deletions, insertions, and single nucleotide substitutions in cloned β-globin genes and new polymorphic nucleotide substitutions in β-globin genes in a Japanese population using ribonuclease cleavage at mismatches in RNA: DNA duplexes

International Nuclear Information System (INIS)

Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

1990-08-01

The applicability of ribonuclease (RNase) cleavage at mismatches in RNA:DNA duplexes (the RNase cleavage method) for determining nucleotide variant rates was examined in a Japanese population. DNA segments of various lengths obtained from four different regions of one normal and three thalassemic cloned human β-globin genes were inserted into transcription vectors. Sense and antisense RNA probes uniformly labeled with 32 P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of one (G), four(TTCT), five (ATTTT), and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C at position 666 of the second intervening sequence (IVS2-666) of the β-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allene T) was 0.52. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. We conclude that it would be feasible to use the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA. (J.P.N.)

Position dependent mismatch discrimination on DNA microarrays – experiments and model

Directory of Open Access Journals (Sweden)

Michel Wolfgang

2008-12-01

Full Text Available Abstract Background The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study. Results We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results. Conclusion Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.

Full-Duplex MIMO Small-Cell Networks: Performance Analysis

OpenAIRE

Atzeni, Italo; Kountouris, Marios

2015-01-01

Full-duplex small-cell relays with multiple antennas constitute a core element of the envisioned 5G network architecture. In this paper, we use stochastic geometry to analyze the performance of wireless networks with full-duplex multiple-antenna small cells, with particular emphasis on the probability of successful transmission. To achieve this goal, we additionally characterize the distribution of the self-interference power of the full-duplex nodes. The proposed framework reveals useful ins...

Electrochemical impedance-based DNA sensor using a modified single walled carbon nanotube electrode

Energy Technology Data Exchange (ETDEWEB)

Weber, Jessica E. [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Pillai, Shreekumar [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States); Ram, Manoj Kumar, E-mail: mkram@usf.edu [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Kumar, Ashok [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Singh, Shree R. [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States)

2011-07-20

Carbon nanotubes have become promising functional materials for the development of advanced electrochemical biosensors with novel features which could promote electron-transfer with various redox active biomolecules. This paper presents the detection of Salmonella enterica serovar Typhimurium using chemically modified single walled carbon nanotubes (SWNTs) with single stranded DNA (ssDNA) on a polished glassy carbon electrode. Hybridization with the corresponding complementary ssDNA has shown a shift in the impedance studies due to a higher charge transfer in ssDNA. The developed biosensor has revealed an excellent specificity for the appropriate targeted DNA strand. The methodologies to prepare and functionalize the electrode could be adopted in the development of DNA hybridization biosensor.

Complex DNA structures and structures of DNA complexes

International Nuclear Information System (INIS)

Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J.

1994-01-01

Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe 1 H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful

Complex DNA structures and structures of DNA complexes

Energy Technology Data Exchange (ETDEWEB)

Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J. [Scripps Research Institute, La Jolla, CA (United States)

1994-12-01

Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.

Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNA

DEFF Research Database (Denmark)

Jensen, Kaj Frank; Nes, Ingolf F.; Wells, Robert D.

1976-01-01

Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100–200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced...

Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

Directory of Open Access Journals (Sweden)

Ma G

2013-03-01

Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

Science.gov (United States)

Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E

2016-08-16

Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

Joint duplex mode selection, channel allocation, and power control for full-duplex cognitive femtocell networks

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Mingjie Feng

2015-02-01

Full Text Available In this paper, we aim to maximize the sum rate of a full-duplex cognitive femtocell network (FDCFN as well as guaranteeing the quality of service (QoS of users in the form of a required signal to interference plus noise ratios (SINR. We first consider the case of a pair of channels, and develop optimum-achieving power control solutions. Then, for the case of multiple channels, we formulate joint duplex model selection, power control, and channel allocation as a mixed integer nonlinear problem (MINLP, and propose an iterative framework to solve it. The proposed iterative framework consists of a duplex mode selection scheme, a near-optimal distributed power control algorithm, and a greedy channel allocation algorithm. We prove the convergence of the proposed iterative framework as well as a lower bound for the greedy channel allocation algorithm. Numerical results show that the proposed schemes effectively improve the sum rate of FDCFNs.

Improper Signaling for Virtual Full-Duplex Relay Systems

KAUST Repository

Gaafar, Mohamed; Amin, Osama; Schaefer, Rafael F.; Alouini, Mohamed-Slim

2017-01-01

Virtual full-duplex (VFD) is a powerful solution to compensate the rate loss of half-duplex relaying without the need to full-duplex capable nodes. Inter-relay interference (IRI) challenges the operation of VFD relaying systems. Recently, improper signaling is employed at both relays of the VFD to mitigate the IRI by imposing the same signal characteristics for both relays. To further boost the achievable rate performance, asymmetric time sharing VFD relaying system is adopted with different improper signals at the half-duplex relays. The joint tuning of the three design parameters improves the achievable rate performance at different ranges of IRI and different relays locations. Extensive simulation results are presented and analyzed to show the achievable rate gain of the proposed system and understand the system behavior.

Improper Signaling for Virtual Full-Duplex Relay Systems

KAUST Repository

Gaafar, Mohamed

2017-02-14

Virtual full-duplex (VFD) is a powerful solution to compensate the rate loss of half-duplex relaying without the need to full-duplex capable nodes. Inter-relay interference (IRI) challenges the operation of VFD relaying systems. Recently, improper signaling is employed at both relays of the VFD to mitigate the IRI by imposing the same signal characteristics for both relays. To further boost the achievable rate performance, asymmetric time sharing VFD relaying system is adopted with different improper signals at the half-duplex relays. The joint tuning of the three design parameters improves the achievable rate performance at different ranges of IRI and different relays locations. Extensive simulation results are presented and analyzed to show the achievable rate gain of the proposed system and understand the system behavior.

Molecular mechanisms of DNA photodamage

International Nuclear Information System (INIS)

Starrs, S.M.

2000-05-01

Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA) n and (GA) n , and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a dimeric

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insert].

Science.gov (United States)

Kupriushkin, M S; Pyshnyĭ, D V

2012-01-01

Non-nucleotide phosporamidites were synthetized, having branched backbone with different position of functional groups. Obtained phosphoramidite monomers contain intercalator moiety--6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with tert-butyldimethylsilyl group for post-synthetic modification. Synthesized oligothymidilates contain one or more modified units in different positions of sequence. Melting temperature and thermodynamic parameters of formation of complementary duplexes formed by modified oligonucleotides was defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.

Ultra-short silicon MMI duplexer

Science.gov (United States)

Yi, Huaxiang; Huang, Yawen; Wang, Xingjun; Zhou, Zhiping

2012-11-01

The fiber-to-the-home (FTTH) systems are growing fast these days, where two different wavelengths are used for upstream and downstream traffic, typically 1310nm and 1490nm. The duplexers are the key elements to separate these wavelengths into different path in central offices (CO) and optical network unit (ONU) in passive optical network (PON). Multimode interference (MMI) has some benefits to be a duplexer including large fabrication tolerance, low-temperature dependence, and low-polarization dependence, but its size is too large to integrate in conventional case. Based on the silicon photonics platform, ultra-short silicon MMI duplexer was demonstrated to separate the 1310nm and 1490nm lights. By studying the theory of self-image phenomena in MMI, the first order images are adopted in order to keep the device short. A cascaded MMI structure was investigated to implement the wavelength splitting, where both the light of 1310nm and 1490nm was input from the same port, and the 1490nm light was coupling cross the first MMI and output at the cross-port in the device while the 1310nm light was coupling through the first and second MMI and output at the bar-port in the device. The experiment was carried on with the SOI wafer of 340nm top silicon. The cascaded MMI was investigated to fold the length of the duplexer as short as 117μm with the extinct ratio over 10dB.

Application of a genetic algorithm in the conformational analysis of methylene-acetal-linked thymine dimers in DNA: Comparison with distance geometry calculations

International Nuclear Information System (INIS)

Beckers, Mischa L.M.; Buydens, Lutgarde M.C.; Pikkemaat, Jeroen A.; Altona, Cornelis

1997-01-01

The three-dimensional spatial structure of a methylene-acetal-linked thymine dimer present in a 10 base-pair (bp) sense-antisense DNA duplex was studied with a genetic algorithm designed to interpret NOE distance restraints. Trial solutions were represented by torsion angles. This means that bond angles for the dimer trial structures are kept fixed during the genetic algorithm optimization. Bond angle values were extracted from a 10 bp sense-antisense duplex model that was subjected to energy minimization by means of a modified AMBER force field. A set of 63 proton-proton distance restraints defining the methylene-acetal-linked thymine dimer was available. The genetic algorithm minimizes the difference between distances in the trial structures and distance restraints. A large conformational search space could be covered in the genetic algorithm optimization by allowing a wide range of torsion angles. The genetic algorithm optimization in all cases led to one family of structures. This family of the methylene-acetal-linked thymine dimer in the duplex differs from the family that was suggested from distance geometry calculations. It is demonstrated that the bond angle geometry around the methylene-acetal linkage plays an important role in the optimization

Programmed self-assembly of DNA/RNA for biomedical applications

Science.gov (United States)

Wang, Pengfei

Three self-assembly strategies were utilized for assembly of novel functional DNA/RNA nanostructures. RNA-DNA hybrid origami method was developed to fabricate nano-objects (ribbon, rectangle, and triangle) with precisely controlled geometry. Unlike conventional DNA origami which use long DNA single strand as scaffold, a long RNA single strand was used instead, which was folded by short DNA single strands (staples) into prescribed objects through sequence specific hybridization between RNA and DNA. Single stranded tiles (SST) and RNA-DNA hybrid origami were utilized to fabricate a variety of barcode-like nanostructures with unique patterns by expanding a plain rectangle via introducing spacers (10-bp dsDNA segment) between parallel duplexes. Finally, complex 2D array and 3D polyhedrons with multiple patterns within one structure were assembled from simple DNA motifs. Two demonstrations of biomedical applications of DNA nanotechnology were presented. Firstly, lambda-DNA was used as template to direct the fabrication of multi-component magnetic nanoparticle chains. Nuclear magnetic relaxation (NMR) characterization showed superb magnetic relaxativity of the nanoparticle chains which have large potential to be utilized as MRI contrast agents. Secondly, DNA nanotechnology was introduced into the conformational study of a routinely used catalytic DNAzyme, the RNA-cleaving 10-23 DNAzyme. The relative angle between two flanking duplexes of the catalytic core was determined (94.8°), which shall be able to provide a clue to further understanding of the cleaving mechanism of this DNAzyme from a conformational perspective.

Full-duplex optical communication system

Science.gov (United States)

Shay, Thomas M. (Inventor); Hazzard, David A. (Inventor); Horan, Stephen (Inventor); Payne, Jason A. (Inventor)

2004-01-01

A method of full-duplex electromagnetic communication wherein a pair of data modulation formats are selected for the forward and return data links respectively such that the forward data electro-magnetic beam serves as a carrier for the return data. A method of encoding optical information is used wherein right-hand and left-hand circular polarizations are assigned to optical information to represent binary states. An application for an earth to low earth orbit optical communications system is presented which implements the full-duplex communication and circular polarization keying modulation format.

l-Proline and RNA Duplex m-Value Temperature Dependence.

Science.gov (United States)

Schwinefus, Jeffrey J; Baka, Nadia L; Modi, Kalpit; Billmeyer, Kaylyn N; Lu, Shutian; Haase, Lucas R; Menssen, Ryan J

2017-08-03

The temperature dependence of l-proline interactions with the RNA dodecamer duplex surface exposed after unfolding was quantified using thermal and isothermal titration denaturation monitored by uv-absorbance. The m-value quantifying proline interactions with the RNA duplex surface area exposed after unfolding was measured using RNA duplexes with GC content ranging between 17 and 83%. The m-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, m-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The m-value from isothermal titration denaturation for a 50% GC RNA duplex decreased (became more negative) as the temperature increased and was in nearly exact agreement with the m-value from thermal denaturation. Since RNA duplex transition temperatures increased with GC content, the more favorable proline interactions with the high GC content duplex surface area observed from thermal denaturation resulted from the temperature dependence of proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the m-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the m-value was positive and increased with temperature. Our results will facilitate proline's use as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures.

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms

Directory of Open Access Journals (Sweden)

Olga A. Kladova

2018-03-01

Full Text Available Endonuclease III (Endo III or Nth is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO, a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5–37 °C. Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van’t Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU recognition and desolvation-accompanied entropy-driven adjustment of the enzyme–substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme–DNA complex formation.

The role of duplex stainless steels for downhole tubulars

International Nuclear Information System (INIS)

Francis, R.

1993-01-01

In sour conditions there is an increasing trend to turn to corrosion resistant alloys for downhole tubulars. The most commonly used CRA tubular is 13Cr, and there are thousands of feet in service. However, there are limits to the use of 13Cr, ie., the risk of sulphide stress corrosion cracking at high H 2 S levels, and the possibility of pitting or high corrosion rates in waters with high chloride contents. Where the service conditions are felt to be too severe for 13Cr alloys it has been traditional to switch to nickel base alloys such as alloys 825 and C-276 (UNS N08825 and N10276). The alloys are much more expensive than 13Cr, and in recent years the duplex stainless steels have been selected as alloys with superior corrosion and SSCC resistance compared with 13Cr, and having lower cost than nickel alloys. Originally the 22Cr duplex alloy (UNS 31803) was used, but more recently the 25Cr super duplex alloys (UNS S32760 and S32750) have become more available. The present paper reviews the data available for 13Cr and the limits of applicability. Data is also presented for laboratory tests for both the 22Cr and 25Cr super duplex alloys. There is extensive service experience with both 22Cr and 25Cr super duplex in the North Sea, covering both downhole tubulars, manifold and post wellhead equipment. Data is presented showing some of the sour condition being experienced in the North Sea by super duplex alloys. These results show that there is a substantial gap between the limits of use for 13Cr and the 25Cr super duplex stainless steel alloys. This means that in many sour environments super duplex stainless steel provides a cost effective alternative to nickel-base alloys

Transcranial Duplex Sonography Predicts Outcome following an Intracerebral Hemorrhage.

Science.gov (United States)

Camps-Renom, P; Méndez, J; Granell, E; Casoni, F; Prats-Sánchez, L; Martínez-Domeño, A; Guisado-Alonso, D; Martí-Fàbregas, J; Delgado-Mederos, R

2017-08-01

Several radiologic features such as hematoma volume are related to poor outcome following an intracerebral hemorrhage and can be measured with transcranial duplex sonography. We sought to determine the prognostic value of transcranial duplex sonography in patients with intracerebral hemorrhage. We conducted a prospective study of patients diagnosed with spontaneous intracerebral hemorrhage. Transcranial duplex sonography examinations were performed within 2 hours of baseline CT, and we recorded the following variables: hematoma volume, midline shift, third ventricle and lateral ventricle diameters, and the pulsatility index in both MCAs. We correlated these data with the CT scans and assessed the prognostic value of the transcranial duplex sonography measurements. We assessed early neurologic deterioration during hospitalization and mortality at 1-month follow-up. We included 35 patients with a mean age of 72.2 ± 12.8 years. Median baseline hematoma volume was 9.85 mL (interquartile range, 2.74-68.29 mL). We found good agreement and excellent correlation between transcranial duplex sonography and CT when measuring hematoma volume ( r = 0.791; P duplex sonography measurements showed that hematoma volume was an independent predictor of early neurologic deterioration (OR, 1.078; 95% CI, 1.023-1.135) and mortality (OR, 1.089; 95% CI, 1.020-1.160). A second regression analysis with CT variables also demonstrated that hematoma volume was associated with early neurologic deterioration and mortality. When we compared the rating operation curves of both models, their predictive power was similar. Transcranial duplex sonography showed an excellent correlation with CT in assessing hematoma volume and midline shift in patients with intracerebral hemorrhage. Hematoma volume measured with transcranial duplex sonography was an independent predictor of poor outcome. © 2017 by American Journal of Neuroradiology.

Increasing the analytical sensitivity by oligonucleotides modified with para- and ortho-twisted intercalating nucleic acids--TINA.

Directory of Open Access Journals (Sweden)

Uffe V Schneider

Full Text Available The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide, with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm, unless placed directly adjacent to the mismatch--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules. We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.

A most spectrum-efficient duplexing system: CDD

Science.gov (United States)

Lee, William C. Y.

2001-10-01

The game to play in wireless communications when it comes to increasing spectrum efficiency is to eliminate interference. Currently, all cellular systems use FDD (Frequency Division Duplexing) in an attempt to eliminate the interference from the adjacent cells. Through the use of many technologies only one type of interference remains and that is the adjacent base-tohome mobile interference. TDD (Time Division Duplexing) has not been used for mobile cellular systems, not only because of the adjacent base-to-home mobile interference, but also because of the additional adjacent base-to-home base interference, and adjacent mobile-to-home mobile interference. Therefore, TDD can only be used for small, confined area systems. CDD (Code Division Duplexing) can eliminate all three kinds of interference; the adjacent base-to-home mobile, the adjacent baseto-home base, and the adjacent mobile- to- home in cellular systems. Eliminating each of these interferences makes CDD the most spectrum efficient duplexing system. This talk will elaborate on a set of smart codes, which will make an efficient CDD system a reality.

Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles.

Science.gov (United States)

Hakker, Lauren; Marchi, Alexandria N; Harris, Kimberly A; LaBean, Thomas H; Agris, Paul F

2014-01-01

DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson-Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure's thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2'-deoxyribothymidines, 2'-O-methylribouridines, or abasic 1',2'-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2'-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

Molecular dynamics analysis of stabilities of the telomeric Watson-Crick duplex and the associated i-motif as a function of pH and temperature.

Science.gov (United States)

Panczyk, Tomasz; Wolski, Pawel

2018-06-01

This work deals with a molecular dynamics analysis of the protonated and deprotonated states of the natural sequence d[(CCCTAA) 3 CCCT] of the telomeric DNA forming the intercalated i-motif or paired with the sequence d[(CCCTAA) 3 CCCT] and forming the Watson-Crick (WC) duplex. By utilizing the amber force field for nucleic acids we built the i-motif and the WC duplex either with native cytosines or using their protonated forms. We studied, by applying molecular dynamics simulations, the role of hydrogen bonds between cytosines or in cytosine-guanine pairs in the stabilization of both structures in the physiological fluid. We found that hydrogen bonds exist in the case of protonated i-motif and in the standard form of the WC duplex. They, however, vanish in the case of the deprotonated i-motif and protonated form of the WC duplex. By determining potentials of mean force in the enforced unwrapping of these structures we found that the protonated i-motif is thermodynamically the most stable. Its deprotonation leads to spontaneous and observed directly in the unbiased calculations unfolding of the i-motif to the hairpin structure at normal temperature. The WC duplex is stable in its standard form and its slight destabilization is observed at the acidic pH. However, the protonated WC duplex unwraps very slowly at 310 K and its decomposition was not observed in the unbiased calculations. At higher temperatures (ca. 400 K or more) the WC duplex unwraps spontaneously. Copyright © 2018. Published by Elsevier B.V.

Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame

Science.gov (United States)

Rajendran, Arivazhagan; Endo, Masayuki; Hidaka, Kumi; Lan Thao Tran, Phong; Mergny, Jean-Louis; Sugiyama, Hiroshi

2013-01-01

Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex. PMID:23863846

Reducing duplex examinations in patients with iatrogenic pseudoaneurysms.

Science.gov (United States)

Stone, Patrick A; Aburahma, Ali F; Flaherty, Sarah K

2006-06-01

Ultrasound-guided thrombin injection has become the initial treatment of choice for femoral access-related pseudoaneurysms. Patients typically undergo serial duplex examinations to assess for spontaneous resolution of small iatrogenic pseudoaneurysms (IPSAs) (IPSAs (>2.5 cm). We evaluated the impact of a revised treatment algorithm that includes primary treatment of both small (2.5 cm), rather than observation of smaller ones, and attempts to establish a single duplex examination via a point-of-care treatment strategy. We reviewed 105 consecutive patients treated with ultrasound-guided thrombin injection from July 2001 through September 2004. Patient, IPSAs, characteristics, and treatment methods were examined. The number of duplex examinations per patient was evaluated over the treatment interval. Also, published cost data were used to compare primary treatment of small ISPAs vs observation with serial duplex examinations. Successful thrombosis occurred in 103 (98.1%) of 105 treated pseudoaneurysms. No minor or major complications occurred after thrombin injection in either small or large ISPAs, and both failures requiring operation were in the large aneurysm group. The recurrence rate for the series was 1.9% (2/105), and both recurrences were successfully treated with an additional thrombin injection. A single injection was successful in treating 43 (97.7%) of 44 small (IPSAs, and one required a second injection. Patients had an average of 3.3 duplex examinations in our first year of treatment experience, which declined to 1.5 by our third year with the institution of a point-of-care service model for all pseudoaneurysms. Based on this decreased use of duplex examination and an average treatment cohort of 35 IPSA patients per year our institution, we determined this results in a reduction of 35 hours of laboratory time and nearly 70 ultrasounds per year. Similarly for small pseudoaneurysms, a point-of-service primary treatment program rather than observation

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

NARCIS (Netherlands)

Hamdan, Samir M.; Loparo, Joseph J.; Takahashi, Masateru; Richardson, Charles C.; Oijen, Antoine M. van

2009-01-01

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to

Downlink Error Rates of Half-duplex Users in Full-duplex Networks over a Laplacian Inter-User Interference Limited and EGK fading

KAUST Repository

Soury, Hamza; Elsawy, Hesham; Alouini, Mohamed-Slim

2017-01-01

This paper develops a mathematical framework to study downlink error rates and throughput for half-duplex (HD) terminals served by a full-duplex (FD) base station (BS). The developed model is used to motivate long term pairing for users that have

High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

DEFF Research Database (Denmark)

Hansen, Mads E; Bentin, Thomas; Nielsen, Peter E

2009-01-01

While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes-mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine subs...

Sequence specificity of alkali-labile DNA damage photosensitized by suprofen.

Science.gov (United States)

Starrs, S M; Davies, R J

2000-09-01

On irradiation at UVB wavelengths, in aerated neutral aqueous solution, the anti-inflammatory drug suprofen (SP) photosensitizes the production of alkali-labile cleavage sites in DNA much more efficiently than direct strand breaks. It is active at submillimolar concentrations despite having no significant binding affinity for DNA. Gel sequencing studies utilizing 32P-end-labeled oligonucleotides have revealed that piperidine-sensitive lesions are formed predominantly at the positions of guanine (G) bases, with the extent of modification being UV dose- and SP concentration-dependent. Quite distinct patterns of G-specific damage are observed in single-stranded and duplex DNA molecules. The uniform attack at all G residues in single-stranded DNA, which is enhanced in D2O, is compatible with a Type-II mechanism. SP is a known generator of singlet oxygen whose participation in the reaction is supported by the effects of quenchers and scavengers. In duplex DNA, piperidine-induced cleavage occurs with high selectivity at the 5'-G of GG and (less prominently) GA doublets. This behavior is characteristic of a Type-I process involving electron transfer from DNA to photoexcited SP molecules. The ability of SP to sensitize the formation of Type-I and Type-II photo-oxidation products from 2'-deoxyguanosine attests to the feasibility of competing mechanisms in DNA.

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR.

Science.gov (United States)

Sakalar, Ergün; Ergün, Seyma Özçirak; Akar, Emine

2015-01-01

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex real-time PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system

International Nuclear Information System (INIS)

Brown, W.C.; Romano, L.J.

1991-01-01

The authors have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. They show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, they present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, they observe an increase in synthesis but to a lesser extent than observed on oligonucleotide-primed synthesis. Finally, they have examined DNA synthesis on duplex templates and show the B[a]P adducts inhibit synthesis by the T7 DNA polymerase and gene 4 protein to the same extent regardless of whether the adducts are positioned in the leading or lagging strand, while synthesis by the polymerase alone is inhibited only when the adducts are in the template strand

One-electron oxidation of DNA: mechanism and consequences.

Science.gov (United States)

Schuster, Gary B

2009-01-01

All living organisms store the information necessary to maintain life in their DNA. Any process that damages DNA and causes loss or corruption of that information threatens the viability of the organism. One-electron oxidation is such a process. Loss of an electron from DNA generates a radical cation that is located primarily on its nucleobases. The radical cation migrates reversibly through duplex DNA by hopping until it is eventually trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In its normal aqueous solutions, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counter ions (typically Na(+)) to the phosphate groups play an important role in facilitating both the migration of the radical cation and in its eventual reaction with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA that is comprised of the four common DNA nucleobases, reaction occurs most commonly at a guanine and results in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step primarily by a tandem process. The general outcomes revealed in the one-electron oxidation of DNA oligomers in solution appear to be generally valid also for more complex DNA structures and for the cellular DNA of living organisms.

The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5' Flaps and the Removal of 5' Adenylylated Products of Aborted Nick Ligation.

Science.gov (United States)

Uson, Maria Loressa; Ghosh, Shreya; Shuman, Stewart

2017-09-01

We characterize Mycobacterium smegmatis FenA as a manganese-dependent 5'-flap endonuclease homologous to the 5'-exonuclease of DNA polymerase I. FenA incises a nicked 5' flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3' single strand is eliminated to create a 5'-tailed duplex, FenA incision shifts to between the second and third nucleotides. A double-flap substrate with a mobile junction (mimicking limited strand displacement synthesis during gap repair) is preferentially incised as the 1-nucleotide 3'-flap isomer, with the scissile phosphodiester shifted by one nucleotide versus a static double flap. FenA efficiently removes the 5' App(dN) terminus of an aborted nick ligation reaction intermediate, thereby highlighting FenA as an agent of repair of such lesions, which are formed under a variety of circumstances by bacterial NAD + -dependent DNA ligases and especially by mycobacterial DNA ligases D and C. IMPORTANCE Structure-specific DNA endonucleases are implicated in bacterial DNA replication, repair, and recombination, yet there is scant knowledge of the roster and catalytic repertoire of such nucleases in Mycobacteria This study identifies M. smegmatis FenA as a stand-alone endonuclease homologous to the 5'-exonuclease domain of mycobacterial DNA polymerase 1. FenA incises 5' flaps, 5' nicks, and 5' App(dN) intermediates of aborted nick ligation. The isolated N-terminal domain of M. smegmatis Pol1 is also shown to be a flap endonuclease. Copyright © 2017 American Society for Microbiology.

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry.

Science.gov (United States)

Mariappan, S V Santhana; Cheng, Xun; van Breemen, Richard B; Silks, Louis A; Gupta, Goutam

2004-11-15

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.

Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

Science.gov (United States)

Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

2015-10-01

Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

Full-duplex wireless communications systems self-interference cancellation

CERN Document Server

Le-Ngoc, Tho

2017-01-01

This book introduces the development of self-interference (SI)-cancellation techniques for full-duplex wireless communication systems. The authors rely on estimation theory and signal processing to develop SI-cancellation algorithms by generating an estimate of the received SI and subtracting it from the received signal. The authors also cover two new SI-cancellation methods using the new concept of active signal injection (ASI) for full-duplex MIMO-OFDM systems. The ASI approach adds an appropriate cancelling signal to each transmitted signal such that the combined signals from transmit antennas attenuate the SI at the receive antennas. The authors illustrate that the SI-pre-cancelling signal does not affect the data-bearing signal. This book is for researchers and professionals working in wireless communications and engineers willing to understand the challenges of deploying full-duplex and practical solutions to implement a full-duplex system. Advanced-level students in electrical engineering and computer ...

Tensile properties of duplex UNS S32205 and lean duplex UNS S32304 steels and the influence of short duration 475 ºC aging

Directory of Open Access Journals (Sweden)

Sérgio Souto Maior Tavares

2012-12-01

Full Text Available Duplex stainless steels are high strength and corrosion resistant steels extensively used in the petrochemical and chemical industries. The aging at 475 ºC for long periods of time provokes embrittlement and deterioration of corrosion resistance. However, short duration aging at 475 ºC may be used as heat treatment to improve mechanical resistance with small decrease in the other properties. In this work the flow stress curves of lean duplex UNS S32304 and duplex UNS S32205 steels were modeled with Hollomon's equation and work hardening exponents (n were determined. The analyses were conducted in specimens annealed and heat treated at 475 ºC for short periods of time. The aging at 475 ºC for 4 hours, 8 hours and 12 hours promoted significant hardening with small decrease of ductility. The work hardening exponents of both steels were compared, being higher in the duplex steel than in the lean duplex grade.

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

Science.gov (United States)

Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin

2014-09-25

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Clinical utility of carotid duplex ultrasound prior to cardiac surgery.

Science.gov (United States)

Lin, Judith C; Kabbani, Loay S; Peterson, Edward L; Masabni, Khalil; Morgan, Jeffrey A; Brooks, Sara; Wertella, Kathleen P; Paone, Gaetano

2016-03-01

Clinical utility and cost-effectiveness of carotid duplex examination prior to cardiac surgery have been questioned by the multidisciplinary committee creating the 2012 Appropriate Use Criteria for Peripheral Vascular Laboratory Testing. We report the clinical outcomes and postoperative neurologic symptoms in patients who underwent carotid duplex ultrasound prior to open heart surgery at a tertiary institution. Using the combined databases from our clinical vascular laboratory and the Society of Thoracic Surgery, a retrospective analysis of all patients who underwent carotid duplex ultrasound within 13 months prior to open heart surgery from March 2005 to March 2013 was performed. The outcomes between those who underwent carotid duplex scanning (group A) and those who did not (group B) were compared. Among 3233 patients in the cohort who underwent cardiac surgery, 515 (15.9%) patients underwent a carotid duplex ultrasound preoperatively, and 2718 patients did not (84.1%). Among the patients who underwent carotid screening vs no screening, there was no statistically significant difference in the risk factors of cerebrovascular disease (10.9% vs 12.7%; P = .26), prior stroke (8.2% vs 7.2%; P = .41), and prior transient ischemic attack (2.9% vs 3.3%; P = .24). For those undergoing isolated coronary artery bypass grafting (CABG), 306 (17.8%) of 1723 patients underwent preoperative carotid duplex ultrasound. Among patients who had carotid screening prior to CABG, the incidence of carotid disease was low: 249 (81.4%) had minimal or mild stenosis (duplex scanning and those who did not. Primary outcomes of patients who underwent open heart surgery also showed no difference in the perioperative mortality (5.1% vs 6.9%; P = .14) and stroke (2.6% vs 2.4%; P = .85) between patients undergoing preoperative duplex scanning and those who did not. Operative intervention of severe carotid stenosis prior to isolated CABG occurred in 2 of the 17 patients (11.8%) identified who

Perancangan dan Implementasi Duplexer Mikrostrip untuk Frekuensi LTE pada band ke-7

Directory of Open Access Journals (Sweden)

ENCENG SULAEMAN

2013-07-01

The necessary of mobile communication has increased in technology, now it released the new of technology is Long Term Evolution (LTE. Type of duplex is Frequency Division Duplex (FDD, designed a duplexer which it is capable for transmitting and receiving process at the same time with the different of frequency between uplink and downlink. Research of designed and implemented a duplexer using Hybrid Coupler and Bandstop filters based on Split Ring Resonators for the frequency band 7th on LTE technology at Base Transceiver Station (BTS. Duplexer is made by combining two coupler and two bandstop filter. The results of duplexer measurements, when Tx port transmit the signal to antenna port with attenuation at the downlink frequency center at 3.168 dB. Attenuation from antenna port to Rx port about 6 dB. Isolation between Tx port and Rx port about 15 dB. Keywords: Duplexer, Bandstop Filter, Split Ring Resonator, Hybrid Coupler, and Long Term Evolution.

Formation of (DNA)2-LNA triplet with recombinant base recognition: A quantum mechanical study

Science.gov (United States)

Mall, Vijaya Shri; Tiwari, Rakesh Kumar

2018-05-01

The formation of DNA triple helix offers the verity of new possibilities in molecular biology. However its applications are limited to purine and pyrimidine rich sequences recognized by forming Hoogsteen/Reverse Hoogsteen triplets in major groove sites of DNA duplex. To overcome this drawback modification in bases backbone and glucose of nucleotide unit of DNA have been proposed so that the third strand base recognized by both the bases of DNA duplex by forming Recombinant type(R-type) of bonding in mixed sequences. Here we performed Quanrum Mechanical (Hartree-Fock and DFT) methodology on natural DNA and Locked Nucleic Acids(LNA) triplets using 6-31G and some other new advance basis sets. Study suggests energetically stable conformation has been observed for recombinant triplets in order of G-C*G > A-T*A > G-C*C > T-A*T for both type of triplets. Interestingly LNA leads to more stable conformation in all set of triplets, clearly suggests an important biological tool to overcome above mentioned drawbacks.

Unusual hydrogen bonding patterns in AF [aminofluorene] and AAF [acetylaminofluorene] modified DNA

International Nuclear Information System (INIS)

Broyde, S.; Hingerty, B.E.; Shapiro, R.; Norman, D.; Oak Ridge National Lab., TN; New York Univ., NY; Columbia Univ., New York, NY

1989-01-01

New structures are presented for AF and AAF modified DNAs that place the carcinogen in the minor groove of a B-DNA helix. These structures employ non-Watson-Crick base pairing schemes with syn guanine at the modification site. 32 refs., 9 figs

Probing the DNA Structural Requirements for Facilitated Diffusion

Science.gov (United States)

2015-01-01

DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein. PMID:25495964

Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

KAUST Repository

Wang, Zhijuan

2011-05-01

Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

Electron Beam Welding of Duplex Steels with using Heat Treatment

Science.gov (United States)

Schwarz, Ladislav; Vrtochová, Tatiana; Ulrich, Koloman

2010-01-01

This contribution presents characteristics, metallurgy and weldability of duplex steels with using concentrated energy source. The first part of the article describes metallurgy of duplex steels and the influence of nitrogen on their solidification. The second part focuses on weldability of duplex steels with using electron beam aimed on acceptable structure and corrosion resistance performed by multiple runs of defocused beam over the penetration weld.

Perancangan dan Implementasi Duplexer Mikrostrip untuk Frekuensi LTE pada band ke-7

Directory of Open Access Journals (Sweden)

ENCENG SULAEMAN

2017-06-01

Full Text Available ABSTRAK Kebutuhan akan komunikasi bergerak semakin mengalami peningkatan dalam dunia teknologi yang ada saat ini, maka hadirlah teknologi terbaru yaitu Long Term Evolution (LTE. Dengan teknik duplex jenis Frequency Division Duplex (FDD, dirancanglah duplexer yang mampu melakukan proses pengiriman dan penerimaan dalam satu waktu dengan frekuensi yang berbeda antara uplink dan downlink. Pada penelitian ini dirancang dan diimplementasikan sebuah duplekser dengan menggunakan metoda Hybrid Coupler dan Bandstop Filter berbasis Split Ring Resonator untuk band frekuensi ke-7 pada teknologi LTE di sisi Base Transceiver Station (BTS. Duplekser dibuat dengan menggabungkan dua buah hybrid Coupler dan dua buah Bandstop Filter. Hasil pengukuran duplexer saat port Tx mengirimkan sinyal ke antena dengan nilai redaman pada frekuensi tengah downlink sebesar 3.168 dB. Lalu pada saat antena menerima sinyal untuk diteruskan ke Rx terdapat redaman di frekuensi tengah uplink sebesar 6 dB. Sedangkan untuk isolasi dari port Tx-port Rx dihasilkan sebesar 15 dB. Kata kunci: Duplexer, Bandstop Filter, Split Ring Resonator, Hybrid Coupler, dan Long Term Evolution. ABSTRACT The necessary of mobile communication has increased in technology, now it released the new of technology is Long Term Evolution (LTE. Type of duplex is Frequency Division Duplex (FDD, designed a duplexer which it is capable for transmitting and receiving process at the same time with the different of frequency between uplink and downlink. Research of designed and implemented a duplexer using Hybrid Coupler and Bandstop filters based on Split Ring Resonators for the frequency band 7th on LTE technology at Base Transceiver Station (BTS. Duplexer is made by combining two coupler and two bandstop filter. The results of duplexer measurements, when Tx port transmit the signal to antenna port with attenuation at the downlink frequency center at 3.168 dB. Attenuation from antenna port to Rx port about 6 d

[Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

Science.gov (United States)

Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

2014-03-01

The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure, and the application of comet assay will be expanded. The endonuclease

Fish DNA-modified clays: Towards highly flame retardant polymer nanocomposite with improved interfacial and mechanical performance

Science.gov (United States)

Zabihi, Omid; Ahmadi, Mojtaba; Khayyam, Hamid; Naebe, Minoo

2016-12-01

Deoxyribonucleic Acid (DNA) has been recently found to be an efficient renewable and environmentally-friendly flame retardant. In this work, for the first time, we have used waste DNA from fishing industry to modify clay structure in order to increase the clay interactions with epoxy resin and take benefit of its additional thermal property effect on thermo-physical properties of epoxy-clay nanocomposites. Intercalation of DNA within the clay layers was accomplished in a one-step approach confirmed by FT-IR, XPS, TGA, and XRD analyses, indicating that d-space of clay layers was expanded from ~1.2 nm for pristine clay to ~1.9 nm for clay modified with DNA (d-clay). Compared to epoxy nanocomposite containing 2.5%wt of Nanomer I.28E organoclay (m-clay), it was found that at 2.5%wt d-clay loading, significant enhancements of ~14%, ~6% and ~26% in tensile strength, tensile modulus, and fracture toughness of epoxy nanocomposite can be achieved, respectively. Effect of DNA as clay modifier on thermal performance of epoxy nanocomposite containing 2.5%wt d-clay was evaluated using TGA and cone calorimetry analysis, revealing significant decreases of ~4000 kJ/m2 and ~78 kW/m2 in total heat release and peak of heat release rate, respectively, in comparison to that containing 2.5%wt of m-clay.

Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I

International Nuclear Information System (INIS)

O'Connor, D.; Stoehrer, G.

1985-01-01

Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation. By a combination of primers and nick-mers, conditions of single-strand-directed DNA synthesis and nick-translation could be created. The results show that the polymerase can bypass both types of lesions. Bypass occurs on a single-stranded template but is facilitated on a nicked, double-stranded template. Only purines, with guanine more favored than adenine, are incorporated across both lesions. The results indicate that site-specifically modified oligonucleotides can be sensitive probes for the action of polymerases on damaged templates. They also suggest a function for polymerase I, in its nick-translation capacity, during DNA repair and mutagenesis

Adenine radicals generated in alternating AT duplexes by direct absorption of low-energy UV radiation.

Science.gov (United States)

Banyasz, Akos; Ketola, Tiia; Martínez-Fernández, Lara; Improta, Roberto; Markovitsi, Dimitra

2018-04-17

There is increasing evidence that the direct absorption of photons with energies that are lower than the ionization potential of nucleobases may result in oxidative damage to DNA. The present work, which combines nanosecond transient absorption spectroscopy and quantum mechanical calculations, studies this process in alternating adenine-thymine duplexes (AT)n. We show that the one-photon ionization quantum yield of (AT)10 at 266 nm (4.66 eV) is (1.5 ± 0.3) × 10-3. According to our PCM/TD-DFT calculations carried out on model duplexes composed of two base pairs, (AT)1 and (TA)1, simultaneous base pairing and stacking does not induce important changes in the absorption spectra of the adenine radical cation and deprotonated radical. The adenine radicals, thus identified in the time-resolved spectra, disappear with a lifetime of 2.5 ms, giving rise to a reaction product that absorbs at 350 nm. In parallel, the fingerprint of reaction intermediates other than radicals, formed directly from singlet excited states and assigned to AT/TA dimers, is detected at shorter wavelengths. PCM/TD-DFT calculations are carried out to map the pathways leading to such species and to characterize their absorption spectra; we find that, in addition to the path leading to the well-known TA* photoproduct, an AT photo-dimerization path may be operative in duplexes.

Capability of ds-DNA duplex structure in growing fluorescent silver nanoclusters

International Nuclear Information System (INIS)

Wu, Tao; Lin, Fan; Hu, Yuehua; Wang, Ying; Zhou, Xiaoshun; Shao, Yong

2016-01-01

Silver nanoclusters (AgNCs) have attracted wide interests in variant fields due to their easy synthesis and practical tunability in fluorescence properties. DNA has been generally used as the host to grow AgNCs due to the sequence-dependent fluorescence behavior. Actually, in such DNA, various ss-DNA segments that are structurally confined by the rigid ds-DNA counterparts have been used as the AgNCsГ—Ві growth sites. However, whether the ds-DNA structure plays somewhat role in AgNCsГ—Ві creation has not been well elucidated. Herein, we found that ds-DNA can also accommodate the growth of fluorescent AgNCs. The fluorescent AgNCs grown on ds-DNA should be separated each other and the G/C base pairs with right context sequences are the growth sites of fluorescent AgNCs. The intermediate A/T base pair among the continuous G/C ones seems to quench the growth of fluorescent AgNCs. For the repeat sequences, the fluorescence band position of AgNCs is not changed but the intensity is enhanced upon increasing the ds-DNA length, which is different from the results obtained with the previously reported ss-DNAs. AgNCs should be grown on the ds-DNA major groove, as convinced by the cytosine methylation experiment. Our work demonstrates that besides the ss-DNA role in defining AgNCs, one should also take into account the critical role of the ds-DNA segment in tuning the AgNCsГ—Ві fluorescence property.

The Duplex Society.

Science.gov (United States)

Schorr, Alvin L.

1984-01-01

The duplex society, in which the poor live in close proximity to others but in a separate compartment, is already with us. Unless something deeply changes about family income, more than one-third of future generations will come to adulthood having spent a portion of their childhood in official poverty. (RM)

Enantiospecific kinking of DNA by a partially intercalating metal complex

KAUST Repository

Reymer, Anna; Nordé n, Bengt

2012-01-01

Opposite enantiomers of [Ru(phenanthroline) 3] 2+ affect the persistence length of DNA differently, a long speculated effect of helix kinking. Our molecular dynamics simulations confirm a substantial change of duplex secondary structure produced

On the Performance of In-Band Full-Duplex Cooperative Communications

KAUST Repository

Khafagy, Mohammad Galal

2016-06-01

In-band full-duplex, by which radios may simultaneously transmit and receive over the same channel, has been always considered practically-unfeasible due to the prohibitively strong self-interference. Indeed, a freshly-generated transmit signal power is typically ten orders of magnitude higher than that of a naturally-attenuated received signal. While unable to manage such an overwhelming interference, wireless communications resorted to half-duplex operation, transmitting and receiving over orthogonal channel resources. Recent research has demonstrated the practical feasibility of full-duplexing via successive sophisticated stages of signal suppression/cancellation, bringing this long-held assumption down and reviving the promising full-duplex potentials. Full-duplex relaying (FDR), where intermediate nodes may now support source-destination communication via simultaneous listening/forwarding, represents one of two full-duplex settings currently recommended for deployment in future fifth-generation (5G) systems. Theoretically, it has been widely accepted that FDR potentially doubles the channel capacity when compared to its half-duplex counterpart. Although FDR doubles the multiplexing gain, the effective signal-to-noise ratio (SNR) can be significantly degraded due to the residual self-interference (RSI) if not properly handled. In this work, efficient protocols are devised for different FDR settings. Selective cooperation is proposed for the canonical three-terminal FDR channel with RSI, which exploits the cooperative diversity offered by the independently fading source/relay message replicas arriving at the destination. Closed-form expressions are derived for the end-to-end SNR cumulative distribution function (CDF) under Rayleigh and Nakagami-m fading. Further, the offered diversity gain is presented as a function of the RSI scaling trend with the relay power. We show that the existing diversity problem in simple FDR protocols can be considerably fixed via

On full duplex Gaussian relay channels with self-interference

KAUST Repository

Behboodi, Arash; Chaaban, Anas; Mathar, Rudolf; Alouini, Mohamed-Slim

2016-01-01

Self interference (SI) in full duplex (FD) systems is the interference caused by the transmission stream on the reception stream. Being one of the main restrictive factors for performance of practical full duplex systems, however, not too much

Efficacy of duplex ultrasound surveillance after infrainguinal vein bypass may be enhanced by identification of characteristics predictive of graft stenosis development.

Science.gov (United States)

Tinder, Chelsey N; Chavanpun, Joe P; Bandyk, Dennis F; Armstrong, Paul A; Back, Martin R; Johnson, Brad L; Shames, Murray L

2008-09-01

(15%) limbs, the bypass graft failed and 20 (6%) limbs required amputation. The efficacy of duplex surveillance after infrainguinal vein bypass may be enhanced by modifying testing protocols, eg, rigorous surveillance for "higher risk" bypasses, based on the initial duplex scan results and other characteristics (warfarin therapy, non- single segment saphenous vein conduit, redo bypass) predictive for stenosis development.

Optimization of welding variables for duplex stainless steel by GTAW and SMAW

International Nuclear Information System (INIS)

Ajmal, M.; Anwar, M.Y.; Nawaz, A.

2006-01-01

The main problems faced during the welding of duplex stainless steels are cleanliness and slag inclusions. In the present work the methods to eliminate these problems were studied during the welding of duplex stainless steel by Gas Tungsten Arc Welding (GTAW) and Shielded Metal Arc Welding (SMAW). Since the duplex stainless steel is an expensive material, the initial experiments for optimization of welding variables were. carried out on low carbon steel (CS) plates with duplex consumables. Welding of butt groove joints on CS plates was carried with various sets of welding variables i.e. current, voltage and arc energy using duplex consumables. The. radiographic inspection, micro-structural observations and hardness testing of the welds suggested the welding variables that will produce a sound weld on CS plate. These optimized variables were then used for the welding of edge groove joint and T -joint on duplex stainless steel by GTAW and SMAW processes. The hardness and micro-structural study of the joints produced on duplex stainless steel by GTAW and SMAW with duplex consumables were also studied. No slag inclusions and porosity were observed in the microstructure of these weldments and their properties were found similar to the parent metal. (author)

Molecular mechanisms of DNA photodamage

Energy Technology Data Exchange (ETDEWEB)

Starrs, S.M

2000-05-01

Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA){sub n} and (GA){sub n}, and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a

Direct voltammetric analysis of DNA modified with enzymatically incorporated 7-deazapurines

Czech Academy of Sciences Publication Activity Database

Pivoňková, Hana; Horáková Brázdilová, Petra; Fojtová, Miloslava; Fojta, Miroslav

2010-01-01

Roč. 82, č. 16 (2010), s. 6807-6813 ISSN 0003-2700 R&D Projects: GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : modified DNA * 7-deazapurines * voltammetric analysis Subject RIV: BO - Biophysics Impact factor: 5.874, year: 2010

Duplex/quadruplex oligonucleotides: Role of the duplex domain in the stabilization of a new generation of highly effective anti-thrombin aptamers.

Science.gov (United States)

Russo Krauss, Irene; Napolitano, Valeria; Petraccone, Luigi; Troisi, Romualdo; Spiridonova, Vera; Mattia, Carlo Andrea; Sica, Filomena

2018-02-01

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

Science.gov (United States)

Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

2014-01-01

DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

Comparative analysis of the end-joining activity of several DNA ligases.

Directory of Open Access Journals (Sweden)

Robert J Bauer

Full Text Available DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1 DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3'- and 5'- single base overhangs, and 5'-two base overhangs. This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5'-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3'-single base overhangs and 2-base overhangs effectively with little blunt or 5'- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5'-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.

The extension of a DNA double helix by an additional Watson-Crick base pair on the same backbone

DEFF Research Database (Denmark)

Kumar, P.; Sharma, P. K.; Madsen, Charlotte S.

2013-01-01

Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand.......Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand....

Isolation/separation of plasmid DNA using hemoglobin modified magnetic nanocomposites as solid-phase adsorbent.

Science.gov (United States)

Chen, Xu-Wei; Mao, Quan-Xing; Liu, Jia-Wei; Wang, Jian-Hua

2012-10-15

Hemoglobin (Hb) modified magnetic nanocomposites are prepared by immobilization of Hb onto the surface of amino-functionalized Fe(3)O(4)@SiO(2) magnetic nanoparticles via covalent bonding with glutaraldehyde as cross-linker. The obtained nanocomposites are characterized with FT-IR, SEM, XRD and surface charge analysis. A direct solid-phase extraction procedure for the isolation/separation of plasmid DNA using this nanocomposite as a novel adsorbent is thus developed. Some important experimental parameters governing the sorption efficiency, i.e., the pH of sample solution and the ionic strength, are investigated. The Hb modified magnetic nanocomposites provide a sorption capacity of 27.86 mg g(-1) for DNA. By using 2.0mg of the nanocomposites as sorption medium and a suitable acidity of pH 6.1, a sorption efficiency of 93% is achieved for 25 μg mL(-1) of DNA in 1.0 mL of sample solution. Afterwards, the absorbed DNA could be readily recovered by using 1.0 mL of Tris-HCl buffer (pH 8.9, 0.01 mol L(-1)), giving rise to a recovery of ca. 68.3%. The present solid-phased extraction protocol is applied for the isolation of plasmid DNA from Escherichia coli culture, resulting in comparable yield and purity of plasmid DNA with respect to those obtained by using commercial kits. Copyright © 2012 Elsevier B.V. All rights reserved.

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

DEFF Research Database (Denmark)

Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

2012-01-01

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...

EFFECT OF INTERMETALLIC PHASES ON CORROSION BEHAVIOR AND MECHANICAL PROPERTIES OF DUPLEX STAINLESS STEEL AND SUPER-DUPLEX STAINLESS STEEL

OpenAIRE

Prabhu Paulraj; Rajnish Garg

2015-01-01

Duplex Stainless Steels (DSS) and Super Duplex Stainless Steel (SDSS) have excellent integration of mechanical and corrosion properties. However, the formation of intermetallic phases is a major problem in their usage. The mechanical and corrosion properties are deteriorated due to the presence of intermetallic phases. These phases are induced during welding, prolonged exposure to high temperatures, and improper heat treatments. The main emphasis of this review article is on intermetallic pha...

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR

OpenAIRE

Sakalar, Erg?n; Erg?n, Seyma ?z?irak; Akar, Emine

2015-01-01

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN? Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks...

Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

DEFF Research Database (Denmark)

Trujillo, L E; Pupo, E; Miranda, F

1996-01-01

We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

Water-evaporation reduction by duplex films: application to the human tear film.

Science.gov (United States)

Cerretani, Colin F; Ho, Nghia H; Radke, C J

2013-09-01

Water-evaporation reduction by duplex-oil films is especially important to understand the physiology of the human tear film. Secreted lipids, called meibum, form a duplex film that coats the aqueous tear film and purportedly reduces tear evaporation. Lipid-layer deficiency is correlated with the occurrence of dry-eye disease; however, in-vitro experiments fail to show water-evaporation reduction by tear-lipid duplex films. We review the available literature on water-evaporation reduction by duplex-oil films and outline the theoretical underpinnings of spreading and evaporation kinetics that govern behavior of these systems. A dissolution-diffusion model unifies the data reported in the literature and identifies dewetting of duplex films into lenses as a key challenge to obtaining significant evaporation reduction. We develop an improved apparatus for measuring evaporation reduction by duplex-oil films including simultaneous assessment of film coverage, stability, and temperature, all under controlled external mass transfer. New data reported in this study fit into the larger body of work conducted on water-evaporation reduction by duplex-oil films. Duplex-oil films of oxidized mineral oil/mucin (MOx/BSM), human meibum (HM), and bovine meibum (BM) reduce water evaporation by a dissolution-diffusion mechanism, as confirmed by agreement between measurement and theory. The water permeability of oxidized-mineral-oil duplex films agrees with those reported in the literature, after correction for the presence of mucin. We find that duplex-oil films of bovine and human meibum at physiologic temperature reduce water evaporation only 6-8% for a 100-nm film thickness pertinent to the human tear film. Comparison to in-vivo human tear-evaporation measurements is inconclusive because evaporation from a clean-water surface is not measured and because the mass-transfer resistance is not characterized. Copyright © 2013 Elsevier B.V. All rights reserved.

Physical-Layer Security of a Buffer-Aided Full-Duplex Relaying System

KAUST Repository

El Shafie, Ahmed

2016-07-07

This letter proposes a novel hybrid half-/full-duplex relaying scheme to enhance the relay channel security. A source node (Alice) communicates with her destination node (Bob) in the presence of a buffer-aided full-duplex relay node (Rooney) and a potential eavesdropper (Eve). Rooney adopts two different relaying, namely randomize-and-forward and decode-andforward relaying strategies, to improve the security of the legitimate system. In the first relaying strategy, Rooney uses a codebook different from that used at Alice. In the second relaying strategy, Rooney and Alice use the same codebooks. In addition, Rooney switches between half-duplex and full-duplex modes to further enhance the security of the legitimate system. The numerical results demonstrate that our proposed scheme achieves a significant average secrecy end-to-end throughput improvement relative to the conventional bufferless full-duplex relaying scheme.

Physical-Layer Security of a Buffer-Aided Full-Duplex Relaying System

KAUST Repository

El Shafie, Ahmed; Salem, Ahmed Sultan; Al-Dhahir, Naofal

2016-01-01

This letter proposes a novel hybrid half-/full-duplex relaying scheme to enhance the relay channel security. A source node (Alice) communicates with her destination node (Bob) in the presence of a buffer-aided full-duplex relay node (Rooney) and a potential eavesdropper (Eve). Rooney adopts two different relaying, namely randomize-and-forward and decode-andforward relaying strategies, to improve the security of the legitimate system. In the first relaying strategy, Rooney uses a codebook different from that used at Alice. In the second relaying strategy, Rooney and Alice use the same codebooks. In addition, Rooney switches between half-duplex and full-duplex modes to further enhance the security of the legitimate system. The numerical results demonstrate that our proposed scheme achieves a significant average secrecy end-to-end throughput improvement relative to the conventional bufferless full-duplex relaying scheme.

Enantiospecific kinking of DNA by a partially intercalating metal complex

KAUST Repository

Reymer, Anna

2012-01-01

Opposite enantiomers of [Ru(phenanthroline) 3] 2+ affect the persistence length of DNA differently, a long speculated effect of helix kinking. Our molecular dynamics simulations confirm a substantial change of duplex secondary structure produced by wedge-intercalation of one but not the other enantiomer. This effect is exploited by several classes of DNA operative proteins. © The Royal Society of Chemistry 2012.

Effect of Temperature on the Intrinsic Flexibility of DNA and Its Interaction with Architectural Proteins

NARCIS (Netherlands)

Driessen, R.P.C.; Sitter, G.; Moolenaar, G.F.; Wuite, G.J.L.; Goosen, N.; Dame, R.T.

2014-01-01

The helical structure of double-stranded DNA is destabilized by increasing temperature. Above a critical temperature (the melting temperature), the two strands in duplex DNA become fully separated. Below this temperature, the structural effects are localized. Using tethered particle motion in a

Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.

Science.gov (United States)

Dziegielewska, Barbara; Beerman, Terry A; Bianco, Piero R

2006-09-01

To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.

Characterization of thermal aging of duplex stainless steel by SQUID

International Nuclear Information System (INIS)

Isobe, Y.; Kamimura, A.; Aoki, K.; Nakayasu, F.

1995-01-01

Thermal aging is a growing concern for long-term-aged duplex stainless steel piping in nuclear power plants. Superconducting QUantum Interference Device (SQUID) was used for the detection of thermal aging of SUS329 rolled duplex stainless steel and SCS16 cast duplex stainless steel. It was found that the SQUID output signal pattern in the presence of AC magnetic field applied to the specimen was sensitive to the changes in electromagnetic properties due to thermal aging

Hydrogen peroxide biosensor based on DNA-Hb modified gold electrode

International Nuclear Information System (INIS)

Kafi, A.K.M.; Fan Yin; Shin, Hoon-Kyu; Kwon, Young-Soo

2006-01-01

A hydrogen peroxide (H 2 O 2 ) biosensor based on DNA-hemoglobin (Hb) modified electrode is described in this paper. The sensor was designed by DNA and hemoglobin dropletting onto gold electrode surface layer by layer. The sensor based on the direct electron transfer of iron of hemoglobin showed a well electrocatalytic response to the reduction of the H 2 O 2 . This sensor offered an excellent electrochemical response for H 2 O 2 concentration below micromole level with high sensitivity and selectivity and short response time. Experimental conditions influencing the biosensor performance such as, pH, potential were optimized and assessed. The levels of the RSD's ( 2 O 2 was observed from 10 to 120 μM with the detection limit of 0.4 μM (based on the S/N = 3)

DNA extraction methods for detecting genetically modified foods: A comparative study.

Science.gov (United States)

Elsanhoty, Rafaat M; Ramadan, Mohamed Fawzy; Jany, Klaus Dieter

2011-06-15

The work presented in this manuscript was achieved to compare six different methods for extracting DNA from raw maize and its derived products. The methods that gave higher yield and quality of DNA were chosen to detect the genetic modification in the samples collected from the Egyptian market. The different methods used were evaluated for extracting DNA from maize kernels (without treatment), maize flour (mechanical treatment), canned maize (sweet corn), frozen maize (sweet corn), maize starch, extruded maize, popcorn, corn flacks, maize snacks, and bread made from corn flour (mechanical and thermal treatments). The quality and quantity of the DNA extracted from the standards, containing known percentages of GMO material and from the different food products were evaluated. For qualitative detection of the GMO varieties in foods, the GMOScreen 35S/NOS test kit was used, to screen the genetic modification in the samples. The positive samples for the 35S promoter and/or the NOS terminator were identified by the standard methods adopted by EU. All of the used methods extracted yielded good DNA quality. However, we noted that the purest DNA extract were obtained using the DNA extraction kit (Roche) and this generally was the best method for extracting DNA from most of the maize-derived foods. We have noted that the yield of DNA extracted from maize-derived foods was generally lower in the processed products. The results indicated that 17 samples were positive for the presence of 35S promoter, while 34% from the samples were positive for the genetically modified maize line Bt-176. Copyright © 2010 Elsevier Ltd. All rights reserved.

Development of DNA marker for Fusarium resistance in Pisang Berangan

International Nuclear Information System (INIS)

Affrida Abu Hassan; Mohd Nazir Basiran; Rosmawati Shaharuddin

2000-01-01

Fusarium wilt (Panama disease), a disease caused by a soil-bome fungus Fusarium oxysporum f. sp. cubense, is regarded as one of the most significant threats to banana (Musa spp.) production worldwide. In Malaysia, it is affecting the Cavendish as well as Pisang Berangan which are widely planted for export as well as for local consumption. Pisang Berangan mutant line (MB96) which was obtained through induced mutation by gamma irradiation has showed certain degree of tolerance towards the disease. Attempts were made to utilise Polymerase Chain Reaction (PCR) based techniques i.e. RAPD (Random Amplified Polymorphic DNA) to screen for unique DNA sequences that are associated or closely linked to these tolerance characteristics. Four single 1 Obp primers and five duplex 1 Obp primers combinations were used to detect polymorphism between the DNA of control and 4 mutant lines micropropagated from MB96. As further control, DNA of Pisang Mas was included. Duplex arbitrary primer combinations 11-89 and single primer OPA-3 have produced DNA fragments that are polymorphic between cultivar, Pisang Berangan and Pisang Mas. However the RAPD analysis failed to show any polymorphism between the control and the mutant lines or in between the mutant lines

Duplex ultrasound: Indications and findings in a newly created ...

African Journals Online (AJOL)

Duplex ultrasound: Indications and findings in a newly created facility at the University of Calabar Teaching Hospital, Calabar. ... It is recommended that timely referrals be made, and mobile Doppler units be acquired to save more lives and limbs in the developing world. Keywords: Calabar, deep venous thrombosis, duplex ...

The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

Directory of Open Access Journals (Sweden)

Elaine Ann Moore

Full Text Available The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

Science.gov (United States)

Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M; Hiasa, Hiroshi; Marks, Kevin R; Kerns, Robert J; Berger, James M; Drlica, Karl

2014-05-02

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

Effect of gold nanoparticle on stability of the DNA molecule: A study of molecular dynamics simulation.

Science.gov (United States)

Izanloo, Cobra

2017-09-02

An understanding of the mechanism of DNA interactions with gold nanoparticles is useful in today medicine applications. We have performed a molecular dynamics simulation on a B-DNA duplex (CCTCAGGCCTCC) in the vicinity of a gold nanoparticle with a truncated octahedron structure composed of 201 gold atoms (diameter ∼1.8 nm) to investigate gold nanoparticle (GNP) effects on the stability of DNA. During simulation, the nanoparticle is closed to DNA and phosphate groups direct the particles into the major grooves of the DNA molecule. Because of peeling and untwisting states that are occur at end of DNA, the nucleotide base lies flat on the surface of GNP. The configuration entropy is estimated using the covariance matrix of atom-positional fluctuations for different bases. The results show that when a gold nanoparticle has interaction with DNA, entropy increases. The results of conformational energy and the hydrogen bond numbers for DNA indicated that DNA becomes unstable in the vicinity of a gold nanoparticle. The radial distribution function was calculated for water hydrogen-phosphate oxygen pairs. Almost for all nucleotide, the presence of a nanoparticle around DNA caused water molecules to be released from the DNA duplex and cations were close to the DNA.

DNA Duplex Length and Salt Concentration Dependence of Enthalpy−Entropy Compensation Parameters for DNA Melting

KAUST Repository

Starikov, E. B.

2009-08-20

Systematical differential calorimetry experiments on DNA oligomers with different lengths and placed in water solutions with various added salt concentrations may, in principle, unravel important information about the structure and dynamics of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated that the application of the latter could enable direct estimation of thermodynamic parameters of the microphase transitions connected to the changes in DNA dynamical regimes versus the length of the biopolymers and the ionic strengths of their water solutions, and this calls for much more systematical experimental and theoretical studies in this field. © 2009 American Chemical Society.

Direct on-chip DNA synthesis using electrochemically modified gold electrodes as solid support

Science.gov (United States)

Levrie, Karen; Jans, Karolien; Schepers, Guy; Vos, Rita; Van Dorpe, Pol; Lagae, Liesbet; Van Hoof, Chris; Van Aerschot, Arthur; Stakenborg, Tim

2018-04-01

DNA microarrays have propelled important advancements in the field of genomic research by enabling the monitoring of thousands of genes in parallel. The throughput can be increased even further by scaling down the microarray feature size. In this respect, microelectronics-based DNA arrays are promising as they can leverage semiconductor processing techniques with lithographic resolutions. We propose a method that enables the use of metal electrodes for de novo DNA synthesis without the need for an insulating support. By electrochemically functionalizing gold electrodes, these electrodes can act as solid support for phosphoramidite-based synthesis. The proposed method relies on the electrochemical reduction of diazonium salts, enabling site-specific incorporation of hydroxyl groups onto the metal electrodes. An automated DNA synthesizer was used to couple phosphoramidite moieties directly onto the OH-modified electrodes to obtain the desired oligonucleotide sequence. Characterization was done via cyclic voltammetry and fluorescence microscopy. Our results present a valuable proof-of-concept for the integration of solid-phase DNA synthesis with microelectronics.

Study on the wear of TiN/Ti duplex and multilayer coatings in microabrasion tests

International Nuclear Information System (INIS)

Flores, M; De Las Heras, E; Ruelas, R; Rodriguez, E; Bautista, A; Pazos, L; Corengia, P

2008-01-01

Ionic nitriding, on steels, is used to harden the surface of components improving resistance to corrosion and wear and increasing the stress life. Duplex treatments are an alternative for resolving the limitations of very hard coatings on less hard substrates. In this case the duplex treatment consists of an ionic nitriding treatment followed by a single or multilayer coatings deposited by means of the PVD technique. This work presents the influence of the variation in the severity of contact on the kind of wear present in the microabrasion test used to measure the wear coefficient of duplex coatings, consisting of a layer nitrided by DC-pulsed plasma plus TiN coatings and multilayers of TiN/Ti deposited on non-nitrided and nitrided AISI 316L stainless steel and H13 steel. The severity of contact was modified by varying the charge (0.25 to 1 N). The abrasives used were a suspension of 0.1μm diameter diamond particles and a suspension of an average 5 μm diameter aluminum particles. The influence of the presence of relatively soft metallic layers on the determination of the wear coefficient was analyzed in the metal-ceramic multilayers. Two sphere revolving speeds of de 0.05 and 0.154 m/s were used on two microabrasion machines: one commercial and the other built in the UdeG laboratory. The wear marks were photographed and measured with an optic microscope. The value of the critical charge at which the transition occurs between the methods of wear of the substrates and the nitrited samples was determined. Resistance to the AISI 316L steel's microabrasive wear increases with the ionic nitriding treatment. The duplex coatings increase resistance to the wear from the nitrited samples. The wear resistance of the samples with multilayer coatings surpassed that of the duplex samples with multilayer coatings. The greater resistance of the multilayers may be explained by an increase in the resistance to the fracture and not by a increase in surface hardness. The transition

Criteria for the Segmentation of Vowels on Duplex Oscillograms.

Science.gov (United States)

Naeser, Margaret A.

This paper develops criteria for the segmentation of vowels on duplex oscillograms. Previous vowel duration studies have primarily used sound spectrograms. The use of duplex oscillograms, rather than sound spectrograms, permits faster production (real time) at less expense (adding machine paper may be used). The speech signal can be more spread…

NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

Science.gov (United States)

Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

2015-01-01

Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

Duplex-guided percutaneous transluminal angioplasty in iliac arterial occlusive disease.

Science.gov (United States)

Krasznai, A G; Sigterman, T A; Welten, R J; Heijboer, R; Sikkink, C J J M; van de Akker, L H J M; Bouwman, L H

2013-11-01

Chronic renal insufficiency (CRI) is a growing global problem. PTA can be performed without nephrotoxic contrast, utilizing Doppler-ultrasound (Duplex) guidance. Duplex-guided infra-inguinal interventions and access-related interventions have been reported. Duplex-guided iliac interventions have not been performed to any extent because of the anatomic location. In our study we evaluated the safety and efficacy of Duplex-guided percutaneous transluminal angioplasty (DuPTA) in iliac arteries. From June 2012 until February 2013, 31 patients (35 iliac lesions), underwent DuPTA. Indications ranged from Rutherford 3 to 5. Preoperative evaluation included Ankle Brachial Index (ABI), Duplex and MRA. Procedural success was defined as crossing the lesion with a guidewire and dilating or stenting the lesion. Clinical success was defined as 50% reduction in peak systolic velocity (PSV) or clinical improvement. PSV was evaluated after PTA, then at 2 weeks. Clinical results were assessed 2 weeks after the procedure. Procedural success was achieved in 94% of patients (33/35), all of whom also had clinical success. Post-procedural PSV reduction showed an average improvement of 63% (431 cm/s to 153 cm/s). Mean preoperative ABI was 0.72 and improved to 0.88 postoperatively. PTA using Duplex-guidance in significant iliac stenosis is a safe method with major advantages in patients at high risk for developing contrast-induced nephropathy. Copyright © 2013 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

A Full-Duplex MAC Tailored for 5G Wireless Networks

Directory of Open Access Journals (Sweden)

Lucas de Melo Guimarães

2018-01-01

Full Text Available The increasing demands for high-data rate traffic stimulated the development of the fifth-generation (5G mobile networks. The envisioned 5G network is expected to meet its challenge by devising means to further improve spectrum usage. Many alternatives to enhance spectrum usage are being researched, such as massive MIMO, operation in mmWave frequency, cognitive radio, and the employment of full-duplex antennas. Efficient utilization of the potential of any of these technologies faces a set of challenges related to medium access control (MAC schemes. This work focuses on MAC schemes tailored for full-duplex antennas, since they are expected to play a major role in the foreseeable 5G networks. In this context, this paper presents a MAC layer technique to improve total transmission time when full-duplex antennas are employed. Several evaluations in different scenarios are conducted to assess the proposed MAC scheme. Numerical results show that the proposed scheme provides gains up to 156% when compared to a state-of-the-art full-duplex antenna MAC protocol. Compared to traditional half-duplex antenna MAC protocols, the proposed scheme yields gain up to 412%.

Analysing Self Interference Cancellation in Full Duplex Radios

DEFF Research Database (Denmark)

Mahmood, Nurul Huda; Shafique Ansari, Imran; Berardinelli, Gilberto

2016-01-01

Full duplex communication promises a theoretical $100\\%$ throughput gain by doubling the number of simultaneous transmissions. Such compelling gains are conditioned on perfect cancellation of the self interference power resulting from simultaneous transmission and reception. Generally, self...... interference power is modelled as a noise-like constant level interference floor. However, experimental validations have shown that the self interference power is in practice a random variable depending on a number of factors such as the surrounding wireless environment and the degree of interference...... cancellation. In this study, we derive an analytical model for the residual self interference power, and demonstrate various applications of the derived model in analysing the performance of a Full Duplex radio. In general, full duplex communication is found to provide only modest throughput gains over half...

Development of oxide dispersion strengthened 2205 duplex stainless steel composite

Directory of Open Access Journals (Sweden)

Oladayo OLANIRAN

2015-05-01

Full Text Available Composites of duplex stainless steel were produced by oxide dispersion strengthening with comparatively improved mechanical properties by hot press sintering of partially stabilized Zirconia (PSZ, 3% yttria, mole fraction dispersion in 2205 duplex stainless steels. Ceramic oxide was added as reinforcement, while chromium (Cr and Nickel (Ni were incorporated to maintain the austenitic/ferritic phase balance of the duplex stainless steel. The powders and sintered were characterized in detail using scanning electron microscopy (SEM and X-ray diffraction (XRD. The microstructural evolution and phase formation during oxide dispersion strengthening of duplex stainless steel composites were investigated. The influence of composition variation of the reinforcements on the microstructural and corrosion behaviour in simulated mine water of the composites were investigated. In this manuscript, it was established that composition has great influence on the structure/properties relationship of the composites developed.

Energy Landscape and Pathways for Transitions between Watson-Crick and Hoogsteen Base Pairing in DNA.

Science.gov (United States)

Chakraborty, Debayan; Wales, David J

2018-01-04

The recent discovery that Hoogsteen (HG) base pairs are widespread in DNA across diverse sequences and positional contexts could have important implications for understanding DNA replication and DNA-protein recognition. While evidence is emerging that the Hoogsteen conformation could be a thermodynamically accessible conformation of the DNA duplex and provide a means to expand its functionality, relatively little is known about the molecular mechanism underlying the Watson-Crick (WC) to HG transition. In this Perspective, we describe pathways and kinetics for this transition at an atomic level of detail, using the energy landscape perspective. We show that competition between the duplex conformations results in a double funnel landscape, which explains some recent experimental observations. The interconversion pathways feature a number of intermediates, with a variable number of WC and HG base pairs. The relatively slow kinetics, with possible deviations from two-state behavior, suggest that this conformational switch is likely to be a challenging target for both simulation and experiment.

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein.

Science.gov (United States)

Plchova, Helena; Hartung, Frank; Puchta, Holger

2003-11-07

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.

DNA Duplex Length and Salt Concentration Dependence of Enthalpy−Entropy Compensation Parameters for DNA Melting

KAUST Repository

Starikov, E. B.; Nordén, Bengt

2009-01-01

of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated

Secondary Hardening Behavior in Super Duplex Stainless Steels during LCF in Dynamic Strain Ageing Regime

OpenAIRE

Chai, Guocai; Andersson, Marcus

2013-01-01

Cyclic deformation behaviors in five modified duplex stainless steel S32705 grades have been studied at 20 °C, 200 °C, 250° and 350 °C. The influence of temperature and nitrogen concentration on the occurrence of the second hardening phenomenon, in the stress response curve was focused. An increase in nitrogen concentration can have a positive effect on dynamic strain ageing by increasing the first hardening and also the second hardening behavior during cyclic deformation. Furthermore, an inc...

Measuring secondary phases in duplex stainless steels

Science.gov (United States)

Calliari, I.; Brunelli, K.; Dabalà, M.; Ramous, E.

2009-01-01

The use of duplex stainless steels is limited by their susceptibility to the formation of dangerous intermetallic phases resulting in detrimental effects on impact toughness and corrosion resistance. This precipitation and the quantitative determinations of the phases have received considerable attention and different precipitation sequences (σ phase, χ phase, and carbides) have been suggested. This study investigates the phase transformation during continuous cooling and isothermal treatments in commercial duplex stainless steel grades and the effects on alloy properties, and compares the most common techniques of analysis.

"Off-on" electrochemical hairpin-DNA-based genosensor for cancer diagnostics.

Science.gov (United States)

Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt; Ferapontova, Elena E

2011-03-01

A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

Roles of the Amino Group of Purine Bases in the Thermodynamic Stability of DNA Base Pairing

Directory of Open Access Journals (Sweden)

Shu-ichi Nakano

2014-08-01

Full Text Available The energetic aspects of hydrogen-bonded base-pair interactions are important for the design of functional nucleotide analogs and for practical applications of oligonucleotides. The present study investigated the contribution of the 2-amino group of DNA purine bases to the thermodynamic stability of oligonucleotide duplexes under different salt and solvent conditions, using 2'-deoxyriboinosine (I and 2'-deoxyribo-2,6-diaminopurine (D as non-canonical nucleotides. The stability of DNA duplexes was changed by substitution of a single base pair in the following order: G•C > D•T ≈ I•C > A•T > G•T > I•T. The apparent stabilization energy due to the presence of the 2-amino group of G and D varied depending on the salt concentration, and decreased in the water-ethanol mixed solvent. The effects of salt concentration on the thermodynamics of DNA duplexes were found to be partially sequence-dependent, and the 2-amino group of the purine bases might have an influence on the binding of ions to DNA through the formation of a stable base-paired structure. Our results also showed that physiological salt conditions were energetically favorable for complementary base recognition, and conversely, low salt concentration media and ethanol-containing solvents were effective for low stringency oligonucleotide hybridization, in the context of conditions employed in this study.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

Energy Technology Data Exchange (ETDEWEB)

Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

2013-03-15

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

International Nuclear Information System (INIS)

Grierson, Patrick M.; Acharya, Samir; Groden, Joanna

2013-01-01

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription

Duplex tab exhaust nozzle

Science.gov (United States)

Gutmark, Ephraim Jeff (Inventor); Martens, Steven (nmn) (Inventor)

2012-01-01

An exhaust nozzle includes a conical duct terminating in an annular outlet. A row of vortex generating duplex tabs are mounted in the outlet. The tabs have compound radial and circumferential aft inclination inside the outlet for generating streamwise vortices for attenuating exhaust noise while reducing performance loss.

Qualitative comparison of duplex Stirling and absorption refrigerators in domestic applications

Energy Technology Data Exchange (ETDEWEB)

Shao, H. [Global Cooling BV, Zutphen (Netherlands)

2000-07-01

A qualitative comparison has been carried out between the duplex Stirling and the absorption refrigerator for domestic applications. The duplex Stirling has many advantages over the absorption refrigerator on efficiency, modulation, suitability, operating costs, pollution reduction. Based on the state of the art of free-piston gas-bearing and linear-motor Stirling engines and coolers, it appears technically and economically feasible to develop the duplex Stirling to compete with the absorption refrigerator for heat-driven domestic refrigeration. (orig.)

A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine

Energy Technology Data Exchange (ETDEWEB)

Oskuee, Reza Kazemi [Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Dosti, Fatemeh [School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Gholami, Leila [Targeted Drug Delivery Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Malaekeh-Nikouei, Bizhan, E-mail: malaekehb@mums.ac.ir [Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of)

2015-04-01

Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65 kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250 nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15 kDa had better transfection efficiency than the vectors made of PAA 65 kDa. In conclusion, results of the present study indicated that grafting PAA 15 kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity. - Highlights: • The modified polyallylamine was synthesized as a gene carrier. • Modification of polyallylamine (15 kDa) with high percentages of hexyl acrylate improved transfection activity remarkably. • Grafting of polymer with acrylate derivatives reduced polymer cytotoxicity especially at percentages of

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

Science.gov (United States)

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

Science.gov (United States)

Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

2017-11-01

5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of Solution Annealing Temperature on the Galvanic Corrosion Behavior of the Super Duplex Stainless Steels

Science.gov (United States)

Lee, Jun-Seob; Jeon, Soon-Hyeok; Park, Yong-Soo

2013-02-01

This study investigated the active dissolution of super duplex stainless steel (SDSS) at various solution annealing temperatures. The active dissolutions of the α-phase and γ-phase were compared, and the effects of the surface area ratio on the active dissolutions of both phases were investigated. There were two peaks in the active-passive transition region in the potentiodynamic test in the modified green-death solution. The two peaks changed as the solution annealing temperature was increased from 1050 to 1150 °C. The solution annealing temperature difference affected the critical anodic current densities. This provides useful information for determining the appropriate solution annealing temperature in the modified green-death solution for SDSS.

Degradation of transgene DNA in genetically modified herbicide-tolerant rice during food processing.

Science.gov (United States)

Song, Shangxin; Zhou, Guanghong; Gao, Feng; Zhang, Wei; Qiu, Liangyan; Dai, Sifa; Xu, Xinglian; Xiao, Hongmei

2011-12-01

In order to assess the effect of food processing on the degradation of exogenous DNA components in sweet rice wine and rice crackers made from genetically modified (GM) rice (Oryza sativa L.), we developed genomic DNA extraction methods and compared the effect of different food processing procedures on DNA degradation. It was found that the purity, quantity and quality of DNA by alkaline lysis method were higher than by CTAB (cetyltrimethylammonium bromide) method. For sweet rice wine, CAMV35S (cauliflower mosaic virus 35S) promoter and NOS (nopaline synthase) terminator were degraded by the third day, whereas the exogenous gene Bar (bialaphos resistance) remained unaffected. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. These results indicated that the stability of DNA in GM rice was different under different processing conditions. For sweet rice wine, Bar was most stable, followed by NOS, CAMV35S, and SPS. For rice crackers, CAMV35S was most stable, followed by SPS, NOS, and Bar. Copyright © 2011 Elsevier Ltd. All rights reserved.

Procedure for normalization of cDNA libraries

Science.gov (United States)

Bonaldo, Maria DeFatima; Soares, Marcelo Bento

1997-01-01

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

Full-Duplex opportunistic relay selection in future spectrum-sharing networks

KAUST Repository

Khafagy, Mohammad Galal; Alouini, Mohamed-Slim; Aï ssa, Sonia

2015-01-01

We propose and analyze the performance of full-duplex relay selection in primary/secondary spectrum-sharing networks. Contrary to half-duplex relaying, full-duplex relaying (FDR) enables simultaneous listening/forwarding at the secondary relay, thereby allowing for a higher spectral efficiency. However, since the source and relay simultaneously transmit in FDR, their superimposed signal at the primary receiver should now satisfy the existing interference constraint which can considerably limit the secondary network throughput. In this regard, relay selection can offer an adequate solution to boost the secondary throughput while satisfying the imposed interference limit. We first analyze the performance of opportunistic relay selection among a cluster of full-duplex decode-and-forward relays with self-interference by deriving the exact cumulative distribution function of its end-to-end signal-to-noise ratio. Second, we evaluate the end-to-end performance of relay selection with interference constraints due to the presence of a primary receiver. Finally, the presented exact theoretical findings are verified by numerical simulations.

Full-Duplex opportunistic relay selection in future spectrum-sharing networks

KAUST Repository

Khafagy, Mohammad Galal

2015-06-01

We propose and analyze the performance of full-duplex relay selection in primary/secondary spectrum-sharing networks. Contrary to half-duplex relaying, full-duplex relaying (FDR) enables simultaneous listening/forwarding at the secondary relay, thereby allowing for a higher spectral efficiency. However, since the source and relay simultaneously transmit in FDR, their superimposed signal at the primary receiver should now satisfy the existing interference constraint which can considerably limit the secondary network throughput. In this regard, relay selection can offer an adequate solution to boost the secondary throughput while satisfying the imposed interference limit. We first analyze the performance of opportunistic relay selection among a cluster of full-duplex decode-and-forward relays with self-interference by deriving the exact cumulative distribution function of its end-to-end signal-to-noise ratio. Second, we evaluate the end-to-end performance of relay selection with interference constraints due to the presence of a primary receiver. Finally, the presented exact theoretical findings are verified by numerical simulations.

Duplex Ultrasonography Has Limited Utility in Detection of Postoperative DVT After Primary Total Joint Arthroplasty.

Science.gov (United States)

Vira, Shaleen; Ramme, Austin J; Alaia, Michael J; Steiger, David; Vigdorchik, Jonathan M; Jaffe, Frederick

2016-07-01

Duplex ultrasound is routinely used to evaluate suspected deep venous thrombosis after total joint arthroplasty. When there is a clinical suspicion for a pulmonary embolism, a chest angiogram (chest CTA) is concomitantly obtained. Two questions were addressed: First, for the population of patients who receive duplex ultrasound after total joint arthroplasty, what is the rate of positive results? Second, for these patients, how many of these also undergo chest CTA for clinical suspicion of pulmonary embolus and how many of these tests are positive? Furthermore, what is the correlation between duplex ultrasound results and chest CTA results? A retrospective chart review was conducted of total joint replacement patients in 2011 at a single institution. Inclusion criteria were adult patients who underwent a postoperative duplex ultrasonography for clinical suspicion of deep venous thrombosis (DVT). Demographic data, result of duplex scan, clinical indications for obtaining the duplex scan, and DVT prophylaxis used were recorded. Additionally, if a chest CTA was obtained for clinical suspicion for pulmonary embolus, results and clinical indication for obtaining the test were recorded. The rate of positive results for duplex ultrasonography and chest CTA was computed and correlated based on clinical indications. Two hundred ninety-five patients underwent duplex ultrasonography of which only 0.7% were positive for a DVT. One hundred three patients underwent a chest CTA for clinical suspicion of a pulmonary embolism (PE) of which 26 revealed a pulmonary embolus, none of which had a positive duplex ultrasound. Postoperative duplex scans have a low rate of positive results. A substantial number of patients with negative duplex results subsequently underwent chest CTA for clinical suspicion for which a pulmonary embolus was found, presumably resulting from a DVT despite negative duplex ultrasound result. A negative duplex ultrasonography should not rule out the presence of a

Development of a lean duplex stainless steel

Energy Technology Data Exchange (ETDEWEB)

Liljas, M.; Johansson, P.; Liu Hui-Ping; Olsson, C.O.A. [Avesta Research Centre, Avesta (Sweden). Outokumpu Stainless

2008-06-15

The classic series of duplex stainless steels shows very high corrosion resistance and can be used for very demanding applications. A new lean duplex steel, LDX 2101 {sup registered} (EN 1.4162, UNS S32101), has been developed with corrosion resistance on a par with standard austenitic grades. Application areas include: structural components, chemical industry, tanks and containers. The steel was designed to have equal amounts of ferrite and austenite in annealed condition and with an austenite that is stable against strain-induced martensite. Thanks to its high nitrogen content, the steel has a fast austenite reformation when subjected to thermal cycling, e.g. welding. Unlike conventional duplex grades, the formation of intermetallic phase is very sluggish, although precipitation of nitrides and carbides has a certain impact on material properties after exposure in the temperature range 600 to 800 C. The precipitation behaviour after different isothermal treatments is described and its influence on different product properties is shown. A good agreement was found between impact toughness and corrosion resistance for a wide range of thermal treatments. (orig.)

Full-Duplex Communications in Large-Scale Cellular Networks

KAUST Repository

AlAmmouri, Ahmad

2016-04-01

In-band full-duplex (FD) communications have been optimistically promoted to improve the spectrum utilization and efficiency. However, the penetration of FD communications to the cellular networks domain is challenging due to the imposed uplink/downlink interference. This thesis presents a tractable framework, based on stochastic geometry, to study FD communications in multi-tier cellular networks. Particularly, we assess the FD communications effect on the network performance and quantify the associated gains. The study proves the vulnerability of the uplink to the downlink interference and shows that the improved FD rate gains harvested in the downlink (up to 97%) comes at the expense of a significant degradation in the uplink rate (up to 94%). Therefore, we propose a novel fine-grained duplexing scheme, denoted as α-duplex scheme, which allows a partial overlap between the uplink and the downlink frequency bands. We derive the required conditions to harvest rate gains from the α-duplex scheme and show its superiority to both the FD and half-duplex (HD) schemes. In particular, we show that the α-duplex scheme provides a simultaneous improvement of 28% for the downlink rate and 56% for the uplink rate. We also show that the amount of the overlap can be optimized based on the network design objective. Moreover, backward compatibility is an essential ingredient for the success of new technologies. In the context of in-band FD communication, FD base stations (BSs) should support HD users\\' equipment (UEs) without sacrificing the foreseen FD gains. The results show that FD-UEs are not necessarily required to harvest rate gains from FD-BSs. In particular, the results show that adding FD-UEs to FD-BSs offers a maximum of 5% rate gain over FD-BSs and HD-UEs case, which is a marginal gain compared to the burden required to implement FD transceivers at the UEs\\' side. To this end, we shed light on practical scenarios where HD-UEs operation with FD-BSs outperforms the

HMGB1-mediated DNA bending: Distinct roles in increasing p53 binding to DNA and the transactivation of p53-responsive gene promoters.

Science.gov (United States)

Štros, Michal; Kučírek, Martin; Sani, Soodabeh Abbasi; Polanská, Eva

2018-03-01

HMGB1 is a chromatin-associated protein that has been implicated in many important biological processes such as transcription, recombination, DNA repair, and genome stability. These functions include the enhancement of binding of a number of transcription factors, including the tumor suppressor protein p53, to their specific DNA-binding sites. HMGB1 is composed of two highly conserved HMG boxes, linked to an intrinsically disordered acidic C-terminal tail. Previous reports have suggested that the ability of HMGB1 to bend DNA may explain the in vitro HMGB1-mediated increase in sequence-specific DNA binding by p53. The aim of this study was to reinvestigate the importance of HMGB1-induced DNA bending in relationship to the ability of the protein to promote the specific binding of p53 to short DNA duplexes in vitro, and to transactivate two major p53-regulated human genes: Mdm2 and p21/WAF1. Using a number of HMGB1 mutants, we report that the HMGB1-mediated increase in sequence-specific p53 binding to DNA duplexes in vitro depends very little on HMGB1-mediated DNA bending. The presence of the acidic C-terminal tail of HMGB1 and/or the oxidation of the protein can reduce the HMGB1-mediated p53 binding. Interestingly, the induction of transactivation of p53-responsive gene promoters by HMGB1 requires both the ability of the protein to bend DNA and the acidic C-terminal tail, and is promoter-specific. We propose that the efficient transactivation of p53-responsive gene promoters by HMGB1 depends on complex events, rather than solely on the promotion of p53 binding to its DNA cognate sites. Copyright © 2018 Elsevier B.V. All rights reserved.

Considerations in Duplex Investment.

Science.gov (United States)

Wright, Arthur; Goen, Tom

Problems of duplex investment are noted in the introduction to this booklet designed to provide a technique by which the investment decision can be approached, develop estimates of typical costs and returns under differing conditions, and encourage investors to analyze objectives and conditions before the decision to buy or build is made. A…

Renal pelvis urothelial carcinoma of the upper moiety in complete right renal duplex: a case report.

Science.gov (United States)

Zhang, Yiran; Yu, Quanfeng; Zhang, Zhihong; Liu, Ranlu; Xu, Yong

2015-01-01

Urothelial carcinoma (UC) originated from renal pelvis is the common tumor of the urinary system, however, neoplasia of the renal pelvis in duplex kidneys is extremely rare, especially in the complete renal and ureteral duplex cases. We present the first case of renal pelvis UC of the upper moiety in a complete right renal duplex. This male patient has bilateral complete renal and ureteral duplex. To the best of our knowledge, this is the first reported case of renal pelvis UC in a complete renal duplex system. After this experience we feel that the diagnosis of renal pelvis UC in duplex kidneys is not so easy, and once the diagnosis is determined, the whole renal duplex units and bladder cuff or ectopic orifice should be excised radically.

AFFINITY BIOSENSOR BASED ON SCREEN-PRINTED ELECTRODE MODIFIED WITH DNA FOR GENOTOXIC COMPOUNDS DETECTION

Directory of Open Access Journals (Sweden)

Bambang Kuswandi

2010-06-01

Full Text Available An electrochemical method for the detection of the genotoxic compounds using a DNA-modified electrode was developed. This electrode was successfully used for the electrochemical detection of genotoxic compounds in water samples. The electrochemical results clearly demonstrated that, the development is related to the molecular interaction between the surface-linked DNA obtained from calf thymus and the target compounds, such as pollutants, in order to develop a simple device for rapid screening of genotoxic compounds in environmental samples. The detection of such compounds was measured by their effect on the oxidation signal of the guanine peak of the DNA immobilised on the surface of carbon based Screen-Printed Electrode (SPE in disposable mode, and monitored by square-wave voltametric analysis. The DNA biosensor is able to detect known intercalating and groove-binding genotoxic compounds such as Dioxin, Bisphenol A, PCBs, and Phtalates. Application to real water samples is discussed and reported.   Keywords: electrochemical, screen-printed electrode, DNA biosensor, genotoxic compounds

An NMR study of the covalent and noncovalent interactions of CC-1065 and DNA

International Nuclear Information System (INIS)

Scahill, T.A.; Jensen, R.M.; Swenson, D.H.; Hatzenbuhler, N.T.; Petzold, G.; Wierenga, W.; Brahme, N.D.

1990-01-01

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR ( 1 H and 13 C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13 C DEPT spectra. An octamer duplex, d(CGATTAGC·GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3' → 5' end in relation to strand A [d(CGATTA 6 GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies

Full-Duplex Relay Selection in Cognitive Underlay Networks

KAUST Repository

Khafagy, Mohammad Galal

2017-09-30

In this work, we analyze the performance of full-duplex relay selection (FDRS) in spectrum-sharing networks. Contrary to half-duplex relaying, full-duplex relaying (FDR) enables simultaneous listening/forwarding at the secondary relay(s), thereby allowing for a higher spectral efficiency. However, since the source and relay simultaneously transmit in FDR, their superimposed signal at the primary receiver should now satisfy the existing interference constraint, which can considerably limit the secondary network throughput. In this regard, relay selection can offer an adequate solution to boost the secondary throughput while satisfying the imposed interference limit. We first analyze the performance of opportunistic FDRS with residual self-interference (RSI) by deriving the exact cumulative distribution function of its end-to-end signal-to-interference-plus-noise ratio under Nakagami-m fading. We also evaluate the offered diversity gain of relay selection for different full-duplex cooperation schemes in the presence/absence of a direct source-destination link. When the adopted RSI link gain model is sublinear in the relay power, which agrees with recent research findings, we show that remarkable diversity gain can be recovered even in the presence of an interfering direct link. Second, we evaluate the end-to-end performance of FDRS with interference constraints due to the presence of a primary receiver. Finally, the presented exact theoretical findings are verified by numerical simulations.

THE INFLUENCE OF POSTHEAT TREATMENT ON FERRITE REDISTRIBUTION IN DUPLEX STEELS ELECTRON BEAM WELDS

OpenAIRE

Zita Iždinská; František Kolenič

2009-01-01

The duplex stainless steel is two-phase steel with the structure composed of austenite and ferrite with optimum austenite/ferrite proportion 50%. At present, classical arc processes for welding duplex steels are generally regarded as acceptable. On the other hand electron and laser beam welding is up to now considered less suitable for welding duplex steels. The submitted work presents the results of testing various thermal conditions at welding duplex stainless steel with electron beam. It w...

In-Band α-Duplex Scheme for Cellular Networks: A Stochastic Geometry Approach

KAUST Repository

Alammouri, Ahmad

2016-07-13

In-band full-duplex (FD) communications have been optimistically promoted to improve the spectrum utilization and efficiency. However, the penetration of FD communications to the cellular networks domain is challenging due to the imposed uplink/downlink interference. This paper presents a tractable framework, based on stochastic geometry, to study FD communications in cellular networks. Particularly, we assess the FD communications effect on the network performance and quantify the associated gains. The study proves the vulnerability of the uplink to the downlink interference and shows that FD rate gains harvested in the downlink (up to 97%) come at the expense of a significant degradation in the uplink rate (up to 94%). Therefore, we propose a novel fine-grained duplexing scheme, denoted as -duplex scheme, which allows a partial overlap between the uplink and the downlink frequency bands. We derive the required conditions to harvest rate gains from the -duplex scheme and show its superiority to both the FD and half-duplex (HD) schemes. In particular, we show that the -duplex scheme provides a simultaneous improvement of 28% for the downlink rate and 56% for the uplink rate. Finally, we show that the amount of the overlap can be optimized based on the network design objective.

An evaluation of nuclear design characteristics of duplex burnable poison rods for extended cycle core

International Nuclear Information System (INIS)

Lee, D. J.; Kim, M. H.; Song, K. W.

2003-01-01

Nuclear design characteristics of duplex burnable poison rod were evaluated for three integral type burnable absorbers; Gadolinia, Erbia and IFBA. Inter-comparison was done for both 12 and 24 month cycle for Korean Standard Nuclear Plant. Fuel assemblies with duplex BP was designed to the equivalent assembly with 8 and 16 gadolinia BP 2 . Duplex BP is composed of inner region of natural U-Gd 2 O 3 , and outer shell of, UO 2 -Er2O 3 . In order to evaluate the duplex BP, assemblies with erbia and IFBA were compared with alternative options. A sensitivity studies were performed to the size of region, compositions and location of duplex BPs. It was shown that duplex BP gave favorable k-infinite curve to burnup, but IFBA provided the least residual reactivity penalty as EOC. Erbia was good for more negative MTCs. IFBA and erbia had better neutronic performance than gadolinia od duplex BP in the aspect of pin power peaking

Chemically modified DNA : methods and applications

NARCIS (Netherlands)

Gjonaj, Lorina

2013-01-01

Over het toepassen van DNA in de chemie DNA is een fascinerend biopolymeer dat gemakkelijk gemanipuleerd kan worden. Doel van het onderzoek van Lorina Gjonaj was het ontwikkelen van nieuwe methodologie om DNA chemisch te modificeren. Verschillende functionaliteiten zijn aan DNA gekoppeld. Deze

Synthesis and conformational properties of oligonucleotides incorporating 2'-O-phosphorylated ribonucleotides as structural motifs of pre-tRNA splicing intermediates.

Science.gov (United States)

Tsuruoka, H; Shohda, K; Wada, T; Sekine, M

2000-11-03

To synthesize oligonucleotides containing 2'-O-phosphate groups, four kinds of ribonucleoside 3'-phosphoramidite building blocks 6a-d having the bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) group were prepared according to our previous phosphorylation procedure. These phosphoramidite units 6a-d were not contaminated with 3'-regioisomers and were successfully applied to solid-phase synthesis to give oligodeoxyuridylates 15, 16 and oligouridylates 21, 22. Self-complementary Drew-Dickerson DNA 12mers 24-28 replaced by a 2'-O-phosphorylated ribonucleotide at various positions were similarly synthesized. In these syntheses, it turned out that KI(3) was the most effective reagent for oxidative desulfurization of the initially generated thiophosphate group to the phosphate group on polymer supports. Without using this conversion step, a tridecadeoxyuridylate 17 incorporating a 2'-O-thiophosphorylated uridine derivative was also synthesized. To investigate the effect of the 2'-phosphate group on the thermal stability and 3D-structure of DNA(RNA) duplexes, T(m) measurement of the self-complementary oligonucleotides obtained and MD simulation of heptamer duplexes 33-36 were carried out. According to these analyses, it was suggested that the nucleoside ribose moiety phosphorylated at the 2'-hydroxyl function predominantly preferred C2'-endo to C3'-endo conformation in DNA duplexes so that it did not significantly affect the stability of the DNA duplex. On the other hand, the 2'-modified ribose moiety was expelled to give a C3'-endo conformation in RNA duplexes so that the RNA duplexes were extremely destabilized.

Intersegmental interactions in supercoiled DNA: atomic force microscope study

Energy Technology Data Exchange (ETDEWEB)

Shlyakhtenko, Luda S.; Miloseska, Lela; Potaman, Vladimir N.; Sinden, Richard R.; Lyubchenko, Yuri L

2003-10-15

Intersegmental interactions in DNA facilitated by the neutralization of electrostatic repulsion was studied as a function of salt concentration and DNA supercoiling. DNA samples with defined superhelical densities were deposited onto aminopropyl mica at different ionic conditions and imaged in air after drying of the samples. Similar to hydrodynamic data, we did not observe a collapse of supercoiled DNA, as proposed earlier by cryo-EM studies. Instead, the formation of the contacts between DNA helices within supercoiled loops with no visible space between the duplexes was observed. The length of such close contacts increased upon increasing NaCl concentration. DNA supercoiling was a critical factor for the stabilization of intersegmental contacts. Implications of the observed effect for understanding DNA compaction in the cell and for regulation DNA transactions via interaction of distantly separated DNA regions are discussed.

Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: Standard and Fpg-modified comet assay

International Nuclear Information System (INIS)

Gajski, Goran; Garaj-Vrhovac, Vera; Orescanin, Visnja

2008-01-01

To investigate the genotoxic potential of atorvastatin on human lymphocytes in vitro standard comet assay was used in the evaluation of basal DNA damage and to investigate possible oxidative DNA damage produced by reactive oxygen species (ROS) Fpg-modified version of comet assay was also conducted. In addition to these techniques the new criteria for scoring micronucleus test were applied for more complete detection of baseline damage in binuclear lymphocytes exposed to atorvastatin 80 mg/day in different time periods by virtue of measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. All parameters obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in the treated than in control lymphocytes. The Fpg-modified comet assay showed a significantly greater tail length, tail intensity, and tail moment in all treated lymphocytes than did the standard comet assay, which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay indicates that some other mechanism is also involved. In addition to the comet assay, a total number of micronuclei, nucleoplasmic bridges and nuclear buds were significantly higher in the exposed than in controlled lymphocytes. Regression analyses showed a positive correlation between the results obtained by the comet (Fpg-modified and standard) and micronucleus assay. Overall, the study demonstrated that atorvastatin in its highest dose is capable of producing damage on the level of DNA molecule and cell

Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: Standard and Fpg-modified comet assay

Energy Technology Data Exchange (ETDEWEB)

Gajski, Goran [Institute for Medical Research and Occupational Health, Mutagenesis Unit, 10000 Zagreb (Croatia); Garaj-Vrhovac, Vera [Institute for Medical Research and Occupational Health, Mutagenesis Unit, 10000 Zagreb (Croatia); Orescanin, Visnja [Ruder Boskovic Institute, 10000 Zagreb (Croatia)

2008-08-15

To investigate the genotoxic potential of atorvastatin on human lymphocytes in vitro standard comet assay was used in the evaluation of basal DNA damage and to investigate possible oxidative DNA damage produced by reactive oxygen species (ROS) Fpg-modified version of comet assay was also conducted. In addition to these techniques the new criteria for scoring micronucleus test were applied for more complete detection of baseline damage in binuclear lymphocytes exposed to atorvastatin 80 mg/day in different time periods by virtue of measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. All parameters obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in the treated than in control lymphocytes. The Fpg-modified comet assay showed a significantly greater tail length, tail intensity, and tail moment in all treated lymphocytes than did the standard comet assay, which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay indicates that some other mechanism is also involved. In addition to the comet assay, a total number of micronuclei, nucleoplasmic bridges and nuclear buds were significantly higher in the exposed than in controlled lymphocytes. Regression analyses showed a positive correlation between the results obtained by the comet (Fpg-modified and standard) and micronucleus assay. Overall, the study demonstrated that atorvastatin in its highest dose is capable of producing damage on the level of DNA molecule and cell.