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Sample records for oligonucleotide phosphate acceptor

  1. Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells

    SciTech Connect

    Plesner, P.; Goodchild, J.; Kalckar, H.M.; Zamecnik, P.C.

    1987-04-01

    Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with /sup 32/Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The /sup 32/P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The /sup 32/P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.

  2. Phosphate acceptor amino acid residues in structural proteins of rhabdoviruses.

    PubMed

    Sokol, F; Tan, K B; McFalls, M L; Madore, P

    1974-07-01

    Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses. PMID:4365328

  3. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation.

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

    Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD(+), respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP(+), respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  4. Isomer-selected photoelectron spectroscopy of isolated DNA oligonucleotides: phosphate and nucleobase deprotonation at high negative charge states.

    PubMed

    Vonderach, Matthias; Ehrler, Oli T; Matheis, Katerina; Weis, Patrick; Kappes, Manfred M

    2012-05-01

    Fractionation according to ion mobility and mass-to-charge ratio has been used to select individual isomers of deprotonated DNA oligonucleotide multianions for subsequent isomer-resolved photoelectron spectroscopy (PES) in the gas phase. Isomer-resolved PE spectra have been recorded for tetranucleotides, pentanucleotides, and hexanucleotides. These were studied primarily in their highest accessible negative charge states (3-, 4-, and 5-, respectively), as provided by electrospraying from room temperature solutions. In particular, the PE spectra obtained for pentanucleotide tetraanions show evidence for two coexisting classes of gas-phase isomeric structures. We suggest that these two classes comprise: (i) species with excess electrons localized exclusively at deprotonated phosphate backbone sites and (ii) species with at least one deprotonated base (in addition to several deprotonated phosphates). By permuting the sequence of bases in various [A(5-x)T(x)](4-) and [GT(4)](4-) pentanucleotides, we have established that the second type of isomer is most likely to occur if the deprotonated base is located at the first or last position in the sequence. We have used a combination of molecular mechanics and semiempirical calculations together with a simple electrostatic model to explore the photodetachment mechanism underlying our photoelectron spectra. Comparison of predicted to measured photoelectron spectra suggests that a significant fraction of the detected electrons originates from the DNA bases (both deprotonated and neutral).

  5. Isomer-selected photoelectron spectroscopy of isolated DNA oligonucleotides: phosphate and nucleobase deprotonation at high negative charge states.

    PubMed

    Vonderach, Matthias; Ehrler, Oli T; Matheis, Katerina; Weis, Patrick; Kappes, Manfred M

    2012-05-01

    Fractionation according to ion mobility and mass-to-charge ratio has been used to select individual isomers of deprotonated DNA oligonucleotide multianions for subsequent isomer-resolved photoelectron spectroscopy (PES) in the gas phase. Isomer-resolved PE spectra have been recorded for tetranucleotides, pentanucleotides, and hexanucleotides. These were studied primarily in their highest accessible negative charge states (3-, 4-, and 5-, respectively), as provided by electrospraying from room temperature solutions. In particular, the PE spectra obtained for pentanucleotide tetraanions show evidence for two coexisting classes of gas-phase isomeric structures. We suggest that these two classes comprise: (i) species with excess electrons localized exclusively at deprotonated phosphate backbone sites and (ii) species with at least one deprotonated base (in addition to several deprotonated phosphates). By permuting the sequence of bases in various [A(5-x)T(x)](4-) and [GT(4)](4-) pentanucleotides, we have established that the second type of isomer is most likely to occur if the deprotonated base is located at the first or last position in the sequence. We have used a combination of molecular mechanics and semiempirical calculations together with a simple electrostatic model to explore the photodetachment mechanism underlying our photoelectron spectra. Comparison of predicted to measured photoelectron spectra suggests that a significant fraction of the detected electrons originates from the DNA bases (both deprotonated and neutral). PMID:22524691

  6. Effect of phosphate activating group on oligonucleotide formation on montmorillonite: the regioselective formation of 3',5'-linked oligoadenylates

    NASA Technical Reports Server (NTRS)

    Prabahar, K. J.; Cole, T. D.; Ferris, J. P.

    1994-01-01

    The effects of amine structure on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidates of adenosine are investigated. 4-Aminopyridine derivatives yielded oligoadenylates as long as dodecamers with a regioselectivity for 3',5'-phosphodiester bond formation averaging 88%. Linear and cyclic oligomers are obtained and no A5'ppA-containing products are detected. Oligomers as long as the hexanucleotide are obtained using 2-aminobenzimidazole as the activating group. A predominance of pA2'pA is detected in the dimer fraction along with cyclic 3',5'-trimer; no A5'ppA-containing oligomers were detected. Little or no oligomer formation was observed when morpholine, piperidine, pyrazole, 1,2,4-triazole, and 2-pyridone are used as phosphate-activating groups. The effects of the structure of the phosphate activating group on the oligomer structure and chain lengths are discussed.

  7. Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition.

    PubMed

    Das, Ushati; Wang, Li Kai; Smith, Paul; Jacewicz, Agata; Shuman, Stewart

    2014-01-01

    Clostridium thermocellum polynucleotide kinase (CthPnk), the 5' end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5'-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg(2+) and a 5'-OH oligonucleotide and a product complex with GDP•Mg(2+) and a 5'-PO4 oligonucleotide. The O5' nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5'-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38-hereby implicated as the essential general base catalyst that abstracts a proton from the 5'-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the 'reverse kinase' reaction by donating a proton to the O5' leaving group of the 5'-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk. PMID:24150947

  8. Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition.

    PubMed

    Das, Ushati; Wang, Li Kai; Smith, Paul; Jacewicz, Agata; Shuman, Stewart

    2014-01-01

    Clostridium thermocellum polynucleotide kinase (CthPnk), the 5' end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5'-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg(2+) and a 5'-OH oligonucleotide and a product complex with GDP•Mg(2+) and a 5'-PO4 oligonucleotide. The O5' nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5'-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38-hereby implicated as the essential general base catalyst that abstracts a proton from the 5'-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the 'reverse kinase' reaction by donating a proton to the O5' leaving group of the 5'-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.

  9. Cytokines and therapeutic oligonucleotides.

    PubMed

    Hartmann, G; Bidlingmaier, M; Eigler, A; Hacker, U; Endres, S

    1997-12-01

    Therapeutic oligonucleotides - short strands of synthetic nucleic acids - encompass antisense and aptamer oligonucleotides. Antisense oligonucleotides are designed to bind to target RNA by complementary base pairing and to inhibit translation of the target protein. Antisense oligonucleotides enable specific inhibition of cytokine synthesis. In contrast, aptamer oligonucleotides are able to bind directly to specific proteins. This binding depends on the sequence of the oligonucleotide. Aptamer oligonucleotides with CpG motifs can exert strong immunostimulatory effects. Both kinds of therapeutic oligonucleotides - antisense and aptamer oligonucleotides - provide promising tools to modulate immunological functions. Recently, therapeutic oligonucleotides have moved towards clinical application. An antisense oligonucleotide directed against the proinflammatory intercellular adhesion molecule 1 (ICAM-1) is currently being tested in clinical trials for therapy of inflammatory disease. Immunostimulatory aptamer oligonucleotides are in preclinical development for immunotherapy. In the present review we summarize the application of therapeutic oligonucleotides to modulate immunological functions. We include technological aspects as well as current therapeutic concepts and clinical studies.

  10. Reaction of phosphorylase-a with α-D-glucose 1-phosphate and maltodextrin acceptors to give products with degree of polymerization 6-89.

    PubMed

    Kazłowski, Bartosz; Ko, Yuan-Tih

    2014-06-15

    A series of linear glucan saccharides (GS) with defined quantity and degree of polymerization (DP) were synthesized from α-d-glucose 1-phosphate (α-d-Glc 1-P) by phosphorylase-a. The GS product fractions with average DP 11, 22, 38, 52, 60, 70, and 79 were measured by HPSEC-ELSD system. Then the same seven fractions were resolved into individual peaks with DP: 6-14, 10-32, 27-55, 37-67, 44-75, 49-83 and 53-89 by HPAEC-PAD system. Results showed that measurement of α-d-Glc 1-P amount consuming during GS synthesis by both systems enable calculation of reaction yield. The reaction yield for the 24h biosynthesis of the GS product was 25.3% (measured by HPSEC-ELSD) or 29.1% (measured by HPAEC-PAD). The HPSEC-ELSD and HPAEC-PAD systems were also successfully used for phosphorylase-a activity measurement in order to perform its kinetic characterization. This study established feasible systems for preparation of various sizes of the GS with defined DP and quantity as well as characterization of phosphorylase-a kinetics.

  11. Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

    PubMed

    Purschke, Werner G; Hoehlig, Kai; Buchner, Klaus; Zboralski, Dirk; Schwoebel, Frank; Vater, Axel; Klussmann, Sven

    2014-08-15

    The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches. PMID:24832383

  12. Associative Memory Acceptors.

    ERIC Educational Resources Information Center

    Card, Roger

    The properties of an associative memory are examined in this paper from the viewpoint of automata theory. A device called an associative memory acceptor is studied under real-time operation. The family "L" of languages accepted by real-time associative memory acceptors is shown to properly contain the family of languages accepted by one-tape,…

  13. The prebiotic synthesis of deoxythymidine oligonucleotides

    NASA Technical Reports Server (NTRS)

    Stephen-Sherwood, E.; Odom, D. G.; Oro, J.

    1974-01-01

    Deoxythymidine 5 prime-triphosphate in the presence of deoxythymidine 5 prime-phosphate, cyanamide and 4-amino-5-imidazole carboxamide polymerizes under drying conditions at moderate temperatures (60 to 90 C) to yield oligonucleotides of up to four units in length. Enzymatic analysis indicated that the majority of these oligomers contained natural 3 prime-5 prime phosphodiester bonds. This reaction offers a possible method for the formation of deoxyoligonucleotides under primitive earth conditions.

  14. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Reck, Folkert; Kudick, Rene; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert; Wippo, Harald

    2001-01-01

    To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an informational Watson-Crick base-pairing system are unique to the pentopyranosyl-(4 + 2)-oligonucleotide isomer derived from the RNA-building block D-ribose, studies on the entire family of diastereoisomeric pyranosyL(4 - Z)-oligonucleotide systems deriving from D-ribose. L-lyxose. D-xylose, and L-arabinose were carried out. The result of these extended studies is unambiguous: not only pyranosyl-RNA, but all members of the pentopyranosyl(4 + 2)-oligonucleotide family are highly efficient Watson-Crick base-pairing systems. Their synthesis and pairing properties will be described in a series of publications in this journal.

  15. Ca2+ enrichment in culture medium potentiates effect of oligonucleotides

    PubMed Central

    Hori, Shin-ichiro; Yamamoto, Tsuyoshi; Waki, Reiko; Wada, Shunsuke; Wada, Fumito; Noda, Mio; Obika, Satoshi

    2015-01-01

    Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca2+ enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ∼100 nm in size are found in Ca2+-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory. PMID:26101258

  16. Ca2+ enrichment in culture medium potentiates effect of oligonucleotides.

    PubMed

    Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Waki, Reiko; Wada, Shunsuke; Wada, Fumito; Noda, Mio; Obika, Satoshi

    2015-10-30

    Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ~100 nm in size are found in Ca(2+)-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory.

  17. Oxygen as Acceptor.

    PubMed

    Borisov, Vitaliy B; Verkhovsky, Michael I

    2015-01-01

    Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate-specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophosphate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and dimethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O₂ is served by two major oxidoreductases (oxidases), cytochrome bo₃ encoded by cyoABCDE and cytochrome bd encoded by cydABX. Terminal oxidases of aerobic respiratory chains of bacteria, which use O₂ as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo₃ and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo₃ and cytochrome bd. The E. coli membrane contains three types of quinones that all have an octaprenyl side chain (C₄₀). It has been proposed that the bo₃ oxidase can have two ubiquinone-binding sites with different affinities. "WHAT'S NEW" IN THE REVISED ARTICLE: The revised article comprises additional information about subunit composition of cytochrome bd and its role in bacterial resistance to nitrosative and oxidative stresses. Also, we present the novel data on the electrogenic function of appBCX-encoded cytochrome bd-II, a second bd-type oxidase that had been thought not to contribute to generation of a proton motive force in E

  18. Oxygen as Acceptor.

    PubMed

    Borisov, Vitaliy B; Verkhovsky, Michael I

    2015-01-01

    Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate-specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophosphate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and dimethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O₂ is served by two major oxidoreductases (oxidases), cytochrome bo₃ encoded by cyoABCDE and cytochrome bd encoded by cydABX. Terminal oxidases of aerobic respiratory chains of bacteria, which use O₂ as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo₃ and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo₃ and cytochrome bd. The E. coli membrane contains three types of quinones that all have an octaprenyl side chain (C₄₀). It has been proposed that the bo₃ oxidase can have two ubiquinone-binding sites with different affinities. "WHAT'S NEW" IN THE REVISED ARTICLE: The revised article comprises additional information about subunit composition of cytochrome bd and its role in bacterial resistance to nitrosative and oxidative stresses. Also, we present the novel data on the electrogenic function of appBCX-encoded cytochrome bd-II, a second bd-type oxidase that had been thought not to contribute to generation of a proton motive force in E

  19. Resonance energy-transfer studies of the conformational change on the adsorption of oligonucleotides to a silica interface.

    PubMed

    Scholes, Colin A; Millar, David P; Gee, Michelle L; Smith, Trevor A

    2011-05-19

    Time-resolved evanescent wave-induced fluorescence studies have been carried out on a series of fluorescently labeled oligonucleotide sequences adsorbed to a silica surface from solution. The fluorescence decay profiles of a fluorescent energy donor group undergoing resonance energy transfer to a nonemissive energy-acceptor molecule have been analyzed in terms of a distribution of donor-acceptor distances to reveal the conformational changes that occur in these oligonucleotides upon adsorption. Evanescent wave-induced time-resolved Förster resonance energy-transfer (EW-TRFRET) measurements indicate that at a high electrolyte concentration, there is localized separation of the oligonucleotide strands, and the helical structure adopts an "unraveled" conformation as a result of adsorption. This is attributed to the flexibility within the oligonucleotide at high electrolyte concentration allowing multiple segments of the oligonucleotide to have direct surface interaction. In contrast, the EW-TRFRET measurements at a lower electrolyte concentration reveal that the oligonucleotide retains its helical conformation in a localized extended state. This behavior implies that the rigidity of the oligonucleotide at this electrolyte concentration restricts direct interaction with the silica to a few segments, which correspondingly introduces kinks in the double helix conformation and results in significant oligonucleotide segmental extension into solution.

  20. Synthesis and triplex-forming properties of cyclic oligonucleotides with (G,A)-antiparallel strands.

    PubMed

    Grimau, Marta G; Aviñó, Anna; Gargallo, Raimundo; Eritja, Ramon

    2005-02-01

    Cyclic oligonucleotides carrying an oligopurine Watson-Crick sequence linked to the corresponding (G,A)- and (G,T)-antiparallel strands were prepared by nonenzymatic template-assisted cyclization of phosphorylated precursors. Cyclization was attempted using 3'-phosphate and 5'-phosphate linear precursors with carbodiimide or BrCN activation. The best results were obtained with the 5'-phosphorylated precursors and carbodiimide activation. Cyclic oligonucleotides bind polypyrimidine target sequence by formation of antiparallel triplexes. We have used UV and circular dichroism (CD) spectroscopy to analyze triplexes formed by cyclic oligonucleotides carrying G and A in the reverse-Hoogsteen strand. The relative stability of the triplexes formed by cyclic and linear oligonucleotides with a common polypyrimidine target was determined by melting experiments. The most-stable triplexes were formed by the cyclic oligonucleotide, followed by the unphosphorylated and phosphorylated oligonucleotide precursors, and, finally, the corresponding hairpin. Although the differences in binding affinity between cyclic oligonucleotides and their corresponding linear precursors are small, the use of cyclic oligonucleotides offers a clear advantage over conventional duplex recognition.

  1. Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide.

    PubMed

    Das, Ushati; Wang, Li Kai; Smith, Paul; Munir, Annum; Shuman, Stewart

    2014-12-01

    Clostridium thermocellum polynucleotide kinase (CthPnk), the 5'-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5'-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg(2+) and a 5'-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2'-hydroxyls of the 5' terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5'-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg(2+) and a 5'-OH DNA yielded a mixed substrate-product complex with GTP-Mg(2+) and 5'-PO4 DNA, wherein the product 5' phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed. PMID:25266383

  2. Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

    PubMed Central

    Grzybowski, J; Will, D W; Randall, R E; Smith, C A; Brown, T

    1993-01-01

    A series of non-nucleoside-based 2,4-dinitrophenyl (DNP) phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid-phase synthesis. The length of spacer arm between the DNP label and the oligonucleotide phosphate backbone, and the number of attached DNP groups have both been varied in order to determine the optimum conditions for anti-DNP antibody binding. Detection using enzyme-linked colorimetric techniques showed sensitivity equivalent to that obtainable using biotinylated oligonucleotides. Images PMID:8493087

  3. The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Zieboll, Gerhard; Orgel, Leslie E.

    1994-01-01

    We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

  4. Template switching between PNA and RNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1995-01-01

    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  5. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    SciTech Connect

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

  6. Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer.

    PubMed

    Jarczewska, Marta; Ziółkowski, Robert; Górski, Łukasz; Malinowska, Elżbieta

    2014-04-01

    The present study aims at the further development of the uranyl oligonucleotide-based voltammetric biosensor, which takes advantage of strong interaction between UO2(2+) and phosphate DNA backbone. Herein we report the optimization of working parameters of previously elaborated electrochemical DNA biosensor. It is shown that the sensor sensitivity is highly dependent on the oligonucleotide probe length and the incubation time of sensor in a sample solution. Consequently, the highest sensitivity was obtained for 10-nucleotide sequence and 60 min incubation time. The lower detection limit towards uranyl cation for developed biosensor was 30 nM. The influence of mixed monolayers and the possibility of developing a non-calibration device were also investigated. The selectivity of the proposed biosensor was significantly improved via elimination of adenine nucleobases from the DNA probe. Moreover, the regeneration procedure was elaborated and tested to prolong the use of the same biosensor for 4 subsequent determinations of UO2(2+).

  7. Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes.

    PubMed Central

    Connolly, B A; Rider, P

    1985-01-01

    Oligonucleotides containing a free sulphydryl group at their 5'-termini have been synthesised and further derivatised with thiol specific probes. The nucleotide sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed using the S-triphenylmethyl O-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan (1) ol or 6-mercaptohexan (1) ol. After cleavage from the resin and removal of the phosphate and base protecting groups, this yields an oligonucleotide containing an S-triphenylmethyl group attached to the 5'-phosphate group via a two, three or six carbon chain. The triphenylmethyl group can be readily removed with silver nitrate to give the free thiol. With the three and six carbon chain oligonucleotides, this thiol can be used, at pH 8, for the attachment of thiol specific probes as illustrated by the reaction with fluorescent conjugates of iodoacetates and maleiimides. However, oligonucleotides containing a thiol attached to the 5'-phosphate group via a two carbon chain are unstable at pH 8 decomposing to the free 5'-phosphate and so are unsuitable for further derivatisation. PMID:4011448

  8. Dual isotope labeling: conjugation of 32P-oligonucleotides with 18F-aryltrifluoroborate via copper(I) catalyzed cycloaddition.

    PubMed

    Li, Ying; Schaffer, Paul; Perrin, David M

    2013-12-01

    A one-pot-two-step labeling of an oligonucleotide with an (18)F-ArBF3(-)(aryltrifluoroborate) radioprosthetic is reported herein. In order to characterize labeling in terms of radiochemistry, phosphorus-32 was also introduced to the 5'-terminus of the oligonucleotide via enzymatic phosphorylation. A pendant azide group was subsequently conjugated to the 5'-phosphate of the oligonucleotide. Copper(I) catalyzed [2+3] cycloaddition was undertaken to conjugate an alkyne-bearing(18)F-ArBF3(-) to the oligonucleotide. Following polyacrylamide gel electrophoresis, this doubly-labeled bioconjugate exhibited decay properties of both the phosphorus-32 and fluorine-18, that were confirmed by autoradiography at selected lengths of time, which in turn provided concrete evidence of successful conjugation. These results are corroborated by HPLC analysis of the labeled material. Taken together this work demonstrates viable use of (18)F-ArBF3(-) prosthetics for labeling oligonucleotides for use in PET imaging. PMID:24144852

  9. Thermodynamics of Oligonucleotide Duplex Melting

    NASA Astrophysics Data System (ADS)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-05-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply rigorous thermodynamic analysis to an important biochemical problem. Because the stacking of base pairs on top of one another is a significant factor in the energetics of oligonucleotide melting, several investigators have applied van't Hoff analysis to melting temperature data using a nearest-neighbor model and have obtained entropies and enthalpies for the stacking of bases. The present article explains how the equilibrium constant for the dissociation of strands from double-stranded oligonucleotides can be expressed in terms of the total strand concentration and thus how the total strand concentration influences the melting temperature. It also presents a simplified analysis based on the entropies and enthalpies of stacking that is manually tractable so that students can work examples to help them understand the thermodynamics of oligonucleotide melting.

  10. Thermodynamics of Oligonucleotide Duplex Melting

    ERIC Educational Resources Information Center

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  11. Acceptors in ZnO

    SciTech Connect

    Mccluskey, Matthew D.; Corolewski, Caleb; Lv, Jinpeng; Tarun, Marianne C.; Teklemichael, Samuel T.; Walter, Eric D.; Norton, M. G.; Harrison, Kale W.; Ha, Su Y.

    2015-03-21

    Zinc oxide (ZnO) has potential for a range of applications in the area of optoelectronics. The quest for p-type ZnO has focused much attention on acceptors. In this paper, Cu, N, and Li acceptor impurities are discussed. Experimental evidence shows that these point defects have acceptor levels 3.2, 1.5, and 0.8 eV above the valence-band maximum, respectively. The levels are deep because the ZnO valence band is quite low compared to conventional, non-oxide semiconductors. Using MoO2 contacts, the electrical resistivity of ZnO:Li was measured and showed behavior consistent with bulk hole conduction for temperatures above 400 K. A photoluminescence peak in ZnO nanocrystals has been attributed to an acceptor, which may involve a zinc vacancy. High field (W-band) electron paramagnetic resonance measurements on the nanocrystals revealed an axial center with g = 2.0033 and g = 2.0075, along with an isotropic center at g = 2.0053.

  12. Acceptors in ZnO

    SciTech Connect

    McCluskey, Matthew D. Corolewski, Caleb D.; Lv, Jinpeng; Tarun, Marianne C.; Teklemichael, Samuel T.; Walter, Eric D.; Norton, M. Grant; Harrison, Kale W.; Ha, Su

    2015-03-21

    Zinc oxide (ZnO) has potential for a range of applications in the area of optoelectronics. The quest for p-type ZnO has focused much attention on acceptors. In this paper, Cu, N, and Li acceptor impurities are discussed. Experimental evidence indicates these point defects have acceptor levels 3.2, 1.4, and 0.8 eV above the valence-band maximum, respectively. The levels are deep because the ZnO valence band is quite low compared to conventional, non-oxide semiconductors. Using MoO{sub 2} contacts, the electrical resistivity of ZnO:Li was measured and showed behavior consistent with bulk hole conduction for temperatures above 400 K. A photoluminescence peak in ZnO nanocrystals is attributed to an acceptor, which may involve a Zn vacancy. High field (W-band) electron paramagnetic resonance measurements on the nanocrystals revealed an axial center with g{sub ⊥} = 2.0015 and g{sub //} = 2.0056, along with an isotropic center at g = 2.0035.

  13. Fluorescent properties of oligonucleotide-conjugated thiazole orange probes.

    PubMed

    Privat, Eric; Melvin, Tracy; Mérola, Fabienne; Schweizer, Gerd; Prodhomme, Sylvie; Asseline, Ulysse; Vigny, Paul

    2002-03-01

    The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (phifl) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT15CAC3']); (phifl values of between 0.06-0.35 were determined for the double-stranded conjugates. The phifl value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA15.d5'[CACT15CAC3'] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher phifl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl

  14. [Study toward practical use of oligonucleotide therapeutics].

    PubMed

    Inoue, Takao; Yoshida, Tokuyuki

    2014-01-01

    Over the past decade, oligonucleotide-based therapeutics such as antisense oligonucleotides and small interfering RNAs (siRNAs) have been developed extensively. For example, mipomersen (Kynamro; ISIS Pharmaceuticals), which is a second-generation antisense oligonucleotide administered by subcutaneous injection, has recently been approved by the FDA for the treatment of homozygous familial hypercholesterolemia. On the other hands, methods for the evaluation of quality, efficacy and safety of oligonucleotide therapeutics have not been fully discussed. Furthermore, the regulatory guidance specific for oligonucleotide therapeutics has not been established yet. Under these circumstances, we started to collaborate with Osaka University and PMDA to discuss regulatory science focused on oligonucleotide therapeutics. Through the collaboration, we would like to propose the possible design of quality evaluation and preclinical safety-evaluation of oligonucleotide therapeutics. PMID:25707197

  15. The synthesis of oligonucleotides containing a primary amino group at the 5'-terminus.

    PubMed Central

    Connolly, B A

    1987-01-01

    Oligonucleotides containing a primary amino group at their 5'-termini have been prepared and further derivatised with amino specific probes. The sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed with N-monomethoxytrityl-0-methoxydiisopropylaminophosphinyl 3-aminopropan(1)ol. After cleavage from the resin, removal of the phosphate and base protecting groups and purification gives a monomethoxytrityl-NH(CH2)3PO4-oligomer. The monomethoxytrityl group can be removed with acetic acid to give the desired amino containing oligomer. The amino group can be further derivatised with amino specific probes yielding fluorescent or biotinylated oligonucleotide products. PMID:3562247

  16. Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1.

    PubMed

    Wu, Chao; Cheng, Yuan-Yuan; Li, Bing-Bing; Li, Wen-Wei; Li, Dao-Bo; Yu, Han-Qing

    2013-05-01

    Shewanella oneidensis MR-1 is an extensively studied dissimilatory metal-reducing bacterium with a great potential for bioremediation and electricity generation. It secretes flavins as electron shuttles which play an important role in extracellular electron transfer. However, the influence of various environmental factors on the secretion of flavins is largely unknown. Here, the effects of electron acceptors, including fumarate, ferrihydrite, Fe(III)-nitrilotriacetic acid (NTA), nitrate and trimethylamine oxide (TMAO), on the secretion of flavins were investigated. The level of riboflavin and riboflavin-5'-phosphate (FMN) secreted by S. oneidensis MR-1 varied considerably with different electron acceptors. While nitrate and ferrihydrite suppressed the secretion of flavins in relative to fumarate, Fe(III)-NTA and TMAO promoted such a secretion and greatly enhanced ferrihydrite reduction and electricity generation. This work clearly demonstrates that electron acceptors could considerably affect the secretion of flavins and consequent microbial EET. Such impacts of electron acceptors in the environment deserve more attention.

  17. Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1.

    PubMed

    Wu, Chao; Cheng, Yuan-Yuan; Li, Bing-Bing; Li, Wen-Wei; Li, Dao-Bo; Yu, Han-Qing

    2013-05-01

    Shewanella oneidensis MR-1 is an extensively studied dissimilatory metal-reducing bacterium with a great potential for bioremediation and electricity generation. It secretes flavins as electron shuttles which play an important role in extracellular electron transfer. However, the influence of various environmental factors on the secretion of flavins is largely unknown. Here, the effects of electron acceptors, including fumarate, ferrihydrite, Fe(III)-nitrilotriacetic acid (NTA), nitrate and trimethylamine oxide (TMAO), on the secretion of flavins were investigated. The level of riboflavin and riboflavin-5'-phosphate (FMN) secreted by S. oneidensis MR-1 varied considerably with different electron acceptors. While nitrate and ferrihydrite suppressed the secretion of flavins in relative to fumarate, Fe(III)-NTA and TMAO promoted such a secretion and greatly enhanced ferrihydrite reduction and electricity generation. This work clearly demonstrates that electron acceptors could considerably affect the secretion of flavins and consequent microbial EET. Such impacts of electron acceptors in the environment deserve more attention. PMID:23558182

  18. Phosphate salts

    MedlinePlus

    ... taken by mouth or used as enemas. Indigestion. Aluminum phosphate and calcium phosphate are FDA-permitted ingredients ... Phosphate salts containing sodium, potassium, aluminum, or calcium are LIKELY SAFE for most people when taken by mouth short-term, when sodium phosphate is inserted into the ...

  19. Biopolymer synthesis on polypropylene supports. I. Oligonucleotides.

    PubMed

    Matson, R S; Rampal, J B; Coassin, P J

    1994-03-01

    We have modified polypropylene to serve as a new solid-phase support for oligonucleotide synthesis. The plastic is first surface aminated by exposure to an ammonia plasma generated by radiofrequency plasma discharge. The aminated polypropylene has been found to be useful as a support for the in situ synthesis of oligonucleotides from monomers. Furthermore, oligonucleotides synthesized on the surface of the plastic remain attached following deprotection and can be used directly for hybridization. PMID:8203760

  20. The prebiotic synthesis of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Oro, J.; Stephen-Sherwood, E.

    1974-01-01

    This paper is primarily a review of recent developments in the abiotic synthesis of nucleotides, short chain oligonucleotides, and their mode of replication in solution. It also presents preliminary results from this laboratory on the prebiotic synthesis of thymidine oligodeoxynucleotides. A discussion, based on the physicochemical properties of RNA and DNA oligomers, relevant to the molecular evolution of these compounds leads to the tentative hypothesis that oligodeoxyribonucleotides of about 12 units may have been of sufficient length to initiate a self replicating coding system. Two models are suggested to account for the synthesis of high molecular weight oligomers using short chain templates and primers.

  1. Inhibition of pancreatic ribonuclease by 2'-5' and 3'-5' oligonucleotides.

    PubMed

    White, M D; Bauer, S; Lapidot, Y

    1977-09-01

    Forty different oligonucleotides were investigated as possible inhibitors of the depolymerizing activity of RNase A. The strongest inhibitors among the diribonucleoside 2'-5' mono- phosphates were: G2'-5'G, C2'-5'G and U2'-5'G, and among the diribonucleoside 3'-5' monophosphates: ApU, ApC and GpU. Of the eight trinucleotides investigated, ApApUp, ApApCp and ApGpUp were the strongest inhibitors. All four dinucleotides studied (ApUp, ApCp, GpUp and GpCp) were very strong inhibitors, ApUp being the strongest one. The results show that the nature of the various bases in the oligonucleotide has an effect on the degree of inhibition, and that the 3' phosphomonoester group increases the binding of the oligonucleotide to RNase A. These inhibitors can be used in physicochemical and biochemical studies of ribonuclease.

  2. Cellular Uptake and Intracellular Trafficking of Oligonucleotides: Implications for Oligonucleotide Pharmacology

    PubMed Central

    Ming, Xin; Carver, Kyle; Laing, Brian

    2014-01-01

    One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed. PMID:24383421

  3. Oligonucleotide therapeutics: chemistry, delivery and clinical progress.

    PubMed

    Sharma, Vivek K; Watts, Jonathan K

    2015-01-01

    Oligonucleotide therapeutics have the potential to become a third pillar of drug development after small molecules and protein therapeutics. However, the three approved oligonucleotide drugs over the past 17 years have not proven to be highly successful in a commercial sense. These trailblazer drugs have nonetheless laid the foundations for entire classes of drug candidates to follow. This review will examine further advances in chemistry that are earlier in the pipeline of oligonucleotide drug candidates. Finally, we consider the possible effect of delivery systems that may provide extra footholds to improve the potency and specificity of oligonucleotide drugs. Our overview focuses on strategies to imbue antisense oligonucleotides with more drug-like properties and their applicability to other nucleic acid therapeutics.

  4. Electron Transfer Dissociation of Oligonucleotide Cations.

    PubMed

    Smith, Suncerae I; Brodbelt, Jennifer S

    2009-06-01

    Electron transfer dissociation (ETD) of multi-protonated 6 - 20-mer oligonucleotides and 12- and 14-mer duplexes is compared to collision activated dissociation (CAD). ETD causes efficient charge reduction of the multi-protonated oligonucleotides in addition to limited backbone cleavages to yield sequence ions of low abundance. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), results in rich fragmentation in terms of w, a, z, and d products, with a marked decrease in the abundance of base loss ions and internal fragments. Complete sequencing was possible for nearly all oligonucleotides studied. ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages, with conservation of weaker non-covalent bonds. PMID:20161288

  5. Synthesis of 5'-Aldehyde Oligonucleotide.

    PubMed

    Lartia, Rémy

    2016-01-01

    Synthesis of oligonucleotide ending with an aldehyde functional group at their 5'-end (5'-AON) is possible for both DNA (5'-AODN) and RNA (5'-AORN) series irrespectively of the nature of the last nucleobase. The 5'-alcohol of on-support ODN is mildly oxidized under Moffat conditions. Transient protection of the resulting aldehyde by N,N'-diphenylethylenediamine derivatives allows cleavage, deprotection, and RP-HPLC purification of the protected 5'-AON. Finally, 5'-AON is deprotected by usual acetic acid treatment. In the aggregates, 5'-AON can be now synthesized and purified as routinely as non-modified ODNs, following procedures similar to the well-known "DMT-On" strategy. PMID:26967469

  6. Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

    NASA Technical Reports Server (NTRS)

    Nealson, K. H.; Moser, D. P.; Saffarini, D. A.

    1995-01-01

    Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.

  7. Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

    PubMed Central

    Barak, Yoram; van Rijn, Jaap

    2000-01-01

    Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H+-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed. PMID

  8. Tandem oligonucleotide synthesis using linker phosphoramidites

    PubMed Central

    Pon, Richard T.; Yu, Shuyuan

    2005-01-01

    Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623–631] is added to the 5′-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3′-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5′- and 3′-terminal OH ends and other oligonucleotides with 5′-phosphorylated and 3′-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated. PMID:15814811

  9. Quantitation of phosphorothioate oligonucleotides in human plasma.

    PubMed

    Leeds, J M; Graham, M J; Truong, L; Cummins, L L

    1996-03-01

    Methods are presented for the extraction of phosphorothioate oligonucleotides from human plasma to permit quantitation by capillary gel electrophoresis. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reverse-phase column to remove salts. A second desalting step, achieved by dialysis utilizing a membrane with a molecular weight cutoff of 2500 Da floating on distilled water, was required to remove residual ionic material from the extracted sample. This method should be generally applicable to the analysis and quantitation of phosphorothioate oligonucleotides. PMID:8850544

  10. Highly parallel microbial diagnostics using oligonucleotide microarrays.

    PubMed

    Loy, Alexander; Bodrossy, Levente

    2006-01-01

    Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simultaneous identification of many different microorganisms and viruses in a single assay. In the past few years, researchers have been confronted with a dramatic increase in the number of studies reporting development and/or improvement of oligonucleotide microarrays for microbial diagnostics, but use of the technology in routine diagnostics is still constrained by a variety of factors. Careful development of microarray essentials (such as oligonucleotide probes, protocols for target preparation and hybridization, etc.) combined with extensive performance testing are thus mandatory requirements for the maturation of diagnostic microarrays from fancy technological gimmicks to robust and routinely applicable tools.

  11. Structural basis for phosphatidylinositol-phosphate biosynthesis

    NASA Astrophysics Data System (ADS)

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-10-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

  12. Structural basis for phosphatidylinositol-phosphate biosynthesis

    PubMed Central

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-01-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis. PMID:26510127

  13. DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

    NASA Astrophysics Data System (ADS)

    Harper, Brett; Neumann, Elizabeth K.; Solouki, Touradj

    2015-08-01

    Collision-induced dissociation (CID) of m/z-isolated w type fragment ions and an intact 5' phosphorylated DNA oligonucleotide generated rearranged product ions. Of the 21 studied w ions of various nucleotide sequences, fragment ion sizes, and charge states, 18 (~86%) generated rearranged product ions upon CID in a Synapt G2-S HDMS (Waters Corporation, Manchester, England, UK) ion mobility-mass spectrometer. Mass spectrometry (MS), ion mobility spectrometry (IMS), and theoretical modeling data suggest that purine bases can attack the free 5' phosphate group in w type ions and 5' phosphorylated DNA to generate sequence permuted [phosphopurine]- fragment ions. We propose and discuss a potential mechanism for generation of rearranged [phosphopurine]- and complementary y-B type product ions.

  14. Bright Solid-State Emission of Disilane-Bridged Donor-Acceptor-Donor and Acceptor-Donor-Acceptor Chromophores.

    PubMed

    Shimada, Masaki; Tsuchiya, Mizuho; Sakamoto, Ryota; Yamanoi, Yoshinori; Nishibori, Eiji; Sugimoto, Kunihisa; Nishihara, Hiroshi

    2016-02-24

    The development of disilane-bridged donor-acceptor-donor (D-Si-Si-A-Si-Si-D) and acceptor-donor-acceptor (A-Si-Si-D-Si-Si-A) compounds is described. Both types of compound showed strong emission (λem =ca. 500 and ca. 400 nm, respectively) in the solid state with high quantum yields (Φ: up to 0.85). Compound 4 exhibited aggregation-induced emission enhancement in solution. X-ray diffraction revealed that the crystal structures of 2, 4, and 12 had no intermolecular π-π interactions to suppress the nonradiative transition in the solid state.

  15. Pyridine as proton acceptor in the concerted proton electron transfer oxidation of phenol.

    PubMed

    Bonin, Julien; Costentin, Cyrille; Robert, Marc; Savéant, Jean-Michel

    2011-06-01

    Taking pyridine as a prototypal example of biologically important nitrogen bases involved in proton-coupled electron transfers, it is shown with the example of the photochemically triggered oxidation of phenol by Ru(III)(bpy)(3) that this proton acceptor partakes in a concerted pathway whose kinetic characteristics can be extracted from the overall kinetic response. The treatment of these data, implemented by the results of a parallel study carried out in heavy water, allowed the determination of the intrinsic kinetic characteristics of this proton acceptor. Comparison of the reorganization energies and of the pre-exponential factors previously derived for hydrogen phosphate and water (in water) as proton acceptors suggests that, in the case of pyridine, the proton charge is delocalized over a primary shell of water molecules firmly bound to the pyridinium cation. PMID:21499600

  16. Pyridine as proton acceptor in the concerted proton electron transfer oxidation of phenol.

    PubMed

    Bonin, Julien; Costentin, Cyrille; Robert, Marc; Savéant, Jean-Michel

    2011-06-01

    Taking pyridine as a prototypal example of biologically important nitrogen bases involved in proton-coupled electron transfers, it is shown with the example of the photochemically triggered oxidation of phenol by Ru(III)(bpy)(3) that this proton acceptor partakes in a concerted pathway whose kinetic characteristics can be extracted from the overall kinetic response. The treatment of these data, implemented by the results of a parallel study carried out in heavy water, allowed the determination of the intrinsic kinetic characteristics of this proton acceptor. Comparison of the reorganization energies and of the pre-exponential factors previously derived for hydrogen phosphate and water (in water) as proton acceptors suggests that, in the case of pyridine, the proton charge is delocalized over a primary shell of water molecules firmly bound to the pyridinium cation.

  17. Gene Assembly from Chip-Synthesized Oligonucleotides

    PubMed Central

    Eroshenko, Nikolai; Kosuri, Sriram; Marblestone, Adam H; Conway, Nicholas; Church, George M.

    2012-01-01

    De novo synthesis of long double-stranded DNA constructs has a myriad of applications in biology and biological engineering. However, its widespread adoption has been hindered by high costs. Cost can be significantly reduced by using oligonucleotides synthesized on high-density DNA chips. However, most methods for using off-chip DNA for gene synthesis have failed to scale due to the high error rates, low yields, and high chemical complexity of the chip-synthesized oligonucleotides. We have recently demonstrated that some commercial DNA chip manufacturers have improved error rates, and that the issues of chemical complexity and low yields can be solved by using barcoded primers to accurately and efficiently amplify subpools of oligonucleotides. This article includes protocols for computationally designing the DNA chip, amplifying the oligonucleotide subpools, and assembling 500-800 basepair (bp) constructs. PMID:25077042

  18. [Phosphate binders].

    PubMed

    Heeb, Rita M

    2016-06-01

    Phosphate binders to treat hyperphosphataemia are part of the medication regime of every dialysis patient. Phosphate binders are taken with every meal (three times a day). Generally, the medication adherence rates of phosphate binders are very low. This is due to inconveniences like their bad taste or their size which makes them hard to swallow. Also nephrologists have differing opinions on phosphate binders as they are aware of the dialysis patients' difficulties to deal with the amount of drugs they are prescribed. Still, phosphate binders are important drugs which have shown potential in reducing mortality by regulating the level of serum phosphate. In order to improve adherence rates, pharmacists have to advise the patients on these drugs' side effects versus the risks associated with omitting their intake. PMID:27439258

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

    PubMed

    Hatters, D M; Wilson, L; Atcliffe, B W; Mulhern, T D; Guzzo-Pernell, N; Howlett, G J

    2001-07-01

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

  20. Synthetic CO.sub.2 acceptor

    DOEpatents

    Lancet, Michael S.; Curran, George P.

    1981-08-18

    A synthetic CO.sub.2 acceptor consisting essentially of at least one compound selected from the group consisting of calcium oxide and calcium carbonate supported in a refractory carrier matrix, the carrier having the general formula Ca.sub.5 (SiO.sub.4).sub.2 CO.sub.3. A method for producing the synthetic CO.sub.2 acceptor is also disclosed.

  1. Caged oligonucleotides for studying biological systems

    PubMed Central

    Ruble, Brittani K.; Yeldell, Sean B.; Dmochowski, Ivan J.

    2015-01-01

    Light-activated (“caged”) compounds have been widely employed for studying biological processes with high spatial and temporal control. In the past decade, several new approaches for caging the structure and function of DNA and RNA oligonucleotides have been developed. This review focuses on caged oligonucleotides that incorporate site-specifically one or two photocleavable linkers, whose photolysis yields oligonucleotides with dramatic structural and functional changes. This technique has been employed by our laboratory and others to photoregulate gene expression in cells and living organisms, typically using near UV-activated organic chromophores. To improve capabilities for in vivo studies, we harnessed the rich inorganic photochemistry of ruthenium bipyridyl complexes to synthesize Ru-caged morpholino antisense oligonucleotides that remain inactive in zebrafish embryos until uncaged with visible light. Expanding into new caged oligonucleotide applications, our lab has developed Transcriptome In Vivo Analysis (TIVA) technology, which provides the first noninvasive, unbiased method for isolating mRNA from single neurons in brain tissues. TIVA-isolated mRNA can be amplified and then analyzed using next-generation sequencing (RNA-seq). PMID:25865001

  2. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field

    PubMed Central

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds’ vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. PMID:26999445

  3. Preparation and Analysis of Oligonucleotides Containing the C4′-Oxidized Abasic Site and Related Mechanistic Probes

    PubMed Central

    Kim, Jaeseung; Kreller, Cortney R.; Greenberg, Marc M.

    2005-01-01

    The C4′-oxidized abasic site (C4-AP) is produced by a variety of DNA damaging agents. This alkali labile lesion can exist in up to four diastereomeric cyclic forms, in addition to the acyclic keto-aldehyde. Synthetic oligonucleotides containing the lesion were prepared from a stable photochemical precursor. Chemical integrity of the lesion containing oligonucleotides was probed using phosphodiesterase lability. Analysis of the 3′,5′-phosphate diester of the monomeric lesion released from single diastereomers of photolabile precursors by 1H NMR indicates that isomerization of the hemiacetal and/or hemiketal is rapid. The syntheses and characterization of oligonucleotides containing configurationally stable analogues of C4-AP, which serve as mechanistic probes for deciphering the structural basis of the biochemical and biological effects of the C4′-oxidized abasic lesion, are also described. PMID:16277338

  4. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    PubMed

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  5. Design and applications of modified oligonucleotides.

    PubMed

    Gallo, M; Montserrat, J M; Iribarren, A M

    2003-02-01

    Oligonucleotides have a wide range of applications in fields such as biotechnology, molecular biology, diagnosis and therapy. However, the spectrum of uses can be broadened by introducing chemical modifications into their structures. The most prolific field in the search for new oligonucleotide analogs is the antisense strategy, where chemical modifications confer appropriate characteristics such as hybridization, resistance to nucleases, cellular uptake, selectivity and, basically, good pharmacokinetic and pharmacodynamic properties. Combinatorial technology is another research area where oligonucleotides and their analogs are extensively employed. Aptamers, new catalytic ribozymes and deoxyribozymes are RNA or DNA molecules individualized from a randomly synthesized library on the basis of a particular property. They are identified by repeated cycles of selection and amplification, using PCR technologies. Modified nucleotides can be introduced either during the amplification procedure or after selection.

  6. Oligosaccharide synthesis by dextransucrase: new unconventional acceptors.

    PubMed

    Demuth, Kristin; Jördening, Hans Joachim; Buchholz, Klaus

    2002-11-01

    The acceptor reactions of dextransucrase offer the potential for a targeted synthesis of a wide range of di-, tri- and higher oligosaccharides by the transfer of a glucosyl group from sucrose to the acceptor. We here report on results which show that the synthetic potential of this enzyme is not restricted to 'normal' saccharides. Additionally functionalized saccharides, such as alditols, aldosuloses, sugar acids, alkyl saccharides, and glycals, and rather unconventional saccharides, such as fructose dianhydride, may also act as acceptors. Some of these acceptors even turned out to be relatively efficient: alpha-D-glucopyranosyl-(1-->5)-D-arabinonic acid, alpha-D-glucopyranosyl-(1-->4)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-mannitol, alpha-D-fructofuranosyl-beta-D-fructofuranosyl-(1,2':2,3')-dianhydride, 1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol ('D-glucal'), and may therefore be of interest for future applications of the dextransucrase acceptor reaction.

  7. Oligonucleotide-based therapy for neurodegenerative diseases.

    PubMed

    Magen, Iddo; Hornstein, Eran

    2014-10-10

    Molecular genetics insight into the pathogenesis of several neurodegenerative diseases, such as Alzheimer׳s disease, Parkinson׳s disease, Huntington׳s disease and amyotrophic lateral sclerosis, encourages direct interference with the activity of neurotoxic genes or the molecular activation of neuroprotective pathways. Oligonucleotide-based therapies are recently emerging as an efficient strategy for drug development and these can be employed as new treatments of neurodegenerative states. Here we review advances in this field in recent years which suggest an encouraging assessment that oligonucleotide technologies for targeting of RNAs will enable the development of new therapies and will contribute to preservation of brain integrity.

  8. Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex.

    PubMed

    Sheehan, J P; Lan, H C

    1998-09-01

    Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 microgram/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (<100 microgram/mL) showed a selective, linear prolongation of the activated partial thromboplastin time (PTT). The rate limiting effect of 50 microgram/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clotting assay. Delaying addition of oligonucleotide until after contact activation failed to correct prolongation of the PTT. The calcium-dependent steps of the intrinsic pathway were individually assessed by adding sufficient activated coagulation factor to correct the PTT in plasma deficient in that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 microgram/mL) did not prolong a modified Russel viper venom time, suggesting no significant inhibition of prothrombinase. Thus, 50 microgram/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase activity (to approximately 35% of control) at clinically relevant oligonucleotide

  9. CD studies on ribonuclease A - oligonucleotides interactions.

    PubMed

    White, M D; Keren-Zur, M; Lapidot, Y

    1977-04-01

    The interaction of ApU, Aps4U, Aps4Up, ApAps4Up and Gps4U with RNase A was studied by CD difference spectroscopy. The use of 4-thiouridine (s4U) containing oligonucleotides enables to distinguish between the interaction of the different components of the ligand with the enzyme. The mode of binding of the oligonucleotides to the enzyme is described. From this mode of binding it is explained why Aps4U, for example, inhibits RNase A, while s4UpA serves as a substrate.

  10. Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

    PubMed Central

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

    2013-01-01

    Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

  11. Donor-acceptor heteroleptic open sandwiches.

    PubMed

    Merino, Gabriel; Beltrán, Hiram I; Vela, Alberto

    2006-02-01

    A series of donor-acceptor heteroleptic open sandwiches with formula CpM-M'Pyl (M = B, Al, Ga; M' = Li, Na; Cp = cyclopentadienyl; Pyl = pentadienyl) has been designed in silico using density functional theory. The most stable complexes are those containing boron as a donor atom. A molecular orbital analysis shows that the s character of the lone pair located at the group 13 element is mainly responsible for the complex stabilization. It is also found that the surrounding medium has a similar effect on these sandwiches such as in the "classical" donor-acceptor complexes, showing a decrement in the group 13 element-alkaline metal bond lengths.

  12. Quantitation of a low level coeluting impurity present in a modified oligonucleotide by both LC-MS and NMR.

    PubMed

    Smith, Marco; Beck, Tony

    2016-01-25

    This paper describes the use of two complementary techniques, LC-MS and NMR, to quantify a low level mono phosphate substituted impurity in an oligonucleotide drug substance. This impurity is the result of a sulphurisation failure, leading to the production of a sequence where a phosphorothioate linkage is replaced by a phosphate. Few quantitative methods are possible to analyse these challenging molecules especially if reversed phase ion pair chromatography, one of the most commonly used techniques for the separation of oligonucleotides, is unable to resolve the impurity in question. With the use of a standard addition method it could be demonstrated that both analytical techniques show equivalency and furthermore, the LC-MS method alone with additional validation has the potential to perform this quantitative assay with a high degree of accuracy. PMID:26512997

  13. Oligonucleotide microarrays in constitutional genetic diagnosis.

    PubMed

    Keren, Boris; Le Caignec, Cedric

    2011-06-01

    Oligonucleotide microarrays such as comparative genomic hybridization arrays and SNP microarrays enable the identification of genomic imbalances - also termed copy-number variants - with increasing resolution. This article will focus on the most significant applications of high-throughput oligonucleotide microarrays, both in genetic diagnosis and research. In genetic diagnosis, the method is becoming a standard tool for investigating patients with unexplained developmental delay/intellectual disability, autism spectrum disorders and/or with multiple congenital anomalies. Oligonucleotide microarray have also been recently applied to the detection of genomic imbalances in prenatal diagnosis either to characterize a chromosomal rearrangement that has previously been identified by standard prenatal karyotyping or to detect a cryptic genomic imbalance in a fetus with ultrasound abnormalities and a normal standard prenatal karyotype. In research, oligonucleotide microarrays have been used for a wide range of applications, such as the identification of new genes responsible for monogenic disorders and the association of a copy-number variant as a predisposing factor to a common disease. Despite its widespread use, the interpretation of results is not always straightforward. We will discuss several unexpected results and ethical issues raised by these new methods.

  14. Oligonucleotides direct synthesis on porous silicon chip.

    PubMed

    De Stefano, Luca; De Tommasi, Edoardo; Rea, Ilaria; Rotiroti, Lucia; Giangrande, Luca; Oliviero, Giorgia; Borbone, Nicola; Galeone, Aldo; Piccialli, Gennaro

    2008-01-01

    A solid phase oligonucleotide (ON) synthesis on porous silicon (PSi) chip is presented. The prepared Si-OH surface were analyzed by FT-IR and the OH functions were quantified by reaction with 3'-phosphoramidite nucleotide building block. Short ONs were synthesized on the chip surface and the coupling yields evaluated. PMID:18776583

  15. Liver as a target for oligonucleotide therapeutics.

    PubMed

    Sehgal, Alfica; Vaishnaw, Akshay; Fitzgerald, Kevin

    2013-12-01

    Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be more akin to small molecule therapeutics because they are small,completely synthetic in origin, do not integrate into the host genome,and have a defined duration of therapeutic activity after which effects recover to baseline. They offer a high degree of specificity at the genetic level, thereby reducing off-target effects.At the same time, they provide a strategy for targeting any gene in the genome, including transcripts that produce mutated proteins.Oligonucleotide-based therapeutics include short interfering RNA (siRNA), that degrade target mRNA through RISC mediated RNAi; anti-miRs, that target miRNAs; miRNA mimics, that regulate target mRNA; antisense oligonucleotides, that may be working through RNAseH mediated mRNA decay; mRNA upregulation,by targeting long non-coding RNAs; and oligonucleotides induced alternative splicing [1]. All these approaches require some minimal degree of homology at the nucleic acid sequence level for them to be functional. The different mechanisms of action and their relevant activity are outlined in Fig. 1. Besides homology,RNA secondary structure has also been exploited in the case of ribozymes and aptamers, which act by binding to nucleic acids or proteins, respectively. While there have been many reports of gene knockdown and gene modulation in cell lines and mice with all these methods, very few have advanced to clinical stages.The main obstacle to date has been the safe and effective intracellular delivery of these compounds in higher species, including humans. Indeed, their action requires direct interaction with DNA/RNA within the target cell so even when one solves the issues of tissue and cellular access, intracellular/intranuclear location represents yet another barrier to

  16. Chemosensitization by antisense oligonucleotides targeting MDM2.

    PubMed

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  17. Electron Donor Acceptor Interactions. Final Progress Report

    SciTech Connect

    2002-08-16

    The Gordon Research Conference (GRC) on Electron Donor Acceptor Interactions was held at Salve Regina University, Newport, Rhode Island, 8/11-16/02. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  18. Core-shell nanospheres for oligonucleotide delivery. V: adsorption/release behavior of 'stealth' nanospheres.

    PubMed

    Tondelli, Luisa; Ballestri, Marco; Magnani, Laura; Vivarelli, Daniela; Fini, Adamo; Cerasi, Aurora; Chiarantini, Laura; Sparnacci, Katia; Laus, Michele

    2003-01-01

    The adsorption/release behavior of oligodeoxynucleotides (ODNs) on new PEGylated core-shell polymethylmethacrylate nanospheres is described. The outer shell consists of alkyl chains containing quaternary ammonium groups and of poly(ethylene glycol) chains, both covalently bound to the inner core. Ion pair formation between negatively charged ODN phosphate groups and positively charged groups on the nanosphere surface is the main interaction mechanism. No cellular toxicity in HL60 cells is observed at nanosphere concentrations required for biologically active ODN delivery. These results indicate that these novel cationic polymeric nanoparticles are safe and represent promising vectors for oligonucleotide delivery.

  19. 5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

    NASA Technical Reports Server (NTRS)

    Frank, Natia L.; Meade, Thomas J.

    2003-01-01

    Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

  20. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis

    PubMed Central

    Genz, Maika; Vickers, Clare; van den Bergh, Tom; Joosten, Henk-Jan; Dörr, Mark; Höhne, Matthias; Bornscheuer, Uwe T.

    2015-01-01

    To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5′-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions. PMID:26569229

  1. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis.

    PubMed

    Genz, Maika; Vickers, Clare; van den Bergh, Tom; Joosten, Henk-Jan; Dörr, Mark; Höhne, Matthias; Bornscheuer, Uwe T

    2015-01-01

    To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5'-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions. PMID:26569229

  2. Antisense oligonucleotides, microRNAs, and antibodies.

    PubMed

    Dávalos, Alberto; Chroni, Angeliki

    2015-01-01

    The specificity of Watson-Crick base pairing and the development of several chemical modifications to oligonucleotides have enabled the development of novel drug classes for the treatment of different human diseases. This review focuses on promising results of recent preclinical or clinical studies on targeting HDL metabolism and function by antisense oligonucleotides and miRNA-based therapies. Although many hurdles regarding basic mechanism of action, delivery, specificity, and toxicity need to be overcome, promising results from recent clinical trials and recent approval of these types of therapy to treat dyslipidemia suggest that the treatment of HDL dysfunction will benefit from these unique clinical opportunities. Moreover, an overview of monoclonal antibodies (mAbs) developed for the treatment of dyslipidemia and cardiovascular disease and currently being tested in clinical studies is provided. Initial studies have shown that these compounds are generally safe and well tolerated, but ongoing large clinical studies will assess their long-term safety and efficacy.

  3. Determination of pKa values for deprotonable nucleobases in short model oligonucleotides.

    PubMed

    González-Olvera, Julio C; Martínez-Reyes, José; González-Jasso, Eva; Pless, Reynaldo C

    2015-11-01

    The deprotonation of ionizable nucleobases centrally placed in short model oligonucleotides was examined under different physical conditions, using UV absorption spectroscopy. The oligonucleotide sequences were designed so that only the central base would be ionized over the pH range examined. pKa values of 9.90±0.01 and 9.34±0.04 were determined for the guanine group in the oligomer d-ACAGCAC and 2'-deoxyguanosine, respectively, both at 25°C and 0.1M NaCl. Lengthening the oligonucleotide up to the tridecamer stage further increases the pKa of the central guanine moiety. Electrolyte concentration, temperature, and mixed water-ethanol solvents affect the acidity of the central base. Changes in the sequence surrounding the central guanine can also have a significant effect, especially in the case of strongly stacking sequences. The pKa values were also determined for the hepta(2'-O-methyl)ribonucleotide and the heptamer PNA of identical sequence, as well as for oligodeoxyribonucleotides with different deprotonable bases, viz. thymine, uracil, or hypoxanthine, in the central position. The results are interpreted in terms of the electric-field effect exerted on the departing proton by the negative electric charges located on the internucleotide phosphate groups, and calculations show this effect to approximately explain the magnitude of the pKa difference observed between the deoxyriboheptanucleotide and its electroneutral PNA analogue. PMID:26188860

  4. One-step insertion of oligonucleotide linkers or adapters to DNA using unphosphorylated oligonucleotides.

    PubMed

    Kang, C; Inouye, M

    1993-10-01

    A simple and efficient method was developed for insertion of oligonucleotide sequences into plasmids. In this method, an unphosphorylated oligonucleotide was ligated to the restriction-digested phagemid DNA. Only the single strand of the oligonucleotide was ligated at the 5' end of the phagemid, and this resulted in the creation of a long self-complementary single-strand overhang. These single-strand overhang-possessing phagemids were used to transform XL-1 cells. This simple ligation and transformation reaction rendered approximately 7.5 x 10(4) to 5 x 10(5) of white colonies per microgram DNA from the isopropyl-beta-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside plate. This number is almost the same or even higher than the number of blue colonies from the control reaction in which ligase was used without the oligonucleotide. By this method we could mutate one enzyme site to another or create ribozyme and substrate phagemid very easily. Fidelity of this method was checked by restriction digestion, DNA sequencing and ribozyme reaction. By DNA sequencing, we observed that 100% of the white colonies contained a single oligonucleotide sequence.

  5. Cyanomethylbenzoic acid: an acceptor for donor-π-acceptor chromophores used in dye-sensitized solar cells.

    PubMed

    Xiang, Wanchun; Gupta, Akhil; Kashif, Muhammad Kalim; Duffy, Noel; Bilic, Ante; Evans, Richard A; Spiccia, Leone; Bach, Udo

    2013-02-01

    Sensing the sun: Incorporation of a cyanomethyl benzoic acid electron acceptor into donor-π-acceptor sensitizers for dye-sensitized-solar cell is shown to lead to devices with improved conversion efficiency when compared with more widely used cyanoacetic acid acceptor.

  6. Penetration of oligonucleotides into mouse organism through mucosa and skin.

    PubMed

    Vlassov, V V; Karamyshev, V N; Yakubov, L A

    1993-08-01

    Benzylamide 5'-32P-oligonucleotide derivatives were shown to penetrate into mice organism when administered by various routes; intranasally, per os, intravaginally and per rectum. In all cases, the compounds are rapidly accumulated in blood and guts. Analysis of the radioactive material from blood and pancreas revealed intact oligonucleotides. Although concentrations of oligonucleotides in tissues differ considerably by the various methods of administration, the efficiency of delivery is sufficient to consider all the routes as being of therapeutic value. Dose effect on the efficiency of oligonucleotide penetration into mice suggests the transport to be a saturable process. Application of an oligonucleotide lotion on mice ear helices results in reproducible accumulation of radioactivity in the animal tissues. Effectiveness of oligonucleotide delivery into mouse through skin can be improved by using electrophoretic procedure.

  7. Template-Directed Ligation of Peptides to Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  8. Oligonucleotide frequencies in DNA follow a Yule distribution.

    PubMed

    Martindale, C; Konopka, A K

    1996-03-01

    We show that ranked oligonucleotide frequencies in both protein-coding and non-coding regions from several genomes fit poorly to the Zipf distribution, but that the same frequency data give excellent fit to the Yule distribution. The parameters of the Yule distribution for oligonucleotide frequencies in exons are the same (within error limits) as the parameters for introns. This precludes application of Yule or Zipf distribution of ranked oligonucleotide frequencies to annotating new genomic sequences.

  9. Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor

    NASA Astrophysics Data System (ADS)

    Zhang, Lili; Yu, Huaina; Zhang, Jianwei; Chen, Tongsheng

    2014-06-01

    We report that binomial distribution depending on acceptor photobleaching degree can be used to characterize the proportions of various kinds of FRET (Fluorescence Resonance Energy Transfer) constructs resulted from partial acceptor photobleaching of multiple-acceptors FRET system. On this basis, we set up a rigorous quantitation theory for multiple-acceptors FRET construct named as Mb-PbFRET which is not affected by the imaging conditions and fluorophore properties. We experimentally validate Mb-PbFRET with FRET constructs consisted of one donor and two or three acceptors inside living cells on confocal and wide-field microscopes.

  10. Alkyl Chlorides as Hydrogen Bond Acceptors

    SciTech Connect

    Nadas, Janos I; Vukovic, Sinisa; Hay, Benjamin

    2012-01-01

    To gain an understanding of the role of an alkyl chloride as a hydrogen bond acceptor, geometries and interaction energies were calculated at the MP2/aug-cc-pVDZ level of theory for complexes between ethyl chloride and representative hydrogen donor groups. The results establish that these donors, which include hydrogen cyanide, methanol, nitrobenzene, pyrrole, acetamide, and N-methylurea, form X-H {hor_ellipsis} Cl hydrogen bonds (X = C, N, O) of weak to moderate strength, with {Delta}E values ranging from -2.8 to -5.3 kcal/mol.

  11. Comparative oligonucleotide fingerprints of three plant viroids.

    PubMed Central

    Gross, H J; Domdey, H; Sänger, H L

    1977-01-01

    5' Phosphorylation in vitro with gamma-32P-ATP and T4 phage induced polynucleotide kinase was used to obtain RNAase A and RNAase T1 fingerprints of three plant viroids: Potato spindle tuber viroid from tomato (PSTV-tom), chrysanthemum stunt viroid from cineraria (ChSV-cin) and citrus exocortis viroid from Gynura aurantiaca (CEV-gyn). These three viroids differ significantly from each other as judged from their oligonucleotide patterns. This supports the concept of individual viroid species. Images PMID:896482

  12. Donor/acceptor chromophores-decorated triazolyl unnatural nucleosides: synthesis, photophysical properties and study of interaction with BSA.

    PubMed

    Bag, Subhendu Sekhar; Talukdar, Sangita; Das, Suman Kalyan; Pradhan, Manoj Kumar; Mukherjee, Soumen

    2016-06-14

    Much effort has been put forth to develop unnatural, stable, hydrophobic base pairs with orthogonal recognition properties and study their effect on DNA duplex stabilisation. Our continuous efforts on the design of fluorescent unnatural biomolecular building blocks lead us to the synthesis of some triazolyl donor/acceptor unnatural nucleosides via an azide-alkyne 1,3-dipolar cycloaddition reaction as a key step, which we want to report herein. We have studied their photophysical properties and found interesting solvatochromic fluorescence for two of the nucleosides. Photophysical interactions among two donor-acceptor β-nucleosides as well as a pair of α/β-nucleosides have also been evaluated. Furthermore, we have exploited one of the fluorescent nucleosides in studying its interaction with BSA with the help of UV-visible and steady state fluorescence techniques. Our design concept is based on the hypothesis that a pair of such donor/acceptor nucleosides might be involved in π-stacking as well as in photophysical interactions, leading to stabilization of the DNA duplex if such nucleosides can be incorporated into short oligonucleotide sequences. Therefore, the designed bases may find application in biophysical studies in the context of DNA. PMID:27181694

  13. An imputation approach for oligonucleotide microarrays.

    PubMed

    Li, Ming; Wen, Yalu; Lu, Qing; Fu, Wenjiang J

    2013-01-01

    Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as "bright spots", "dark clouds", and "shadowy circles", etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.

  14. BIOCONJUGATION OF OLIGONUCLEOTIDES FOR TREATING LIVER FIBROSIS

    PubMed Central

    Ye, Zhaoyang; Hajj Houssein, Houssam S.; Mahato, Ram I.

    2009-01-01

    Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is in urgent need to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remains the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of α1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed. PMID:18154454

  15. Electromigration of microbial electron acceptors and nutrients: (I) transport in synthetic media.

    PubMed

    Lohner, Svenja T; Katzoreck, Daniel; Tiehm, Andreas

    2008-07-01

    Microbiological cleanup is a widely used in situ remediation strategy for organic soil and groundwater contaminations. However, often the availability of electron acceptors and nutrients are limiting factors for microbial pollutant degradation in the field. Electromigration represents a new approach for the transport of microbiological agents in soil. In this study, the electrokinetic transport of the microbial electron acceptors nitrate and sulfate and of the nutrients ammonium and phosphate was compared. All experiments were performed under standardized conditions, i.e. with constant voltage in demineralized water and a model sandy soil. Average transport rates for nitrate, sulfate, poly-phosphate, and ammonium were 1.34 cm/h, 1.91 cm/h, 0.48 cm/h, and 0.40 cm/h, respectively, in single compound studies. Transport velocities were dependent on applied voltage gradient but not on the initial ion concentration. Additionally, electrokinetic transport was studied with ion mixtures. The ion distribution in the soil was significantly influenced by the pH profile and the associated voltage gradient.

  16. The reaction of choline dehydrogenase with some electron acceptors.

    PubMed Central

    Barrett, M C; Dawson, A P

    1975-01-01

    1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme. PMID:1218095

  17. The reaction of choline dehydrogenase with some electron acceptors.

    PubMed

    Barrett, M C; Dawson, A P

    1975-12-01

    1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme.

  18. Electronic structure of acceptor-donor complexes in silicon

    NASA Astrophysics Data System (ADS)

    Atoro, E.; Ohama, Y.; Hayafuji, Y.

    2003-10-01

    The electronic structure of trimer acceptor-donor complexes in silicon Si clusters is studied using the ab initio discrete variational-Xα molecular-orbital (MO) method. The trimer complexes In2D (D=phosphorus P, arsenic As, antimony Sb, or bismuth Bi) consist of two indium In acceptor elements and a centered donor element D from the group V elements. Calculations are performed under the assumption that the three atoms are arranged in the nearest neighbor substitutional trimer configuration. Results indicate that the trimer complexes act as shallower acceptors having smaller ionization activation energies than In acceptor. The potential of In2D as an acceptor in Si is then discussed and In2D is proposed as a promising acceptor for the formation of channels and source/drains in ultralarge scaled integration.

  19. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  20. Disulfide-linked oligonucleotide phosphorothioates - Novel analogues of nucleic acids

    NASA Technical Reports Server (NTRS)

    Wu, Taifeng; Orgel, Leslie E.

    1991-01-01

    The synthesis of phosphorothioate analogs of oligonucleotides by the oxidation of deoxyadenosine 3',5'-bisphosphorothioate (3) was attempted. Cyclization of 3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5'-terminal phosphorotioate group undergoes efficient chain-extension when oxidized in the presence of 3.

  1. Enzymatic Synthesis of Single-Stranded Clonal Pure Oligonucleotides.

    PubMed

    Ducani, Cosimo; Högberg, Björn

    2017-01-01

    Single-stranded oligonucleotides, or oligodeoxyribonucleotides (ODNs), are very important in several fields of science such as molecular biology, diagnostics, nanotechnology, and gene therapy. They are usually chemically synthesized. Here we describe an enzymatic method which enables us to synthesize pure oligonucleotides which can be up to several hundred long bases. PMID:27671934

  2. Antisense oligonucleotides: is the glass half full or half empty?

    PubMed

    Bennett, C F

    1998-01-01

    Antisense oligonucleotides are widely used as tools to explore the pharmacological effects of inhibiting expression of a selected gene product. In addition, they are being investigated as therapeutic agents for the treatment of viral infections, cancers, and inflammatory disorders. Proof that the pharmacological effects produced by the oligonucleotides are attributable to an antisense mechanism of action requires careful experimentation. Central to this problem is the finding that oligonucleotides are capable of interacting with and modulating function of specific proteins in both a sequence-independent and -dependent manner. Despite these undesired interactions, it has been possible to demonstrate that oligonucleotides are capable of binding to a specific RNA in cultured cells, or within tissues, resulting in selective reduction of the targeted gene product and pharmacological activity. In general, these oligonucleotides were identified after a selection process in which multiple oligonucleotides targeting different regions on the RNA were evaluated for direct inhibition of targeted gene product, resulting in the identification of a potent and selective oligonucleotide. Similar to other drug-receptor interactions, selection of the most potent inhibitor results in an increase in the signal-to-noise ratio, yielding increased confidence that activity observed is the result of a desired effect of the inhibitor. With careful selection, proper controls, and careful dose-response curves it is possible to utilize antisense oligonucleotides as effective research tools and potentially as therapeutic agents. PMID:9413924

  3. 2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

    PubMed

    Griffey, R H; Monia, B P; Cummins, L L; Freier, S; Greig, M J; Guinosso, C J; Lesnik, E; Manalili, S M; Mohan, V; Owens, S; Ross, B R; Sasmor, H; Wancewicz, E; Weiler, K; Wheeler, P D; Cook, P D

    1996-12-20

    Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

  4. Predicting oligonucleotide-directed mutagenesis failures in protein engineering.

    PubMed

    Wassman, Christopher D; Tam, Phillip Y; Lathrop, Richard H; Weiss, Gregory A

    2004-01-01

    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed 'cross-hybridization', as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries.

  5. Noncoding oligonucleotides: the belle of the ball in gene therapy.

    PubMed

    Shum, Ka-To; Rossi, John J

    2015-01-01

    Gene therapy carries the promise of cures for many diseases based on manipulating the expression of a person's genes toward the therapeutic goal. The relevance of noncoding oligonucleotides to human disease is attracting widespread attention. Noncoding oligonucleotides are not only involved in gene regulation, but can also be modified into therapeutic tools. There are many strategies that leverage noncoding oligonucleotides for gene therapy, including small interfering RNAs, antisense oligonucleotides, aptamers, ribozymes, decoys, and bacteriophage phi 29 RNAs. In this chapter, we will provide a broad, comprehensive overview of gene therapies that use noncoding oligonucleotides for disease treatment. The mechanism and development of each therapeutic will be described, with a particular focus on its clinical development. Finally, we will discuss the challenges associated with developing nucleic acid therapeutics and the prospects for future success.

  6. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  7. Quantum computing with acceptor spins in silicon.

    PubMed

    Salfi, Joe; Tong, Mengyang; Rogge, Sven; Culcer, Dimitrie

    2016-06-17

    The states of a boron acceptor near a Si/SiO2 interface, which bind two low-energy Kramers pairs, have exceptional properties for encoding quantum information and, with the aid of strain, both heavy hole and light hole-based spin qubits can be designed. Whereas a light-hole spin qubit was introduced recently (arXiv:1508.04259), here we present analytical and numerical results proving that a heavy-hole spin qubit can be reliably initialised, rotated and entangled by electrical means alone. This is due to strong Rashba-like spin-orbit interaction terms enabled by the interface inversion asymmetry. Single qubit rotations rely on electric-dipole spin resonance (EDSR), which is strongly enhanced by interface-induced spin-orbit terms. Entanglement can be accomplished by Coulomb exchange, coupling to a resonator, or spin-orbit induced dipole-dipole interactions. By analysing the qubit sensitivity to charge noise, we demonstrate that interface-induced spin-orbit terms are responsible for sweet spots in the dephasing time [Formula: see text] as a function of the top gate electric field, which are close to maxima in the EDSR strength, where the EDSR gate has high fidelity. We show that both qubits can be described using the same starting Hamiltonian, and by comparing their properties we show that the complex interplay of bulk and interface-induced spin-orbit terms allows a high degree of electrical control and makes acceptors potential candidates for scalable quantum computation in Si. PMID:27171901

  8. Quantum computing with acceptor spins in silicon.

    PubMed

    Salfi, Joe; Tong, Mengyang; Rogge, Sven; Culcer, Dimitrie

    2016-06-17

    The states of a boron acceptor near a Si/SiO2 interface, which bind two low-energy Kramers pairs, have exceptional properties for encoding quantum information and, with the aid of strain, both heavy hole and light hole-based spin qubits can be designed. Whereas a light-hole spin qubit was introduced recently (arXiv:1508.04259), here we present analytical and numerical results proving that a heavy-hole spin qubit can be reliably initialised, rotated and entangled by electrical means alone. This is due to strong Rashba-like spin-orbit interaction terms enabled by the interface inversion asymmetry. Single qubit rotations rely on electric-dipole spin resonance (EDSR), which is strongly enhanced by interface-induced spin-orbit terms. Entanglement can be accomplished by Coulomb exchange, coupling to a resonator, or spin-orbit induced dipole-dipole interactions. By analysing the qubit sensitivity to charge noise, we demonstrate that interface-induced spin-orbit terms are responsible for sweet spots in the dephasing time [Formula: see text] as a function of the top gate electric field, which are close to maxima in the EDSR strength, where the EDSR gate has high fidelity. We show that both qubits can be described using the same starting Hamiltonian, and by comparing their properties we show that the complex interplay of bulk and interface-induced spin-orbit terms allows a high degree of electrical control and makes acceptors potential candidates for scalable quantum computation in Si.

  9. Quantum computing with acceptor spins in silicon

    NASA Astrophysics Data System (ADS)

    Salfi, Joe; Tong, Mengyang; Rogge, Sven; Culcer, Dimitrie

    2016-06-01

    The states of a boron acceptor near a Si/SiO2 interface, which bind two low-energy Kramers pairs, have exceptional properties for encoding quantum information and, with the aid of strain, both heavy hole and light hole-based spin qubits can be designed. Whereas a light-hole spin qubit was introduced recently (arXiv:1508.04259), here we present analytical and numerical results proving that a heavy-hole spin qubit can be reliably initialised, rotated and entangled by electrical means alone. This is due to strong Rashba-like spin-orbit interaction terms enabled by the interface inversion asymmetry. Single qubit rotations rely on electric-dipole spin resonance (EDSR), which is strongly enhanced by interface-induced spin-orbit terms. Entanglement can be accomplished by Coulomb exchange, coupling to a resonator, or spin-orbit induced dipole-dipole interactions. By analysing the qubit sensitivity to charge noise, we demonstrate that interface-induced spin-orbit terms are responsible for sweet spots in the dephasing time {T}2* as a function of the top gate electric field, which are close to maxima in the EDSR strength, where the EDSR gate has high fidelity. We show that both qubits can be described using the same starting Hamiltonian, and by comparing their properties we show that the complex interplay of bulk and interface-induced spin-orbit terms allows a high degree of electrical control and makes acceptors potential candidates for scalable quantum computation in Si.

  10. Comparative gene expression profiling by oligonucleotide fingerprinting.

    PubMed Central

    Meier-Ewert, S; Lange, J; Gerst, H; Herwig, R; Schmitt, A; Freund, J; Elge, T; Mott, R; Herrmann, B; Lehrach, H

    1998-01-01

    The use of hybridisation of synthetic oligonucleotides to cDNAs under high stringency to characterise gene sequences has been demonstrated by a number of groups. We have used two cDNA libraries of 9 and 12 day mouse embryos (24 133 and 34 783 clones respectively) in a pilot study to characterise expressed genes by hybridisation with 110 hybridisation probes. We have identified 33 369 clusters of cDNA clones, that ranged in representation from 1 to 487 copies (0.7%). 737 were assigned to known rodent genes, and a further 13 845 showed significant homologies. A total of 404 clusters were identified as significantly differentially represented (P < 0.01) between the two cDNA libraries. This study demonstrates the utility of the fingerprinting approach for the generation of comparative gene expression profiles through the analysis of cDNAs derived from different biological materials. PMID:9547283

  11. New acceptor-donor-acceptor (A-D-A) type copolymers for efficient organic photovoltaic devices

    NASA Astrophysics Data System (ADS)

    Ghomrasni, S.; Ayachi, S.; Alimi, K.

    2015-01-01

    Three new conjugated systems alternating acceptor-donor-acceptor (A-D-A) type copolymers have been investigated by means of Density Functional Theory (DFT) and Time-Dependent DFT (TD-DFT) at the 6-31g (d) level of theory. 4,4‧-Dimethoxy-chalcone, also called the 1,3-bis(4-methoxyphenyl)prop-2-en-1-one (BMP), has been used as a common acceptor moiety. It forced intra-molecular S⋯O interactions through alternating oligo-thiophene derivatives: 4-AlkylThiophenes (4-ATP), 4-AlkylBithiophenes (4-ABTP) and 4-Thienylene Vinylene (4-TEV) as donor moieties. The band gap, HOMO and LUMO electron distributions as well as optical properties were analyzed for each molecule. The fully optimized resulting copolymers showed low band gaps (2.2-2.8 eV) and deep HOMO energy levels ranging from -4.66 to -4.86 eV. A broad absorption [300-900 nm] covering the solar spectrum and absorption maxima ranges from 486 to 604 nm. In addition, organic photovoltaic cells (OPCs) based on alternating copolymers in bulk heterojunction (BHJ) composites with the 1-(3-methoxycarbonyl) propyl-1-phenyl-[6,6]-C61 (PCBM), as an acceptor, have been optimized. Thus, the band gap decreased to 1.62 eV, the power conversion efficiencies (PCEs) were about 3-5% and the open circuit voltage Voc of the resulting molecules decreased from 1.50 to 1.27 eV.

  12. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    SciTech Connect

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  13. Design and analysis of mismatch probes for long oligonucleotide microarrays

    SciTech Connect

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  14. Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals.

    PubMed

    Seth, Punit P; Jazayeri, Ali; Yu, Jeff; Allerson, Charles R; Bhat, Balkrishen; Swayze, Eric E

    2012-01-01

    We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012. PMID:23344239

  15. Aromatic donor-acceptor interactions in non-polar environments.

    PubMed

    Prentice, Giles M; Pascu, Sofia I; Filip, Sorin V; West, Kevin R; Pantoş, G Dan

    2015-05-14

    We have evaluated the strength of aromatic donor-acceptor interactions between dialkyl naphthalenediimide and dialkoxynaphthalene in non-polar environments. (1)H NMR, UV-vis spectroscopy and isothermal titration calorimetry were used to characterise this interaction. We concluded that the strength of donor-acceptor interactions in heptane is sufficient to drive supramolecular assemblies in this and other aliphatic solvents. PMID:25875729

  16. Acceptor impurity activation in III-nitride light emitting diodes

    SciTech Connect

    Römer, Friedhard Witzigmann, Bernd

    2015-01-12

    In this work, the role of the acceptor doping and the acceptor activation and its impact on the internal quantum efficiency (IQE) of a Gallium Nitride (GaN) based multi-quantum well light emitting diode is studied by microscopic simulation. Acceptor impurities in GaN are subject to a high activation energy which depends on the presence of proximate dopant atoms and the electric field. A combined model for the dopant ionization and activation barrier reduction has been developed and implemented in a semiconductor carrier transport simulator. By model calculations, we demonstrate the impact of the acceptor activation mechanisms on the decay of the IQE at high current densities, which is known as the efficiency droop. A major contributor to the droop is the electron leakage which is largely affected by the acceptor doping.

  17. Method for the preparation of size marker for synthetic oligonucleotides

    SciTech Connect

    Jing, G.Z.; Liu, A.; Leung, W.C.

    1986-01-01

    Terminal deoxynucleotidyltransferase was used for the addition of (..cap alpha..-/sup 32/P)dCTP to the 3'-OH termini of oligo(dT)/sub 12-18/. A collection of oligonucleotides with chain lengths ranging continuously from 13-mer to over 100-mer was generated. The reaction mixture was then mixed with oligo(dT)/sub 12-18/ labeled with (..gamma..-/sup 32/P)ATP by T/sub 4/ polynucleotide kinase. A sequence ladder with the bottom base as 12-mer was then formed. These oligonucleotides served as size marker for the purification and identification of oligonucleotides on polyacrylamide gel.

  18. Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants.

    PubMed

    Sauer, Noel J; Narváez-Vásquez, Javier; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Woodward, Melody J; Mihiret, Yohannes A; Lincoln, Tracey A; Segami, Rosa E; Sanders, Steven L; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-04-01

    Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants. PMID:26864017

  19. Regulation of serum phosphate

    PubMed Central

    Lederer, Eleanor

    2014-01-01

    The regulation of serum phosphate, an acknowledged risk factor for chronic kidney disease and cardiovascular mortality, is poorly understood. The discovery of fibroblast growth factor 23 (FGF23) as a key regulator of renal phosphate handling and activation of vitamin D has revolutionized our comprehension of phosphate homeostasis. Through as yet undetermined mechanisms, circulating and dietary phosphate appear to have a direct effect on FGF23 release by bone cells that, in turn, causes renal phosphate excretion and decreases intestinal phosphate absorption through a decrease in vitamin D production. Thus, the two major phosphaturic hormones, PTH and FGF23, have opposing effects on vitamin D production, placing vitamin D at the nexus of phosphate homeostasis. While our understanding of phosphate homeostasis has advanced, the factors determining regulation of serum phosphate level remain enigmatic. Diet, time of day, season, gender, age and genetics have all been identified as significant contributors to serum phosphate level. The effects of these factors on serum phosphate have major implications for what is understood as ‘normal’ and for studies of phosphate homeostasis and metabolism. Moreover, other hormonal mediators such as dopamine, insulin-like growth factor, and angiotensin II also affect renal handling of phosphate. How the major hormone effects on phosphate handling are regulated and how the effect of these other factors are integrated to yield the measurable serum phosphate are only now beginning to be studied. PMID:24973411

  20. Oligonucleotide-Functionalized Anisotropic Gold Nanoparticles

    NASA Astrophysics Data System (ADS)

    Jones, Matthew Robert

    In this thesis, we describe the properties of oligonucleotide-functionalized gold colloids under the unique set of conditions where the particles are geometrically anisotropic and have nanometer-scale dimensions. While nearly two decades of previous work elucidated numerous unexpected and emergent phenomena arising from the combination of inorganic nanoparticles with surface-bound DNA strands, virtually nothing was known about how these properties are altered when the shape of the nanoparticle core is chosen to be non-spherical. In particular, we are interested in understanding, and ultimately controlling, the ways in which these DNA-conjugated anisotropic nanostructures interact when their attraction is governed by programmable DNA hybridization events. Chapter 1 introduces the field of DNA-based materials assembly by discussing how nanoscale building blocks which present rigid, directional interactions can be thought of as possessing artificial versions of the familiar chemical principles of "bonds" and "valency". In chapter 2 we explore the fundamental interparticle binding thermodynamics of DNA-functionalized spherical and anisotropic nanoparticles, which reveals enormous preferences for collective ligand interactions occurring between flat surfaces over those that occur between curved surfaces. Using these insights, chapter 3 demonstrates that when syntheses produce mixtures of different nanoparticle shapes, the tailorable nature of DNA-mediated interparticle association can be used to selectively crystallize and purify the desired anisotropic nanostructure products, leaving spherical impurity particles behind. Chapter 4 leverages the principle that the flat facets of anisotropic particles generate directional DNA-based hybridization interactions to assemble a variety of tailorable nanoparticle superlattices whose symmetry and dimensionality are a direct consequence of the shape of the nanoparticle building block used in their construction. Chapter 5 explores

  1. PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

    EPA Science Inventory

    Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

  2. Micro- and nano-structure based oligonucleotide sensors.

    PubMed

    Ferrier, David C; Shaver, Michael P; Hands, Philip J W

    2015-06-15

    This paper presents a review of micro- and nano-structure based oligonucleotide detection and quantification techniques. The characteristics of such devices make them very attractive for Point-of-Care or On-Site-Testing biosensing applications. Their small scale means that they can be robust and portable, their compatibility with modern CMOS electronics means that they can easily be incorporated into hand-held devices and their suitability for mass production means that, out of the different approaches to oligonucleotide detection, they are the most suitable for commercialisation. This review discusses the advantages of micro- and nano-structure based sensors and covers the various oligonucleotide detection techniques that have been developed to date. These include: Bulk Acoustic Wave and Surface Acoustic Wave devices, micro- and nano-cantilever sensors, gene Field Effect Transistors, and nanowire and nanopore based sensors. Oligonucleotide immobilisation techniques are also discussed.

  3. Sequence-dependent theory of oligonucleotide hybridization kinetics

    SciTech Connect

    Marimuthu, Karthikeyan; Chakrabarti, Raj E-mail: rajc@andrew.cmu.edu

    2014-05-07

    A theoretical approach to the prediction of the sequence and temperature-dependent rate constants for oligonucleotide hybridization reactions has been developed based on the theory of relaxation kinetics. One-sided and two-sided melting reaction mechanisms for oligonucleotide hybridization reactions have been considered, analyzed, modified, and compared to select a physically consistent as well as robust model for prediction of the relaxation times of DNA hybridization reactions that agrees with the experimental evidence. The temperature- and sequence-dependent parameters of the proposed model have been estimated using available experimental data. The relaxation time model that we developed has been combined with the nearest neighbor model of hybridization thermodynamics to estimate the temperature- and sequence-dependent rate constants of an oligonucleotide hybridization reaction. The model-predicted rate constants are compared to experimentally determined rate constants for the same oligonucleotide hybridization reactions. Finally, we consider a few important applications of kinetically controlled DNA hybridization reactions.

  4. Molecular Selection, Modification and Development of Therapeutic Oligonucleotide Aptamers

    PubMed Central

    Yu, Yuanyuan; Liang, Chao; Lv, Quanxia; Li, Defang; Xu, Xuegong; Liu, Baoqin; Lu, Aiping; Zhang, Ge

    2016-01-01

    Monoclonal antibodies are the dominant agents used in inhibition of biological target molecules for disease therapeutics, but there are concerns of immunogenicity, production, cost and stability. Oligonucleotide aptamers have comparable affinity and specificity to targets with monoclonal antibodies whilst they have minimal immunogenicity, high production, low cost and high stability, thus are promising inhibitors to rival antibodies for disease therapy. In this review, we will compare the detailed advantages and disadvantages of antibodies and aptamers in therapeutic applications and summarize recent progress in aptamer selection and modification approaches. We will present therapeutic oligonucleotide aptamers in preclinical studies for skeletal diseases and further discuss oligonucleotide aptamers in different stages of clinical evaluation for various disease therapies including macular degeneration, cancer, inflammation and coagulation to highlight the bright commercial future and potential challenges of therapeutic oligonucleotide aptamers. PMID:26978355

  5. [Preliminary study on HLA-B genotyping by oligonucleotide chips].

    PubMed

    Lan, Ke; Hu, Shou-Wang; Zhang, Fan; Wang, Hui; Guan, Wei; Ding, Yu; Sun, Ou-Jun; Wang, Sheng-Qi

    2003-04-01

    HLA genes constitute a highly polymorphic multigene system. In the present study, HLA-B oligonucleotide chips were manufactured by using a set of sequence-specific oligonucleotide probes derived from polymorphic regions in exon 2 and exon 3 of HLA-B gene spotted by microarrayer onto the aldehyde modified glass slides. In addition, the sequenced HLA-B gene clones used as standard samples were amplified from exon 2 and exon 3 by PCR. Together with the correct hybridization and wash conditions, the PCR products were bound with the array probes on the chip, and the hybridization patterns were transformed to HLA-B genotypes. The results showed that the genotypes of standard samples by the HLA-B oligonucleotide chips were completely identical with the sequenced clones. In conclusion, the oligonucleotide chip method presented here for HLA-B genotyping is a rapid, accurate, sensitive and attractive high throughput biochemical way.

  6. Long-term release and improved intracellular penetration of oligonucleotide-polyethylenimine complexes entrapped in biodegradable microspheres.

    PubMed

    De Rosa, Giuseppe; Quaglia, Fabiana; Bochot, Amélie; Ungaro, Francesca; Fattal, Elias

    2003-01-01

    The aim of this work was to design a biodegradable delivery system for oligonucleotides providing both a sustained release and an improved intracellular penetration. To this purpose oligonucleotide/polyethylenimine (ON/PEI) complexes at nitrogen to phosphate (N/P) molar ratios of about 15 or 40 were encapsulated into poly(lactide-co-glycolide) microspheres by the multiple emulsion-solvent evaporation technique. ON/PEI complexes were efficiently entrapped inside microspheres. The introduction of salts within the external aqueous phase allowed an improvement of microsphere characteristics. In particular, the use of sodium chloride led to a reduced microsphere porosity and a more homogeneous ON distribution inside the polymeric matrix. These effects were attributed to the reduced flux of water from the external aqueous phase toward the internal aqueous droplets, due to the osmotic effect of sodium chloride. Both, the reduced porosity and the improved ON distribution inside the matrix, were considered responsible for the lower burst effect and the slower ON release rate from microsphere prepared with sodium chloride. ON/PEI complexes encapsulated inside microspheres were also protected toward enzymatic degradation in fetal calf serum. Interestingly, ON/PEI complexes slowly released from microspheres efficiently penetrated inside HeLa cells and oligonucleotides were preferentially located in the nucleus. PMID:12741766

  7. Evaluation of thiazole intercalating dyes as acceptors for quantum dot donors in Förster resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Massey, Melissa; Algar, W. Russ

    2014-05-01

    Fluorescent probes suitable for the selective detection of DNA sequences are important in genomic research, disease diagnostics, and pathogen detection, among many other applications. The unique optical properties of semiconductor quantum dots (QDs) have proven to be highly valuable for development of fluorescent probes and biosensors. We describe preliminary work toward combining QDs with monomeric thiazole dyes for the detection of nucleic acid hybridization. BO, TO, BO3, and TO3 dyes, which span the visible spectrum, were synthesized with undecanoic acid linkers to permit bioconjugation and their fluorescent enhancements in response to DNA oligonucleotides was evaluated. Contrast ratios between single-stranded probe oligonucleotide and double-stranded probe/target hybrids were between 2.5 and 7.5. BO3 and TO3 were used to label a polyhistidine-appended peptide that self-assembled to QDs and were found to be suitable acceptor dyes for Förster resonance energy transfer (FRET) with QD donors that had their peak emission at 540 nm and 625 nm, respectively. We further conjugated a probe oligonucleotide to a polyhistidineappended peptide at an internal site, and this probe also self-assembled to QDs. Mixing these conjugates with BO3 and either complementary DNA target or non-complementary DNA could induce quenching of the QD emission via FRET, but no FRET-sensitized BO3 emission was observed. Experiments suggested that binding of BO3 to the interface of the QDs was in competition with binding to DNA. Our results provide insight into important criteria (e.g., QD surface chemistry) for designing and optimizing a QD-FRET probe for DNA detection that utilizes the fluorescent properties of monomeric thiazole intercalating dyes.

  8. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    SciTech Connect

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S.; Golova, Julia; Perov, Alexander N.; Protic, Miroslava; Robison, Richard; Shipma, Matthew; White, Amanda M.; Willse, Alan R.

    2006-01-01

    A diagnostic, genome-independent microbial fingerprinting method using DNA oligonucleotide microarrays was used for high-resolution differentiation between closely related Bacillus strains, including two strains of Bacillus anthracis that are monomorphic (indistinguishable) via amplified fragment length polymorphism fingerprinting techniques. Replicated hybridizations on 391-probe nonamer arrays were used to construct a prototype fingerprint library for quantitative comparisons. Descriptive analysis of the fingerprints, including phylogenetic reconstruction, is consistent with previous taxonomic organization of the genus. Newly developed statistical analysis methods were used to quantitatively compare and objectively confirm apparent differences in microarray fingerprints with the statistical rigor required for microbial forensics and clinical diagnostics. These data suggest that a relatively simple fingerprinting microarray and statistical analysis method can differentiate between species in the Bacillus cereus complex, and between strains of B. anthracis. A synthetic DNA standard was used to understand underlying microarray and process-level variability, leading to specific recommendations for the development of a standard operating procedure and/or continued technology enhancements for microbial forensics and diagnostics.

  9. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    PubMed

    Gooding, Matt; Malhotra, Meenakshi; Evans, James C; Darcy, Raphael; O'Driscoll, Caitriona M

    2016-10-01

    The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications. PMID:27521696

  10. Oligonucleotide and Long Polymeric DNA Encoding

    SciTech Connect

    Miller, E; Mariella Jr., R P; Christian, A T; Gardner, S N; Williams, J M

    2003-11-24

    This report summarizes the work done at Lawrence Livermore National Laboratory for the Oligonucleotide and Long Polymeric DNA Encoding project, part of the Microelectronic Bioprocesses Program at DARPA. The goal of the project was to develop a process by which long (circa 10,000 base-pair) synthetic DNA molecules could be synthesized in a timely and economic manner. During construction of the long molecule, errors in DNA sequence occur during hybridization and/or the subsequent enzymatic process. The work done on this project has resulted in a novel synthesis scheme that we call the parallel pyramid synthesis protocol, the development of a suit of computational tools to minimize and quantify errors in the synthesized DNA sequence, and experimental proof of this technique. The modeling consists of three interrelated modules: the bioinformatics code which determines the specifics of parallel pyramid synthesis for a given chain of long DNA, the thermodynamics code which tracks the products of DNA hybridization and polymerase extension during the later steps in the process, and the kinetics model which examines the temporal and spatial processes during one thermocycle. Most importantly, we conducted the first successful syntheses of a gene using small starting oligomers (tetramers). The synthesized sequence, 813 base pairs long, contained a 725 base pair gene, modified green fluorescent protein (mGFP), which has been shown to be a functional gene by cloning into cells and observing its green fluorescent product.

  11. Oligonucleotide Therapies: The Past and the Present

    PubMed Central

    Lundin, Karin E.; Gissberg, Olof; Smith, C.I. Edvard

    2015-01-01

    In this review we address the development of oligonucleotide (ON) medicines from a historical perspective by listing the landmark discoveries in this field. The various biological processes that have been targeted and the corresponding ON interventions found in the literature are discussed together with brief updates on some of the more recent developments. Most ON therapies act through antisense mechanisms and are directed against various RNA species, as exemplified by gapmers, steric block ONs, antagomirs, small interfering RNAs (siRNAs), micro-RNA mimics, and splice switching ONs. However, ONs binding to Toll-like receptors and those forming aptamers have completely different modes of action. Similar to other novel medicines, the path to success has been lined with numerous failures, where different therapeutic ONs did not stand the test of time. Since the first ON drug was approved for clinical use in 1998, the therapeutic landscape has changed considerably, but many challenges remain until the expectations for this new form of medicine are met. However, there is room for cautious optimism. PMID:26160334

  12. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    PubMed

    Gooding, Matt; Malhotra, Meenakshi; Evans, James C; Darcy, Raphael; O'Driscoll, Caitriona M

    2016-10-01

    The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications.

  13. Potent Antiscrapie Activities of Degenerate Phosphorothioate Oligonucleotides

    PubMed Central

    Kocisko, David A.; Vaillant, Andrew; Lee, Kil Sun; Arnold, Kevin M.; Bertholet, Nadine; Race, Richard E.; Olsen, Emily A.; Juteau, Jean-Marc; Caughey, Byron

    2006-01-01

    Although transmissible spongiform encephalopathies (TSEs) are incurable, a key therapeutic approach is prevention of conversion of the normal, protease-sensitive form of prion protein (PrP-sen) to the disease-specific protease-resistant form of prion protein (PrP-res). Here degenerate phosphorothioate oligonucleotides (PS-ONs) are introduced as low-nM PrP-res conversion inhibitors with strong antiscrapie activities in vivo. Comparisons of various PS-ON analogs indicated that hydrophobicity and size were important, while base composition was only minimally influential. PS-ONs bound avidly to PrP-sen but could be displaced by sulfated glycan PrP-res inhibitors, indicating the presence of overlapping binding sites. Labeled PS-ONs also bound to PrP-sen on live cells and were internalized. This binding likely accounts for the antiscrapie activity. Prophylactic PS-ON treatments more than tripled scrapie survival periods in mice. Survival times also increased when PS-ONs were mixed with scrapie brain inoculum. With these antiscrapie activities and their much lower anticoagulant activities than that of pentosan polysulfate, degenerate PS-ONs are attractive new compounds for the treatment of TSEs. PMID:16495266

  14. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    PubMed

    Gerard, Xavier; Garanto, Alejandro; Rozet, Jean-Michel; Collin, Rob W J

    2016-01-01

    Inherited retinal dystrophies (IRDs) are an extremely heterogeneous group of genetic diseases for which currently no effective treatment strategies exist. Over the last decade, significant progress has been made utilizing gene augmentation therapy for a few genetic subtypes of IRD, although several technical challenges so far prevent a broad clinical application of this approach for other forms of IRD. Many of the mutations leading to these retinal diseases affect pre-mRNA splicing of the mutated genes . Antisense oligonucleotide (AON)-mediated splice modulation appears to be a powerful approach to correct the consequences of such mutations at the pre-mRNA level , as demonstrated by promising results in clinical trials for several inherited disorders like Duchenne muscular dystrophy, hypercholesterolemia and various types of cancer. In this mini-review, we summarize ongoing pre-clinical research on AON-based therapy for a few genetic subtypes of IRD , speculate on other potential therapeutic targets, and discuss the opportunities and challenges that lie ahead to translate splice modulation therapy for retinal disorders to the clinic.

  15. Photophysical deactivation pathways in adenine oligonucleotides.

    PubMed

    Spata, Vincent A; Matsika, Spiridoula

    2015-12-14

    In this work we study deactivation processes in adenine oligomers after absorption of UV radiation using Quantum Mechanics combined with Molecular Mechanics (QM/MM). Correlated electronic structure methods appropriate for describing the excited states are used to describe a π-stacked dimer of adenine bases incorporated into (dA)20(dT)20. The results of these calculations reveal three different types of excited state minima which play a role in deactivation processes. Within this set of minima there are minima where the excited state is localized on one adenine (monomer-like) as well as minima where the excited state is delocalized on two adenines, forming different types of excimers and bonded excimers of varying but inter-related character. The proximity of their energies reveals that the minima can decay into one another along a flat potential energy surface dependent on the interbase separation. Additionally, analysis of the emissive energies and other physical properties, including theoretical anisotropy calculations, and comparison with fluorescence experiments, provides evidence that excimers play an important role in long-lived signals in adenine oligonucleotides while the subpicosecond decay is attributed to monomer-like minima. The necessity for a close approach of the nucleobases reveals that the deactivation mechanism is tied to macro-molecular motion. PMID:26536353

  16. Microbial solubilization of phosphate

    DOEpatents

    Rogers, R.D.; Wolfram, J.H.

    1993-10-26

    A process is provided for solubilizing phosphate from phosphate containing ore by treatment with microorganisms which comprises forming an aqueous mixture of phosphate ore, microorganisms operable for solubilizing phosphate from the phosphate ore and maintaining the aqueous mixture for a period of time and under conditions operable to effect the microbial solubilization process. An aqueous solution containing soluble phosphorus can be separated from the reacted mixture by precipitation, solvent extraction, selective membrane, exchange resin or gravity methods to recover phosphate from the aqueous solution. 6 figures.

  17. Microbial solubilization of phosphate

    DOEpatents

    Rogers, Robert D.; Wolfram, James H.

    1993-01-01

    A process is provided for solubilizing phosphate from phosphate containing ore by treatment with microorganisms which comprises forming an aqueous mixture of phosphate ore, microorganisms operable for solubilizing phosphate from the phosphate ore and maintaining the aqueous mixture for a period of time and under conditions operable to effect the microbial solubilization process. An aqueous solution containing soluble phosphorous can be separated from the reacted mixture by precipitation, solvent extraction, selective membrane, exchange resin or gravity methods to recover phosphate from the aqueous solution.

  18. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel

    PubMed Central

    Li, Wei

    2015-01-01

    To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution. PMID:26650843

  19. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

    PubMed

    Tang, Weizhong; Zhou, Huafu; Li, Wei

    2015-01-01

    To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.

  20. Three holes bound to a double acceptor - Be(+) in germanium

    NASA Technical Reports Server (NTRS)

    Haller, E. E.; Mcmurray, R. E., Jr.; Falicov, L. M.; Haegel, N. M.; Hansen, W. L.

    1983-01-01

    A double acceptor binding three holes has been observed for the first time with photoconductive far-infrared spectroscopy in beryllium-doped germanium single crystals. This new center, Be(+), has a hole binding energy of about 5 meV and is only present when free holes are generated by ionization of either neutral shallow acceptors or neutral Be double acceptors. The Be(+) center thermally ionizes above 4 K. It disappears at a uniaxial stress higher than about a billion dyn/sq cm parallel to (111) as a result of the lifting of the valence-band degeneracy.

  1. Delivery of antisense oligonucleotide to the cornea by iontophoresis.

    PubMed

    Berdugo, M; Valamanesh, F; Andrieu, C; Klein, C; Benezra, D; Courtois, Y; Behar-Cohen, F

    2003-04-01

    We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the

  2. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  3. Uranium from phosphate ores

    SciTech Connect

    Hurst, F.J.

    1983-01-01

    The following topics are described briefly: the way phosphate fertilizers are made; how uranium is recovered in the phosphate industry; and how to detect covert uranium recovery operations in a phsophate plant.

  4. Nitrogen is a deep acceptor in ZnO

    DOE PAGES

    Tarun, M. C.; Iqbal, M. Zafar; McCluskey, M. D.

    2011-04-14

    Zinc oxide is a promising material for blue and UV solid-state lighting devices, among other applications. Nitrogen has been regarded as a potential p-type dopant for ZnO. However, recent calculations indicate that nitrogen is a deep acceptor. This paper presents experimental evidence that nitrogen is, in fact, a deep acceptor and therefore cannot produce p-type ZnO. A broad photoluminescence (PL) emission band near 1.7 eV, with an excitation onset of ~2.2 eV, was observed, in agreement with the deep-acceptor model of the nitrogen defect. Thus the deep-acceptor behavior can be explained by the low energy of the ZnO valence bandmore » relative to the vacuum level.« less

  5. Nitrogen is a deep acceptor in ZnO

    SciTech Connect

    Tarun, M. C.; Iqbal, M. Zafar; McCluskey, M. D.

    2011-04-14

    Zinc oxide is a promising material for blue and UV solid-state lighting devices, among other applications. Nitrogen has been regarded as a potential p-type dopant for ZnO. However, recent calculations indicate that nitrogen is a deep acceptor. This paper presents experimental evidence that nitrogen is, in fact, a deep acceptor and therefore cannot produce p-type ZnO. A broad photoluminescence (PL) emission band near 1.7 eV, with an excitation onset of ~2.2 eV, was observed, in agreement with the deep-acceptor model of the nitrogen defect. Thus the deep-acceptor behavior can be explained by the low energy of the ZnO valence band relative to the vacuum level.

  6. Electron Transfer Rate Maxima at Large Donor-Acceptor Distances.

    PubMed

    Kuss-Petermann, Martin; Wenger, Oliver S

    2016-02-01

    Because of their low mass, electrons can transfer rapidly over long (>15 Å) distances, but usually reaction rates decrease with increasing donor-acceptor distance. We report here on electron transfer rate maxima at donor-acceptor separations of 30.6 Å, observed for thermal electron transfer between an anthraquinone radical anion and a triarylamine radical cation in three homologous series of rigid-rod-like donor-photosensitizer-acceptor triads with p-xylene bridges. Our experimental observations can be explained by a weak distance dependence of electronic donor-acceptor coupling combined with a strong increase of the (outer-sphere) reorganization energy with increasing distance, as predicted by electron transfer theory more than 30 years ago. The observed effect has important consequences for light-to-chemical energy conversion. PMID:26800279

  7. Mechanism of oligonucleotide release from cationic liposomes.

    PubMed Central

    Zelphati, O; Szoka, F C

    1996-01-01

    We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm. Images Fig. 1 Fig. 3 PMID:8876163

  8. Synthesis, Properties, and Design Principles of Donor-Acceptor Nanohoops.

    PubMed

    Darzi, Evan R; Hirst, Elizabeth S; Weber, Christopher D; Zakharov, Lev N; Lonergan, Mark C; Jasti, Ramesh

    2015-09-23

    We have synthesized a series of aza[8]cycloparaphenylenes containing one, two, and three nitrogens to probe the impact of nitrogen doping on optoelectronic properties and solid state packing. Alkylation of these azananohoops afforded the first donor-acceptor nanohoops where the phenylene backbone acts as the donor and the pyridinium units act as the acceptor. The impact on the optoelectronic properties was then studied experimentally and computationally to provide new insight into the effect of functionalization on nanohoops properties. PMID:27162989

  9. Synthesis, Properties, and Design Principles of Donor–Acceptor Nanohoops

    PubMed Central

    2015-01-01

    We have synthesized a series of aza[8]cycloparaphenylenes containing one, two, and three nitrogens to probe the impact of nitrogen doping on optoelectronic properties and solid state packing. Alkylation of these azananohoops afforded the first donor–acceptor nanohoops where the phenylene backbone acts as the donor and the pyridinium units act as the acceptor. The impact on the optoelectronic properties was then studied experimentally and computationally to provide new insight into the effect of functionalization on nanohoops properties. PMID:27162989

  10. Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Tripathy, Sushant

    Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

  11. Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

    SciTech Connect

    Abdur, Rob; Gerlits, Oksana O.; Gan, Jianhua; Jiang, Jiansheng; Salon, Jozef; Kovalevsky, Andrey Y.; Chumanevich, Alexander A.; Weber, Irene T.; Huang, Zhen

    2014-02-01

    Selenium-derivatized oligonucleotides may facilitate phase determination and high-resolution structure determination for protein–nucleic acid crystallography. The Se atom-specific mutagenesis (SAM) strategy may also enhance the study of nuclease catalysis. The crystal structures of protein–nucleic acid complexes are commonly determined using selenium-derivatized proteins via MAD or SAD phasing. Here, the first protein–nucleic acid complex structure determined using selenium-derivatized nucleic acids is reported. The RNase H–RNA/DNA complex is used as an example to demonstrate the proof of principle. The high-resolution crystal structure indicates that this selenium replacement results in a local subtle unwinding of the RNA/DNA substrate duplex, thereby shifting the RNA scissile phosphate closer to the transition state of the enzyme-catalyzed reaction. It was also observed that the scissile phosphate forms a hydrogen bond to the water nucleophile and helps to position the water molecule in the structure. Consistently, it was discovered that the substitution of a single O atom by a Se atom in a guide DNA sequence can largely accelerate RNase H catalysis. These structural and catalytic studies shed new light on the guide-dependent RNA cleavage.

  12. Surface characterization of oligonucleotides immobilized on polymer surfaces

    NASA Astrophysics Data System (ADS)

    Pham, Duy K.; Ivanova, Elena P.; Wright, Jonathan P.; Grodzinski, Piotr A.; Lenigk, Ralf; Nicolau, Dan V.

    2002-11-01

    The immobilization and hybridization of amino-terminated oligonucleotide strands to cyclo-olefin-copolymer (COC) and polycarbonate (PC) surfaces have been investigated for potential application in micro-PCR devices. The oligonucleotides were covalently bound to the plasma-treated COC and PC surfaces via an N-hydroxy-sulfosuccinimide (NHSS) intermediate. Analysis by AFM showed that the oligonucleotides were present on the surfaces as lumps, and that the size, both vertically and laterally, of these lumps on the COC surface was larger compared to the PC surface. The immobilization efficiency of the former was also higher (15.8 x 1012 molecules / cm2) compared to the latter (3.3 x 1012 molecules / cm2). The higher efficiency of the COC surface is attributed to the more effective NHSS-functionalization and its higher surface roughness. Subsequent hybridization doubled the height of the lumps, while the lateral dimensions remained essentially unchanged. This is explained in terms of organization of the long probe strands used on the surface as flexible, coil-like polymer chains, which allow the complementary oligonucleotides to bind and increase the height of the lumps. The AFM frictional images showed that the hybridization had the effect of reversing hydrophilicity of the oligonucleotide lumps from being more hydrophilic to more hydrophobic, consistent with the hydrophilic bases of the probe strands being shielded as a result of hybridization.

  13. Structure and stability of the complex formed by oligonucleotides.

    PubMed

    Zheng, Cui; Niu, Lin; Yan, Jingjing; Liu, Jie; Luo, Ying; Liang, Dehai

    2012-05-28

    Polycations and cationic lipids have been widely used as non-viral vectors for the delivery of plasmid DNA, siRNA and anti-sense oligonucleotides. To demonstrate that one polycation can form a complex with several types of DNA, we conducted a comparative study on the complexation of poly(L-lysine) (PLL) with 2000 bp salmon testes DNA (dsDNA), 21 bp double-stranded oligonucleotides (ds-oligo), and 21 nt single-stranded oligonucleotides (ss-oligo) in PBS buffer. The complexes are prepared by a titration method and the process is monitored by laser light scattering. It was found that in most cases, ss-oligo and ds-oligo form complexes with higher molecular weights than the complex formed by dsDNA at the same +/- ratio immediately after mixing. More importantly, the complexes formed by oligonucleotides are not stable, the scattered intensity gradually decreases to the level of the solvent in weeks. Atomic force microscopy measurements also indicate that the freshly prepared complex is subject to environmental changes and could dissociate very quickly. The behaviour of oligonucleotides cannot be predicted by the classical polyelectrolyte theories.

  14. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  15. Molecular beacon probes of photodamage in thymine and uracil oligonucleotides.

    PubMed

    Yarasi, Soujanya; McConachie, Cheryl; Loppnow, Glen R

    2005-01-01

    Molecular beacons (MB) are becoming more common as sequence-selective detectors of nucleic acids. Although they can easily detect single-base mismatches, they have never been used to directly detect DNA or RNA damage. To measure the degree of ultraviolet (UV) light damage in oligonucleotides, we report a novel MB approach for general detection of photoproducts in UV-irradiated rU17 and dT17 oligonucleotides. With monochromatic UV light irradiation at ca 280 nm under anoxic conditions, the oligonucleotide absorption decays with a single-exponential time constant of 123+/-1 min for rU17 and with double-exponential time constants of 78+/-0.5 min (99%) and 180+/-5 min (0.05%) for dT17 oligonucleotides. Under the same conditions, the MB fluorescence decays more quickly, with single-exponential time constants of 19+/-2 and 26+/-3 min for rU17 and dT17, respectively. Similar kinetics were observed with broadband UV light irradiation of oligonucleotides. The differences in the UV damage kinetics of dT17 and rU17 and their detection by absorption and fluorescence techniques will be discussed in the context of differential instabilities introduced in the nucleic acid-MB duplex by the different photoproducts formed.

  16. Four Ways to Oligonucleotides Without Phosphoimidazolides.

    PubMed

    Šponer, Judit E; Šponer, Jiří; Di Mauro, Ernesto

    2016-01-01

    Emergence of the very first RNA or RNA-like oligomers from simple nucleotide precursors is one of the most intriguing questions of the origin of life research. In the current paper, we analyse the mechanism of four non-enzymatic template-free scenarios suggested for the oligomerization of chemically non-modified cyclic and acyclic nucleotides in the literature. We show that amines may have a twofold role in these syntheses: due to their high affinity to bind protons they may activate the phosphorus of the phosphate group via proton transfer reactions, or indirectly they may serve as charge compensating species and influence the self-assembling of nucleotides to supramolecular architectures compatible with the oligomerization reactions. Effect of cations and pH on the reactions is also discussed. PMID:26520151

  17. Four Ways to Oligonucleotides Without Phosphoimidazolides.

    PubMed

    Šponer, Judit E; Šponer, Jiří; Di Mauro, Ernesto

    2016-01-01

    Emergence of the very first RNA or RNA-like oligomers from simple nucleotide precursors is one of the most intriguing questions of the origin of life research. In the current paper, we analyse the mechanism of four non-enzymatic template-free scenarios suggested for the oligomerization of chemically non-modified cyclic and acyclic nucleotides in the literature. We show that amines may have a twofold role in these syntheses: due to their high affinity to bind protons they may activate the phosphorus of the phosphate group via proton transfer reactions, or indirectly they may serve as charge compensating species and influence the self-assembling of nucleotides to supramolecular architectures compatible with the oligomerization reactions. Effect of cations and pH on the reactions is also discussed.

  18. Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

    PubMed

    Wang, Fenghua; Zhang, Hefei; Gao, Jingxia; Chen, Fengjiao; Chen, Sijie; Zhang, Cuizhen; Peng, Gang

    2016-01-01

    Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

  19. A novel catechol-based universal support for oligonucleotide synthesis.

    PubMed

    Anderson, Keith M; Jaquinod, Laurent; Jensen, Michael A; Ngo, Nam; Davis, Ronald W

    2007-12-21

    A novel universal support for deoxyribo- and ribonucleic acid synthesis has been developed. The support, constructed from 1,4-dimethoxycatechol, represents an improvement over existing universal supports because of its ability to cleave and deprotect under mild conditions in standard reagents. Because no nonvolatile additives are required for cleavage and deprotection, the synthesized oligonucleotides do not require purification prior to use in biochemical assays. Using reverse phase HPLC and electrospray mass spectroscopy, it was determined that oligonucleotides synthesized on the universal support (UL1) 3'-dephosphorylate quickly (9 h in 28-30% ammonium hydroxide (NH4OH) at 55 degrees C, 2 h in 28-30% NH4OH at 80 degrees C, or <1 h in ammonium hydroxide/methylamine (1:1) (AMA) at 80 degrees C). Oligonucleotides used as primers for the polymerase chain reaction (PCR) assay were found to perform identically to control primers, demonstrating full biological compatibility. In addition, a method was developed for sintering the universal support directly into a filter plug which can be pressure fit into the synthesis column of a commercial synthesizer. The universal support plugs allow the synthesis of high-quality oligonucleotides at least 120 nucleotides in length, with purity comparable to non-universal commercial supports and approximately 50% lower reagent consumption. The universal support plugs are routinely used to synthesize deoxyribo-, ribo-, 3'-modified, 5'-modified, and thioated oligonucleotides. The flexibility of the universal support and the efficiency of 3'-dephosphorylation are expected to increase the use of universal supports in oligonucleotide synthesis.

  20. Oligonucleotide Array for Identification and Detection of Pythium Species†

    PubMed Central

    Tambong, J. T.; de Cock, A. W. A. M.; Tinker, N. A.; Lévesque, C. A.

    2006-01-01

    A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples

  1. Survival of the fittest before the beginning of life: selection of the first oligonucleotide-like polymers by UV light

    PubMed Central

    Mulkidjanian, Armen Y; Cherepanov, Dmitry A; Galperin, Michael Y

    2003-01-01

    Background A key event in the origin of life on this planet has been formation of self-replicating RNA-type molecules, which were complex enough to undergo a Darwinian-type evolution (origin of the "RNA world"). However, so far there has been no explanation of how the first RNA-like biopolymers could originate and survive on the primordial Earth. Results As condensation of sugar phosphates and nitrogenous bases is thermodynamically unfavorable, these compounds, if ever formed, should have undergone rapid hydrolysis. Thus, formation of oligonucleotide-like structures could have happened only if and when these structures had some selective advantage over simpler compounds. It is well known that nitrogenous bases are powerful quenchers of UV quanta and effectively protect the pentose-phosphate backbones of RNA and DNA from UV cleavage. To check if such a protection could play a role in abiogenic evolution on the primordial Earth (in the absence of the UV-protecting ozone layer), we simulated, by using Monte Carlo approach, the formation of the first oligonucleotides under continuous UV illumination. The simulations confirmed that UV irradiation could have worked as a selective factor leading to a relative enrichment of the system in longer sugar-phosphate polymers carrying nitrogenous bases as UV-protectors. Partial funneling of the UV energy into the condensation reactions could provide a further boost for the oligomerization. Conclusion These results suggest that accumulation of the first polynucleotides could be explained by their abiogenic selection as the most UV-resistant biopolymers. PMID:12773209

  2. Genome Engineering Using Targeted Oligonucleotide Libraries and Functional Selection

    PubMed Central

    Diner, Elie J.; Garza-Sánchez, Fernando; Hayes, Christopher S.

    2011-01-01

    The λ phage Red proteins greatly enhance homologous recombination in Escherichia coli. Red-mediated recombination or “recombineering” can be used to construct targeted gene deletions as well as to introduce point mutations into the genome. Here, we describe our method for scanning mutagenesis using recombineered oligonucleotide libraries. This approach entails randomization of specific codons within a target gene, followed by functional selection to isolate mutants. Oligonucleotide library mutagenesis has generated hundreds of novel antibiotic resistance mutations in genes encoding ribosomal proteins, and should be applicable to other systems for which functional selections exist. PMID:21815087

  3. Electron acceptor-dependent respiratory and physiological stratifications in biofilms.

    PubMed

    Yang, Yonggang; Xiang, Yinbo; Sun, Guoping; Wu, Wei-Min; Xu, Meiying

    2015-01-01

    Bacterial respiration is an essential driving force in biogeochemical cycling and bioremediation processes. Electron acceptors respired by bacteria often have solid and soluble forms that typically coexist in the environment. It is important to understand how sessile bacteria attached to solid electron acceptors respond to ambient soluble alternative electron acceptors. Microbial fuel cells (MFCs) provide a useful tool to investigate this interaction. In MFCs with Shewanella decolorationis, azo dye was used as an alternative electron acceptor in the anode chamber. Different respiration patterns were observed for biofilm and planktonic cells, with planktonic cells preferred to respire with azo dye while biofilm cells respired with both the anode and azo dye. The additional azo respiration dissipated the proton accumulation within the anode biofilm. There was a large redox potential gap between the biofilms and anode surface. Changing cathodic conditions caused immediate effects on the anode potential but not on the biofilm potential. Biofilm viability showed an inverse and respiration-dependent profile when respiring with only the anode or azo dye and was enhanced when respiring with both simultaneously. These results provide new insights into the bacterial respiration strategies in environments containing multiple electron acceptors and support an electron-hopping mechanism within Shewanella electrode-respiring biofilms.

  4. Organic solar cells based on acceptor-functionalized diketopyrrolopyrrole derivatives

    NASA Astrophysics Data System (ADS)

    Ghosh, Sanjay S.; Serrano, Luis A.; Ebenhoch, Bernd; Rotello, Vincent M.; Cooke, Graeme; Samuel, Ifor D. W.

    2015-01-01

    The synthesis and characterization of three solution processable diketopyrrolopyrrole (DPP) derivatives featuring acceptor units attached to the core by alkyne linker units is reported. Cyclic voltammetry and density functional theory calculations indicate that the DPP derivatives possess similar HOMO and LUMO energies. Solar cells were fabricated by blending the synthesized DPP derivatives with [6,6]-phenyl-C71-butyrate methyl ester. The influence of donor:acceptor blend ratio, film thickness, annealing temperature, and annealing time on device performance was studied. Differences in device performance were related to atomic force microscopy measurements of the films. The highest power conversion efficiency of 1.76% was achieved for the DPP derivative functionalized with an aldehyde electron-withdrawing group with a 1∶0.7 donor:acceptor ratio when the active layer was annealed for 10 min at 110°C.

  5. Theory of Primary Photoexcitations in Donor-Acceptor Copolymers

    NASA Astrophysics Data System (ADS)

    Aryanpour, Karan; Dutta, Tirthankar; Huynh, Uyen N. V.; Vardeny, Zeev Valy; Mazumdar, Sumit

    2015-12-01

    We present a generic theory of primary photoexcitations in low band gap donor-acceptor conjugated copolymers. Because of the combined effects of strong electron correlations and broken symmetry, there is considerable mixing between a charge-transfer exciton and an energetically proximate triplet-triplet state with an overall spin singlet. The triplet-triplet state, optically forbidden in homopolymers, is allowed in donor-acceptor copolymers. For an intermediate difference in electron affinities of the donor and the acceptor, the triplet-triplet state can have a stronger oscillator strength than the charge-transfer exciton. We discuss the possibility of intramolecular singlet fission from the triplet-triplet state, and how such fission can be detected experimentally.

  6. Zinc phosphate conversion coatings

    DOEpatents

    Sugama, T.

    1997-02-18

    Zinc phosphate conversion coatings for producing metals which exhibit enhanced corrosion prevention characteristics are prepared by the addition of a transition-metal-compound promoter comprising a manganese, iron, cobalt, nickel, or copper compound and an electrolyte such as polyacrylic acid, polymethacrylic acid, polyitaconic acid and poly-L-glutamic acid to a phosphating solution. These coatings are further improved by the incorporation of Fe ions. Thermal treatment of zinc phosphate coatings to generate {alpha}-phase anhydrous zinc phosphate improves the corrosion prevention qualities of the resulting coated metal. 33 figs.

  7. Zinc phosphate conversion coatings

    DOEpatents

    Sugama, Toshifumi

    1997-01-01

    Zinc phosphate conversion coatings for producing metals which exhibit enhanced corrosion prevention characteristics are prepared by the addition of a transition-metal-compound promoter comprising a manganese, iron, cobalt, nickel, or copper compound and an electrolyte such as polyacrylic acid, polymethacrylic acid, polyitaconic acid and poly-L-glutamic acid to a phosphating solution. These coatings are further improved by the incorporation of Fe ions. Thermal treatment of zinc phosphate coatings to generate .alpha.-phase anhydrous zinc phosphate improves the corrosion prevention qualities of the resulting coated metal.

  8. Phosphorus, phosphorous, and phosphate.

    PubMed

    Iheagwara, O Susan; Ing, Todd S; Kjellstrand, Carl M; Lew, Susie Q

    2013-10-01

    This article distinguishes the terms "phosphorus, phosphorous, and phosphate" which are frequently used interchangeably. We point out the difference between phosphorus and phosphate, with an emphasis on the unit of measure. Expressing a value without the proper name or unit of measure may lead to misunderstanding and erroneous conclusions. We indicate why phosphate must be expressed as milligrams per deciliter or millimoles per liter and not as milliequivalents per liter. Therefore, we elucidate the distinction among the terms "phosphorus, phosphorous, and phosphate" and the importance of saying precisely what one really means.

  9. Higher excited states of acceptors in cubic semiconductors

    NASA Astrophysics Data System (ADS)

    Said, M.; Kanehisa, M. A.; Balkanski, M.

    1986-02-01

    For the first time, higher excited states of shallow acceptors up to the 3s and 4s states are calculated based on the Balderschi and Lipari theory including the cubic correction. The eigenvalues and eigenvectors of the effective mass Hamiltonian for shallow acceptor states were obtained by the finite element method. The resultant sparse matrix is diagonalized by a newly developed Saad's method based on Arnoldi's algorithm. Comparison with experimental spectra on ZnTe:Li and ZnTe:P gives best valence band parameters for ZnTe; μ = 0.60 and δ = 0.12.

  10. Donor-acceptor chemistry in the main group.

    PubMed

    Rivard, Eric

    2014-06-21

    This Perspective article summarizes recent progress from our laboratory in the isolation of reactive main group species using a general donor-acceptor protocol. A highlight of this program is the use of carbon-based donors in combination with suitable Lewis acidic acceptors to yield stable complexes of parent Group 14 element hydrides (e.g. GeH2 and H2SiGeH2). It is anticipated that this strategy could be extended to include new synthetic targets from throughout the Periodic Table with possible applications in bottom-up materials synthesis and main group element catalysis envisioned. PMID:24788390

  11. Acceptors in bulk and nanoscale ZnO

    NASA Astrophysics Data System (ADS)

    McCluskey, M. D.

    2012-02-01

    Zinc oxide (ZnO) is a semiconductor that emits bright UV light, with little wasted heat. This intrinsic feature makes it a promising material for energy-efficient white lighting, nano-lasers, and other optical applications. For devices to be competitive, however, it is necessary to develop reliable p-type doping. Although substitutional nitrogen has been considered as a potential p-type dopant for ZnO, theoretical and experimental work indicates that nitrogen is a deep acceptor and will not lead to p-type conductivity. This talk will highlight recent experiments on ZnO:N at low temperatures. A red/near-IR photoluminescence (PL) band is correlated with the presence of deep nitrogen acceptors. PL excitation (PLE) measurements show an absorption threshold of 2.26 eV, in good agreement with theory. Magnetic resonance experiments provide further evidence for this assignment. The results of these studies seem to rule out group-V elements as shallow acceptors in ZnO, contradicting numerous reports in the literature. If these acceptors do not work as advertised, is there a viable alternative? Optical studies on ZnO nanocrystals show some intriguing leads. At liquid-helium temperatures, a series of sharp IR absorption peaks arise from an unknown acceptor impurity. The data are consistent with a hydrogenic acceptor 0.46 eV above the valence band edge. While this binding energy is still too deep for many practical applications, it represents a significant improvement over the ˜ 1.3 eV binding energy for nitrogen acceptors. Nanocrystals present another twist. Due to their high surface-to-volume ratio, surface states are especially important. Specifically, electron-hole recombination at the surface give rises to a red luminescence band. From our PL and IR experiments, we have developed a ``unified'' model that attempts to explain acceptor and surface states in ZnO nanocrystals. This model could provide a useful framework for designing future nanoscale ZnO devices.

  12. Gene expression profiling in peanut using oligonucleotide microarrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently have a moderately significant number of ESTs been released into the public domain. Utilization of these ESTs for the oligonucleotide microarrays provides a means to investigate l...

  13. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

  14. Chromosome-specific painting in Cucumis species using bulked oligonucleotides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a sing...

  15. Optical Spectroscopy of Acceptors in Semiconductors: I. Acceptor Complexes in Neutron Transmutation-Doped Silicon. I. Piezospectroscopy of Beryllium Double Acceptors in Germanium.

    NASA Astrophysics Data System (ADS)

    Labrec, Charles Raymond

    Substitutional group III impurities in group IV elemental semiconductors bind a hole from the valence band and are solid-state analogs of the hydrogen atom; likewise, group II impurities bind two holes and are analogs of the helium atom. In these materials, the electronic transitions from the acceptor s-like ground state to the p-like excited states are infrared active. A high-resolution Fourier transform spectrometer, and a liquid-helium cooled germanium bolometer and glass sample cryostat are ideally suited to study these effects. When silicon is exposed to neutron radiation, a fraction of the atoms are converted to phosphorus. After annealing, these impurity atoms occupy substitutional locations and are thus donors. When the initial crystal is p-type and the final phosphorus concentration is less than that of the acceptor, the sample is left p-type but highly compensated. This results in broadening of the transition lines. However, before complete annealing, it is discovered that a new, extremely shallow acceptor is formed, with an ionization energy of 28.24 meV, which is far shallower than any previously known. Neutral Be in Ge is known to be a double acceptor. Under uniaxial stress, the single hole (1s)^2 to (1s)(np) excitation spectrum shows splittings and polarization effects. These piezospectroscopic effects were observed for a compressive force vec F | (111) and vec F | (100). The phenomenological shear deformation potential constants have been deduced for the ground and excited states of the D transition.

  16. Ligand-bound Structures and Site-directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE*

    PubMed Central

    Syson, Karl; Stevenson, Clare E. M.; Miah, Farzana; Barclay, J. Elaine; Tang, Minhong; Gorelik, Andrii; Rashid, Abdul M.; Lawson, David M.; Bornemann, Stephen

    2016-01-01

    GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential. PMID:27531751

  17. Tetrathiafulvalene-based mixed-valence acceptor-donor-acceptor triads: a joint theoretical and experimental approach.

    PubMed

    Calbo, Joaquín; Aragó, Juan; Otón, Francisco; Lloveras, Vega; Mas-Torrent, Marta; Vidal-Gancedo, José; Veciana, Jaume; Rovira, Concepció; Ortí, Enrique

    2013-12-01

    This work presents a joint theoretical and experimental characterisation of the structural and electronic properties of two tetrathiafulvalene (TTF)-based acceptor-donor-acceptor triads (BQ-TTF-BQ and BTCNQ-TTF-BTCNQ; BQ is naphthoquinone and BTCNQ is benzotetracyano-p-quinodimethane) in their neutral and reduced states. The study is performed with the use of electrochemical, electron paramagnetic resonance (EPR), and UV/Vis/NIR spectroelectrochemical techniques guided by quantum-chemical calculations. Emphasis is placed on the mixed-valence properties of both triads in their radical anion states. The electrochemical and EPR results reveal that both BQ-TTF-BQ and BTCNQ-TTF-BTCNQ triads in their radical anion states behave as class-II mixed-valence compounds with significant electronic communication between the acceptor moieties. Density functional theory calculations (BLYP35/cc-pVTZ), taking into account the solvent effects, predict charge-localised species (BQ(.-)-TTF-BQ and BTCNQ(.-)-TTF-BTCNQ) as the most stable structures for the radical anion states of both triads. A stronger localisation is found both experimentally and theoretically for the BTCNQ-TTF-BTCNQ anion, in accordance with the more electron-withdrawing character of the BTCNQ acceptor. CASSCF/CASPT2 calculations suggest that the low-energy, broad absorption bands observed experimentally for the BQ-TTF-BQ and BTCNQ-TTF-BTCNQ radical anions are associated with the intervalence charge transfer (IV-CT) electronic transition and two nearby donor-to-acceptor CT excitations. The study highlights the molecular efficiency of the electron-donor TTF unit as a molecular wire connecting two acceptor redox centres.

  18. Three Redox States of a Diradical Acceptor-Donor-Acceptor Triad: Gating the Magnetic Coupling and the Electron Delocalization.

    PubMed

    Souto, Manuel; Lloveras, Vega; Vela, Sergi; Fumanal, Maria; Ratera, Imma; Veciana, Jaume

    2016-06-16

    The diradical acceptor-donor-acceptor triad 1(••), based on two polychlorotriphenylmethyl (PTM) radicals connected through a tetrathiafulvalene(TTF)-vinylene bridge, has been synthesized. The generation of the mixed-valence radical anion, 1(•-), and triradical cation species, 1(•••+), obtained upon electrochemical reduction and oxidation, respectively, was monitored by optical and ESR spectroscopy. Interestingly, the modification of electron delocalization and magnetic coupling was observed when the charged species were generated and the changes have been rationalized by theoretical calculations.

  19. CADMIUM PHOSPHATE GLASS

    DOEpatents

    Carpenter, H.W.; Johnson, P.D.

    1963-04-01

    A method of preparing a cadmium phosphate glass that comprises providing a mixture of solid inorganic compounds of cadmuim and phosphate having vaporizable components and heating the resulting composition to a temperature of at least 850 un. Concent 85% C is presented. (AEC)

  20. Covalent non-fused tetrathiafulvalene-acceptor systems.

    PubMed

    Pop, Flavia; Avarvari, Narcis

    2016-06-28

    Covalent donor-acceptor (D-A) systems have significantly contributed to the development of many organic materials and to molecular electronics. Tetrathiafulvalene (TTF) represents one of the most widely studied donor precursors and has been incorporated into the structure of many D-A derivatives with the objective of obtaining redox control and modulation of the intramolecular charge transfer (ICT), in order to address switchable emissive systems and to take advantage of its propensity to form regular stacks in the solid state. In this review, we focus on the main families of non-fused TTF-acceptors, which are classified according to the nature of the acceptor: nitrogen-containing heterocycles, BODIPY, perylenes and electron poor unsaturated hydrocarbons, as well as radical acceptors. We describe herein the most representative members of each family with a brief mention of their synthesis and a special focus on their D-A characteristics. Special attention is given to ICT and its modulation, fluorescence quenching and switching, photoconductivity, bistability and spin distribution by discussing and comparing spectroscopic and electrochemical features, photophysical properties, solid-state properties and theoretical calculations. PMID:27193500

  1. Fine structure of the Mn acceptor in GaAs

    NASA Astrophysics Data System (ADS)

    Krainov, I. V.; Debus, J.; Averkiev, N. S.; Dimitriev, G. S.; Sapega, V. F.; Lähderanta, E.

    2016-06-01

    We reveal the electronic level structure of the Mn acceptor, which consists of a valence-band hole bound to an Mn2 + ion, in presence of applied uniaxial stress and an external magnetic field in bulk GaAs. Resonant spin-flip Raman scattering is used to measure the g factor of the AMn0 center in the ground and excited states with the total angular momenta F =1 and F =2 and characterize the optical selection rules of the spin-flip transitions between these Mn-acceptor states. We determine the random stress fields near the Mn acceptor, the constant of the antiferromagnetic exchange interaction between the valence-band holes and the electrons of the inner Mn2 + shell as well as the deformation potential for the exchange energy. The p -d exchange energy, in particular, decreases significantly with increasing compressive stress. By combining the experimental Raman study with the developed theoretical model on the scattering efficiency, in which also the random local and external uniaxial stresses and magnetic field are considered, the fine structure of the Mn acceptor is determined in full detail.

  2. Does phosphate enhance the natural attenuation of crude oil in groundwater under defined redox conditions?

    PubMed

    Ponsin, Violaine; Mouloubou, Olsen Raïnness; Prudent, Pascale; Höhener, Patrick

    2014-11-15

    After a crude oil spill caused by a broken pipeline in 2009 to a gravel aquifer in southern France, degradation processes under various redox conditions progressively established, but at rates that predict a long life-time of the source under natural attenuation after partial source removal. In this study, we aimed at identifying the rate-limiting factors for each redox condition, with special emphasis on phosphate as limiting nutrient. The study was conducted in laboratory microcosms assembled with material collected on site: sediments, water from monitoring wells, oil and microbial sludge. Redox conditions were promoted by adding electron acceptors (either oxygen, nitrate, limonite (FeO(OH)), cryptomelane (K(Mn(4+),Mn(2+))8O16), or sulfate). For each condition, the role of phosphate was studied by repeated additions for up to 290days. The results showed a very strong stimulation of aerobic and denitrifying rates of oil degradation by phosphate, provided that oxygen and nitrate were repeatedly supplied. Phosphate caused also a marked stimulation of methanogenic degradation, and a relatively small stimulation of metal reduction. These anaerobic processes started only after marked lag phases, and phosphate shortened the lag phase for methanogenic degradation. Degradation of aromatic and aliphatic hydrocarbons with less than 8 carbons, including benzene, was confirmed even under unstimulated conditions. It is concluded that degradation rates at the site are limited by both, availability of electron acceptors and availability of phosphate needed for promoting microbial growth. PMID:24795042

  3. Does phosphate enhance the natural attenuation of crude oil in groundwater under defined redox conditions?

    NASA Astrophysics Data System (ADS)

    Ponsin, Violaine; Mouloubou, Olsen Raïnness; Prudent, Pascale; Höhener, Patrick

    2014-11-01

    After a crude oil spill caused by a broken pipeline in 2009 to a gravel aquifer in southern France, degradation processes under various redox conditions progressively established, but at rates that predict a long life-time of the source under natural attenuation after partial source removal. In this study, we aimed at identifying the rate-limiting factors for each redox condition, with special emphasis on phosphate as limiting nutrient. The study was conducted in laboratory microcosms assembled with material collected on site: sediments, water from monitoring wells, oil and microbial sludge. Redox conditions were promoted by adding electron acceptors (either oxygen, nitrate, limonite (FeO(OH)), cryptomelane (K(Mn4 +,Mn2 +)8O16), or sulfate). For each condition, the role of phosphate was studied by repeated additions for up to 290 days. The results showed a very strong stimulation of aerobic and denitrifying rates of oil degradation by phosphate, provided that oxygen and nitrate were repeatedly supplied. Phosphate caused also a marked stimulation of methanogenic degradation, and a relatively small stimulation of metal reduction. These anaerobic processes started only after marked lag phases, and phosphate shortened the lag phase for methanogenic degradation. Degradation of aromatic and aliphatic hydrocarbons with less than 8 carbons, including benzene, was confirmed even under unstimulated conditions. It is concluded that degradation rates at the site are limited by both, availability of electron acceptors and availability of phosphate needed for promoting microbial growth.

  4. Does phosphate enhance the natural attenuation of crude oil in groundwater under defined redox conditions?

    PubMed

    Ponsin, Violaine; Mouloubou, Olsen Raïnness; Prudent, Pascale; Höhener, Patrick

    2014-11-15

    After a crude oil spill caused by a broken pipeline in 2009 to a gravel aquifer in southern France, degradation processes under various redox conditions progressively established, but at rates that predict a long life-time of the source under natural attenuation after partial source removal. In this study, we aimed at identifying the rate-limiting factors for each redox condition, with special emphasis on phosphate as limiting nutrient. The study was conducted in laboratory microcosms assembled with material collected on site: sediments, water from monitoring wells, oil and microbial sludge. Redox conditions were promoted by adding electron acceptors (either oxygen, nitrate, limonite (FeO(OH)), cryptomelane (K(Mn(4+),Mn(2+))8O16), or sulfate). For each condition, the role of phosphate was studied by repeated additions for up to 290days. The results showed a very strong stimulation of aerobic and denitrifying rates of oil degradation by phosphate, provided that oxygen and nitrate were repeatedly supplied. Phosphate caused also a marked stimulation of methanogenic degradation, and a relatively small stimulation of metal reduction. These anaerobic processes started only after marked lag phases, and phosphate shortened the lag phase for methanogenic degradation. Degradation of aromatic and aliphatic hydrocarbons with less than 8 carbons, including benzene, was confirmed even under unstimulated conditions. It is concluded that degradation rates at the site are limited by both, availability of electron acceptors and availability of phosphate needed for promoting microbial growth.

  5. Development of imide- and imidazole-containing electron acceptors for use in donor-acceptor conjugated compounds and polymers

    NASA Astrophysics Data System (ADS)

    Li, Duo

    Conjugated organic compounds and polymers have attracted significant attention due to their potential application in electronic devices as semiconducting materials, such as organic solar cells (OSCs). In order to tune band gaps, donor-acceptor (D-A) structure is widely used, which has been proved to be one of the most effective strategies. This thesis consists of three parts: 1) design, syntheses and characterization of new weak acceptors based on imides and the systematic study of the structure-property relationship; (2) introduction of weak and strong acceptors in one polymer to achieve a broad coverage of light absorption and improve the power conversion efficiency (PCE); (3) modification of benzothiadiazole (BT) acceptor in order to increase the electron withdrawing ability. Imide-based electron acceptors, 4-(5-bromothiophen-2-y1)-2-(2-ethylhexyl)-9- phenyl- 1H-benzo[f]isoindole-1,3(2H)-dione (BIDO-1) and 4,9-bis(5-bromothiophen-2-yl)-2-(2-ethylhexyl)-benzo[f]isoindole-1,3-dione (BIDO-2), were designed and synthesized. In this design, naphthalene is selected as its main core to maintain a planar structure, and thienyl groups are able to facilitate the bromination reaction and lower the band gap. BIDO-1 and BIDO-2 were successfully coupled with different donors by both Suzuki cross-coupling and Stille cross-coupling reactions. Based on the energy levels and band gaps of the BIDO-containing compounds and polymers, BIDO-1 and BIDO-2 are proved to be weak electron acceptors. Pyromellitic diimide (PMDI) was also studied and found to be a stronger electron acceptor than BIDO . In order to obtain broad absorption coverage, both weak acceptor ( BIDO-2) and strong acceptor diketopyrrolopyrrole (DPP) were introduced in the same polymer. The resulting polymers show two absorption bands at 400 and 600 nm and two emission peaks at 500 and 680 nm. The band gaps of the polymers are around 1.6 eV, which is ideal for OSC application. The PCE of 1.17% was achieved. Finally

  6. Anaerobic methanotrophy in tidal wetland: Effects of electron acceptors

    NASA Astrophysics Data System (ADS)

    Lin, Li-Hung; Yu, Zih-Huei; Wang, Pei-Ling

    2016-04-01

    Wetlands have been considered to represent the largest natural source of methane emission, contributing substantially to intensify greenhouse effect. Despite in situ methanogenesis fueled by organic degradation, methanotrophy also plays a vital role in controlling the exact quantity of methane release across the air-sediment interface. As wetlands constantly experience various disturbances of anthropogenic activities, biological burrowing, tidal inundation, and plant development, rapid elemental turnover would enable various electron acceptors available for anaerobic methanotrophy. The effects of electron acceptors on stimulating anaerobic methanotrophy and the population compositions involved in carbon transformation in wetland sediments are poorly explored. In this study, sediments recovered from tidally influenced, mangrove covered wetland in northern Taiwan were incubated under the static conditions to investigate whether anaerobic methanotrophy could be stimulated by the presence of individual electron acceptors. Our results demonstrated that anaerobic methanotrophy was clearly stimulated in incubations amended with no electron acceptor, sulfate, or Fe-oxyhydroxide. No apparent methane consumption was observed in incubations with nitrate, citrate, fumarate or Mn-oxides. Anaerobic methanotrophy in incubations with no exogenous electron acceptor appears to proceed at the greatest rates, being sequentially followed by incubations with sulfate and Fe-oxyhydroxide. The presence of basal salt solution stimulated methane oxidation by a factor of 2 to 3. In addition to the direct impact of electron acceptor and basal salts, incubations with sediments retrieved from low tide period yielded a lower rate of methane oxidation than from high tide period. Overall, this study demonstrates that anaerobic methanotrophy in wetland sediments could proceed under various treatments of electron acceptors. Low sulfate content is not a critical factor in inhibiting methane

  7. Acceptor conductivity in bulk zinc oxide (0001) crystals

    NASA Astrophysics Data System (ADS)

    Adekore, Bababunmi Tolu

    ZnO is a promising wide bandgap semiconductor. Its renowned and prominent properties as its bandgap of 3.37eV at 4.2K; its very high excitonic binding energy, 60meV; its high melting temperature, 2248K constitute the basis for the recently renewed and sustained scientific interests in the material. In addition to the foregoing, the availability of bulk substrates of industrially relevant sizes provides important opportunities such as homoepitaxial deposition of the material which is a technological asset in the production of efficient optoelectronic and electronic devices. The nemesis of wide bandgap materials cannot be more exemplified than in ZnO. The notorious limitation of asymmetric doping and the haunting plague of electrically active point defects dim the bright future of the material. In this case, the search for reliable and consistent acceptor conductivity in bulk substrates has been hitherto, unsuccessful. In the dissertation that now follows, our efforts have been concerted in the search for a reliable acceptor. We have carefully investigated the science of point defects in the material, especially those responsible for the high donor conductivity. We also investigated and herein report variety of techniques of introducing acceptors into the material. We employ the most relevant and informative characterization techniques in verifying both the intended conductivity and the response of intrinsic crystals to variation in temperature and strain. And finally we explain deviations, where they exist, from ideal acceptor characteristics. Our work on reliable acceptor has been articulated in four papers. The first establishing capacitance based methods of monitoring electrically active donor defects. The second investigates the nature of anion acceptors on the oxygen sublattice. A study similar to the preceding study was conducted for cation acceptors on the zinc sublattice and reported in the third paper. Finally, an analysis of the response of the crystal to

  8. Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants[OPEN

    PubMed Central

    Sauer, Noel J.; Narváez-Vásquez, Javier; Mozoruk, Jerry; Miller, Ryan B.; Warburg, Zachary J.; Woodward, Melody J.; Mihiret, Yohannes A.; Lincoln, Tracey A.; Segami, Rosa E.; Sanders, Steven L.; Walker, Keith A.; Beetham, Peter R.; Schöpke, Christian R.; Gocal, Greg F.W.

    2016-01-01

    Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5′-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants. PMID:26864017

  9. Structural Basis of Duplex Thermodynamic Stability and Enhanced Nuclease Resistance of 5'-C-Methyl Pyrimidine-Modified Oligonucleotides.

    PubMed

    Kel'in, Alexander V; Zlatev, Ivan; Harp, Joel; Jayaraman, Muthusamy; Bisbe, Anna; O'Shea, Jonathan; Taneja, Nate; Manoharan, Rajar M; Khan, Saeed; Charisse, Klaus; Maier, Martin A; Egli, Martin; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2016-03-18

    Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers.

  10. Structural Basis of Duplex Thermodynamic Stability and Enhanced Nuclease Resistance of 5'-C-Methyl Pyrimidine-Modified Oligonucleotides.

    PubMed

    Kel'in, Alexander V; Zlatev, Ivan; Harp, Joel; Jayaraman, Muthusamy; Bisbe, Anna; O'Shea, Jonathan; Taneja, Nate; Manoharan, Rajar M; Khan, Saeed; Charisse, Klaus; Maier, Martin A; Egli, Martin; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2016-03-18

    Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers. PMID:26940174

  11. Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

    PubMed

    Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun; Cha, Hyung Joon

    2014-01-01

    It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes. PMID:24185846

  12. PHOSPHATE MANAGEMENT: FY2010 RESULTS OF PHOSPHATE PRECIPITATION TESTS

    SciTech Connect

    Hay, M.; King, W.

    2011-04-04

    The Phosphate Management program seeks to develop treatment options for caustic phosphate solutions resulting from the caustic leaching of the bismuth phosphate sludge. The SRNL subtask investigated the precipitation of phosphate salts from caustic solutions through addition of fluoride and by crystallization. The scoping tests examined the: precipitation of phosphate by the addition of sodium fluoride to form the sodium fluorophosphate double salt, Na{sub 7}F(PO{sub 4}){sub 2} {center_dot} 19H{sub 2}O, crystallization of phosphate by reducing the temperature of saturated phosphate solutions, and combinations of precipitation and crystallization. A simplified leachate simulant was used in the study produced by dissolving sodium phosphate in 1 M to 3.5 M sodium hydroxide solutions. The results show that all three processes; precipitation with sodium fluoride, crystallization, and combined precipitation/crystallization can be effective for removing large amounts of phosphate from solution. The combined process of precipitation/crystallization showed >90% removal of phosphate at all hydroxide concentrations when cooling a non-saturated phosphate solution from 65 C to 25 C. Based on the measured solubility of sodium phosphate, pH adjustment/caustic addition will also remove large amounts of phosphate from solution (>80%). For all three processes, the phosphate concentration in the caustic solution must be managed to keep the phosphate from becoming too concentrated and thereby potentially forming a solid mass of sodium phosphate after an effective phosphate removal process.

  13. Mechanisms of electron acceptor utilization: Implications for simulating anaerobic biodegradation

    USGS Publications Warehouse

    Schreiber, M.E.; Carey, G.R.; Feinstein, D.T.; Bahr, J.M.

    2004-01-01

    Simulation of biodegradation reactions within a reactive transport framework requires information on mechanisms of terminal electron acceptor processes (TEAPs). In initial modeling efforts, TEAPs were approximated as occurring sequentially, with the highest energy-yielding electron acceptors (e.g. oxygen) consumed before those that yield less energy (e.g., sulfate). Within this framework in a steady state plume, sequential electron acceptor utilization would theoretically produce methane at an organic-rich source and Fe(II) further downgradient, resulting in a limited zone of Fe(II) and methane overlap. However, contaminant plumes often display much more extensive zones of overlapping Fe(II) and methane. The extensive overlap could be caused by several abiotic and biotic processes including vertical mixing of byproducts in long-screened monitoring wells, adsorption of Fe(II) onto aquifer solids, or microscale heterogeneity in Fe(III) concentrations. Alternatively, the overlap could be due to simultaneous utilization of terminal electron acceptors. Because biodegradation rates are controlled by TEAPs, evaluating the mechanisms of electron acceptor utilization is critical for improving prediction of contaminant mass losses due to biodegradation. Using BioRedox-MT3DMS, a three-dimensional, multi-species reactive transport code, we simulated the current configurations of a BTEX plume and TEAP zones at a petroleum- contaminated field site in Wisconsin. Simulation results suggest that BTEX mass loss due to biodegradation is greatest under oxygen-reducing conditions, with smaller but similar contributions to mass loss from biodegradation under Fe(III)-reducing, sulfate-reducing, and methanogenic conditions. Results of sensitivity calculations document that BTEX losses due to biodegradation are most sensitive to the age of the plume, while the shape of the BTEX plume is most sensitive to effective porosity and rate constants for biodegradation under Fe(III)-reducing and

  14. Selective release of multiple DNA oligonucleotides from gold nanorods.

    PubMed

    Wijaya, Andy; Schaffer, Stefan B; Pallares, Ivan G; Hamad-Schifferli, Kimberly

    2009-01-27

    Combination therapy, or the use of multiple drugs, has been proven to be effective for complex diseases, but the differences in chemical properties and pharmacokinetics can be challenging in terms of the loading, delivering, and releasing multiple drugs. Here we demonstrate that we can load and selectively release two different DNA oligonucleotides from two different gold nanorods. DNA was loaded on the nanorods via thiol conjugation. Selective releases were induced by selective melting of gold nanorods via ultrafast laser irradiation at the nanorods' longitudinal surface plasmon resonance peaks. Excitation at one wavelength could selectively melt one type of gold nanorods and selectively release one type of DNA strand. Releases were efficient (50-80%) and externally tunable by laser fluence. Released oligonucleotides were still functional. This proof of concept is potentially a powerful method for multiple-drug delivery strategies.

  15. Mechanism of Oligonucleotide Uptake by Cells: Involvement of Specific receptors?

    NASA Astrophysics Data System (ADS)

    Yakubov, Leonid A.; Deeva, Elena A.; Zarytova, Valentina F.; Ivanova, Eugenia M.; Ryte, Antonina S.; Yurchenko, Lyudmila V.; Vlassov, Valentin V.

    1989-09-01

    We have investigated the interaction of oligonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (< 1 μ M), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single- and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.

  16. Typing of enteroviruses by use of microwell oligonucleotide arrays.

    PubMed

    Susi, P; Hattara, L; Waris, M; Luoma-Aho, T; Siitari, H; Hyypiä, T; Saviranta, P

    2009-06-01

    We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

  17. Palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides.

    PubMed

    Shaughnessy, Kevin H

    2015-05-22

    Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

  18. Synthesis and properties of oligonucleotides containing aminodeoxythymidine units.

    PubMed Central

    Gryaznov, S M; Letsinger, R L

    1992-01-01

    Procedures are described for synthesis via solid support methodology of oligonucleotide analogues derived in part from 3'-amino-3'-deoxythymidine or 5'-amino-5'-deoxythymidine. Oligothymidylate decamers terminated with a 3'-amino group or containing a 3'-NHP(O)(O-)O-5' internucleoside link are found to form unusually stable complexes with poly(dA), poly(A), and oligo(dA). For related derivatives of 5'-amino-5'-deoxythymidine enhancement is less or absent, and in the case of multiple substitution destabilization of the heteroduplex may be observed. That the effect of the 3'-amino group is general for oligonucleotide derivatives is indicated by enhanced Tm values for heteroduplex complexes of the mixed-base oligomer, d(TATTCAGTCAT(NH2)), and the methyl phosphonate derivatives, TmTmTmTmTmTmTmTmTmT(NH2) and d(TmAmTmTmCmAmGmTmCmAmT(NH2)). PMID:1630911

  19. Nanomaterial building blocks based on spider silk-oligonucleotide conjugates.

    PubMed

    Humenik, Martin; Scheibel, Thomas

    2014-02-25

    Self-assembling protein nanofibrils are promising structures for the "bottom-up" fabrication of bionanomaterials. Here, the recombinant protein eADF4(C16), a variant of Araneus diadematus dragline silk ADF4, which self-assembles into nanofibrils, and short oligonucleotides were modified for site-specific azide-alkyne coupling. Corresponding oligonuleotide-eADF4(C16) "click" conjugates were hybridized in linear or branched fashion according to the designed complementarities of the DNA moieties. Self-assembly properties of higher ordered structures of the spider silk-DNA conjugates were dominated by the silk component. Assembled β-sheet rich conjugate fibrils were similar in appearance to fibrils of unmodified eADF4(C16) but enabled the specific attachment of neutravidin-modified gold nanoparticles on their surface directed by complementary biotin-oligonucleotides, providing the basis for functionalization of such conjugates.

  20. Selective release of multiple DNA oligonucleotides from gold nanorods.

    PubMed

    Wijaya, Andy; Schaffer, Stefan B; Pallares, Ivan G; Hamad-Schifferli, Kimberly

    2009-01-27

    Combination therapy, or the use of multiple drugs, has been proven to be effective for complex diseases, but the differences in chemical properties and pharmacokinetics can be challenging in terms of the loading, delivering, and releasing multiple drugs. Here we demonstrate that we can load and selectively release two different DNA oligonucleotides from two different gold nanorods. DNA was loaded on the nanorods via thiol conjugation. Selective releases were induced by selective melting of gold nanorods via ultrafast laser irradiation at the nanorods' longitudinal surface plasmon resonance peaks. Excitation at one wavelength could selectively melt one type of gold nanorods and selectively release one type of DNA strand. Releases were efficient (50-80%) and externally tunable by laser fluence. Released oligonucleotides were still functional. This proof of concept is potentially a powerful method for multiple-drug delivery strategies. PMID:19206252

  1. Experimental design, modeling and optimization of polyplex formation between DNA oligonucleotides and branched polyethylenimine.

    PubMed

    Clima, Lilia; Ursu, Elena L; Cojocaru, Corneliu; Rotaru, Alexandru; Barboiu, Mihail; Pinteala, Mariana

    2015-09-28

    The complexes formed by DNA and polycations have received great attention owing to their potential application in gene therapy. In this study, the binding efficiency between double-stranded oligonucleotides (dsDNA) and branched polyethylenimine (B-PEI) has been quantified by processing of the images captured from the gel electrophoresis assays. The central composite experimental design has been employed to investigate the effects of controllable factors on the binding efficiency. On the basis of experimental data and the response surface methodology, a multivariate regression model has been constructed and statistically validated. The model has enabled us to predict the binding efficiency depending on experimental factors, such as concentrations of dsDNA and B-PEI as well as the initial pH of solution. The optimization of the binding process has been performed using simplex and gradient methods. The optimal conditions determined for polyplex formation have yielded a maximal binding efficiency close to 100%. In order to reveal the mechanism of complex formation at the atomic-scale, a molecular dynamic simulation has been carried out. According to the computation results, B-PEI amine hydrogen atoms have interacted with oxygen atoms from dsDNA phosphate groups. These interactions have led to the formation of hydrogen bonds between macromolecules, stabilizing the polyplex structure.

  2. One-pot synthesis of fluorescent oligonucleotide Ag nanoclusters for specific and sensitive detection of DNA.

    PubMed

    Lan, Guo-Yu; Chen, Wei-Yu; Chang, Huan-Tsung

    2011-01-15

    In this study, we prepared fluorescent, functional oligonucleotide-stabilized silver nanoclusters (FFDNA-Ag NCs) through one-pot synthesis and then employed them as probes for single nucleotide polymorphisms (SNPs). The FFDNA-Ag NCs were obtained through the NaBH(4)-mediated reduction of AgNO(3) in the presence of a DNA strand having the sequence 5'-C(12)-CCAGATACTCACCGG-3'. The specific DNA scaffold combines a fluorescent base motif (C(12)) and a specific sequence (CCAGATACTCACCGG) that recognizes a gene for fumarylacetoacetate hydrolase (FAH). The sensing mechanism of our new probe is based on the FFDNA-Ag NCs having different stabilities (fluorescence intensities) in solutions containing 150 mM NaCl in the absence and presence of perfect match DNA (DNA(pmt)). Under the optimal conditions (150 mM NaCl, 20 mM phosphate solution, pH 7.0), the fluorescence ratios of the FFDNA-Ag NC probes in the presence and absence of DNA(pmt), plotted against the concentration of DNA(pmt), was linear over the range 25-1000 nM (R(2)=0.98), with a limit of detection (S/N=3) of 14 nM. This cost-effective and simple FFDNA-Ag NC probe is sensitive and selective for SNPs of a gene for FAH.

  3. More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Nyakas, Adrien; Eberle, Rahel P.; Stucki, Silvan R.; Schürch, Stefan

    2014-07-01

    Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO-), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO- ion by MS3 revealed a so far unreported consecutive excision of a metaphosphate (PO3 -)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO3 - loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO- and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs.

  4. Cationic carbosilane dendrimers and oligonucleotide binding: an energetic affair

    NASA Astrophysics Data System (ADS)

    Marson, D.; Laurini, E.; Posocco, P.; Fermeglia, M.; Pricl, S.

    2015-02-01

    Generation 2 cationic carbosilane dendrimers hold great promise as internalizing agents for gene therapy as they present low toxicity and retain and internalize the genetic material as an oligonucleotide or siRNA. In this work we carried out complete in silico structural and energetical characterization of the interactions of a set of G2 carbosilane dendrimers, showing different affinity towards two single strand oligonucleotide (ODN) sequences in vitro. Our simulations predict that these four dendrimers and the relevant ODN complexes are characterized by similar size and shape, and that the molecule-specific ODN binding ability can be rationalized only by considering a critical molecular design parameter: the normalized effective binding energy ΔGbind,eff/Neff, i.e. the performance of each active individual dendrimer branch directly involved in a binding interaction.Generation 2 cationic carbosilane dendrimers hold great promise as internalizing agents for gene therapy as they present low toxicity and retain and internalize the genetic material as an oligonucleotide or siRNA. In this work we carried out complete in silico structural and energetical characterization of the interactions of a set of G2 carbosilane dendrimers, showing different affinity towards two single strand oligonucleotide (ODN) sequences in vitro. Our simulations predict that these four dendrimers and the relevant ODN complexes are characterized by similar size and shape, and that the molecule-specific ODN binding ability can be rationalized only by considering a critical molecular design parameter: the normalized effective binding energy ΔGbind,eff/Neff, i.e. the performance of each active individual dendrimer branch directly involved in a binding interaction. Electronic supplementary information (ESI) available: Additional figures and tables. See DOI: 10.1039/c4nr04510f

  5. Thermoplastic polymers surfaces for Dip-Pen Nanolithography of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Suriano, Raffaella; Biella, Serena; Cesura, Federico; Levi, Marinella; Turri, Stefano

    2013-05-01

    Different thermoplastic polymers were spin-coated to prepare smooth surfaces for the direct deposition of end-group modified oligonucleotides by Dip-Pen Nanolithography. A study of the diffusion process was done in order to investigate the dependence of calibration coefficient and quality of deposited features on environmental parameters (temperature, relative humidity) and ink's molecular weight and functionality. The optimization of the process parameters led to the realization of high quality and density nanoarrays on plastics.

  6. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea.

    PubMed

    Druzhinina, Irina S; Kopchinskiy, Alexei G; Komoń, Monika; Bissett, John; Szakacs, George; Kubicek, Christian P

    2005-10-01

    One of the biggest obstructions to studies on Trichoderma has been the incorrect and confused application of species names to isolates used in industry, biocontrol of plant pathogens and ecological surveys, thereby making the comparison of results questionable. Here we provide a convenient, on-line method for the quick molecular identification of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode: a diagnostic combination of several oligonucleotides (hallmarks) specifically allocated within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of the rDNA repeat. The barcode was developed on the basis of 979 sequences of 88 vouchered species which displayed in total 135 ITS1 and 2 haplotypes. Oligonucleotide sequences which are constant in all known ITS1 and 2 of Hypocrea/Trichoderma but different in closely related fungal genera, were used to define genus-specific hallmarks. The library of species-, clade- and genus-specific hallmarks is stored in the MySQL database and integrated in the TrichOKey v. 1.0 - barcode sequence identification program with the web interface located on . TrichOKey v. 1.0 identifies 75 single species, 5 species pairs and 1 species triplet. Verification of the DNA-barcode was done by a blind test on 53 unknown isolates of Trichoderma, collected in Central and South America. The obtained results were in a total agreement with phylogenetic identification based on tef1 (large intron), NCBI BLAST of vouchered records and postum morphological analysis. We conclude that oligonucleotide barcode is a powerful tool for the routine identification of Hypocrea/Trichoderma species and should be useful as a complement to traditional methods.

  7. Metal-phosphate binders

    SciTech Connect

    Howe, Beth Ann; Chaps-Cabrera, Jesus Guadalupe

    2009-05-12

    A metal-phosphate binder is provided. The binder may include an aqueous phosphoric acid solution, a metal-cation donor including a metal other than aluminum, an aluminum-cation donor, and a non-carbohydrate electron donor.

  8. Therapeutic antisense oligonucleotides against cancer: hurdling to the clinic

    PubMed Central

    Moreno, Pedro M. D.; Pêgo, Ana P.

    2014-01-01

    Under clinical development since the early 90's and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics has not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given toward a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field. PMID:25353019

  9. Gene expression profiling in peanut using high density oligonucleotide microarrays

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  10. Particle-Based Microarrays of Oligonucleotides and Oligopeptides

    PubMed Central

    Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K.; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F. Ralf; Breitling, Frank; Loeffler, Felix F.

    2014-01-01

    In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches. PMID:27600347

  11. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

    PubMed

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

    2015-09-22

    Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

  12. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    PubMed Central

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  13. Therapeutic Antisense Oligonucleotides against Cancer: Hurdling to the Clinic

    NASA Astrophysics Data System (ADS)

    Moreno, Pedro; Pêgo, Ana

    2014-10-01

    Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

  14. Microarray oligonucleotide probe designer (MOPeD): A web service

    PubMed Central

    Patel, Viren C; Mondal, Kajari; Shetty, Amol Carl; Horner, Vanessa L; Bedoyan, Jirair K; Martin, Donna; Caspary, Tamara; Cutler, David J; Zwick, Michael E

    2011-01-01

    Methods of genomic selection that combine high-density oligonucleotide microarrays with next-generation DNA sequencing allow investigators to characterize genomic variation in selected portions of complex eukaryotic genomes. Yet choosing which specific oligonucleotides to be use can pose a major technical challenge. To address this issue, we have developed a software package called MOPeD (Microarray Oligonucleotide Probe Designer), which automates the process of designing genomic selection microarrays. This web-based software allows individual investigators to design custom genomic selection microarrays optimized for synthesis with Roche NimbleGen’s maskless photolithography. Design parameters include uniqueness of the probe sequences, melting temperature, hairpin formation, and the presence of single nucleotide polymorphisms. We generated probe databases for the human, mouse, and rhesus macaque genomes and conducted experimental validation of MOPeD-designed microarrays in human samples by sequencing the human X chromosome exome, where relevant sequence metrics indicated superior performance relative to a microarray designed by the Roche NimbleGen proprietary algorithm. We also performed validation in the mouse to identify known mutations contained within a 487-kb region from mouse chromosome 16, the mouse chromosome 16 exome (1.7 Mb), and the mouse chromosome 12 exome (3.3 Mb). Our results suggest that the open source MOPeD software package and website (http://moped.genetics.emory.edu/) will make a valuable resource for investigators in their sequence-based studies of complex eukaryotic genomes. PMID:21379402

  15. Phosphate control in dialysis

    PubMed Central

    Cupisti, Adamasco; Gallieni, Maurizio; Rizzo, Maria Antonietta; Caria, Stefania; Meola, Mario; Bolasco, Piergiorgio

    2013-01-01

    Prevention and correction of hyperphosphatemia is a major goal of chronic kidney disease–mineral and bone disorder (CKD–MBD) management, achievable through avoidance of a positive phosphate balance. To this aim, optimal dialysis removal, careful use of phosphate binders, and dietary phosphate control are needed to optimize the control of phosphate balance in well-nourished patients on a standard three-times-a-week hemodialysis schedule. Using a mixed diffusive–convective hemodialysis tecniques, and increasing the number and/or the duration of dialysis tecniques are all measures able to enhance phosphorus (P) mass removal through dialysis. However, dialytic removal does not equal the high P intake linked to the high dietary protein requirement of dialysis patients; hence, the use of intestinal P binders is mandatory to reduce P net intestinal absorption. Unfortunately, even a large dose of P binders is able to bind approximately 200–300 mg of P on a daily basis, so it is evident that their efficacy is limited in the case of an uncontrolled dietary P load. Hence, limitation of dietary P intake is needed to reach the goal of neutral phosphate balance in dialysis, coupled to an adequate protein intake. To this aim, patients should be informed and educated to avoid foods that are naturally rich in phosphate and also processed food with P-containing preservatives. In addition, patients should preferentially choose food with a low P-to-protein ratio. For example, patients could choose egg white or protein from a vegetable source. Finally, boiling should be the preferred cooking procedure, because it induces food demineralization, including phosphate loss. The integrated approach outlined in this article should be actively adapted as a therapeutic alliance by clinicians, dieticians, and patients for an effective control of phosphate balance in dialysis patients. PMID:24133374

  16. Interface effects on acceptor qubits in silicon and germanium.

    PubMed

    Abadillo-Uriel, J C; Calderón, M J

    2016-01-15

    Dopant-based quantum computing implementations often require the dopants to be situated close to an interface to facilitate qubit manipulation with local gates. Interfaces not only modify the energies of the bound states but also affect their symmetry. Making use of the successful effective mass theory we study the energy spectra of acceptors in Si or Ge taking into account the quantum confinement, the dielectric mismatch and the central cell effects. The presence of an interface puts constraints to the allowed symmetries and leads to the splitting of the ground state in two Kramers doublets (Mol et al 2015 Appl. Phys. Lett. 106 203110). Inversion symmetry breaking also implies parity mixing which affects the allowed optical transitions. Consequences for acceptor qubits are discussed. PMID:26618443

  17. Photoconductivity in donor-acceptor heterojunction organic photovoltaics

    NASA Astrophysics Data System (ADS)

    Renshaw, C. K.; Zimmerman, J. D.; Lassiter, B. E.; Forrest, S. R.

    2012-08-01

    Organic photovoltaics (OPVs) differ from ideal inorganic solar cells due to their pronounced voltage dependence under reverse bias. This feature is commonly modeled in an ad hoc fashion by including a parallel junction resistance (Rp) that bypasses the heterojunction energy barrier between donor and acceptor. The existence of a finite Rp has variously been attributed to rough interfaces, pinhole defects, or to the electric field dependence of the dissociation of polaron pairs that are bound at the heterojunction. Here we show that the voltage dependence of the photocurrent can also arise from photoconductivity resulting from exciton generation followed by dissociation into free polarons within the bulk of the donor and acceptor layers. The presence of photoconductivity of the active layers does not result in an increase in power conversion efficiency, and places a constraint on the maximum fill factor that can be achieved in an OPV cell.

  18. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    NASA Astrophysics Data System (ADS)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

  19. Free Carrier Generation in Organic Photovoltaic Bulk Heterojunctions of Conjugated Polymers with Molecular Acceptors: Planar versus Spherical Acceptors

    SciTech Connect

    Nardes, Alexandre M.; Ferguson, Andrew J.; Wolfer, Pascal; Gui, Kurt; Burn, Paul L.; Meredith, Paul; Kopidakis, Nikos

    2014-03-05

    We present a comparative study of the photophysical performance of the prototypical fullerene derivative PC61BM with a planar small-molecule acceptor in an organic photovoltaic device. The small-molecule planar acceptor is 2-[{7-(9,9-di-n-propyl-9H-fluoren-2-yl)benzo[c][1,2,5]thiadiazol-4-yl}methylene]malononitrile, termed K12. We discuss photoinduced free charge-carrier generation and transport in blends of PC61BM or K12 with poly(3-n-hexylthiophene) (P3HT), surveying literature results for P3HT:PC61BM and presenting new results on P3HT:K12. For both systems we also review previous work on film structure and correlate the structural and photophysical results. In both cases, a disordered mixed phase is formed between P3HT and the acceptor, although the photophysical properties of this mixed phase differ markedly for PC61BM and K12. In the case of PC61BM the mixed phase acts as a free carrier generation region that can efficiently shuttle carriers to the pure polymer and fullerene domains. As a result, the vast majority of excitons quenched in P3HT:PC61BM blends yield free carriers detected by the contactless time-resolved microwave conductivity (TRMC) method. In contrast, approximately 85 % of the excitons quenched in P3HT:K12 do not result in free carriers over the nanosecond timescale of the TRMC experiment. We attribute this to poor electron-transport properties in the mixed P3HT:K12 phase. Here, we propose that the observed differences can be traced to the respective shapes of PC61BM and K12: the three-dimensional nature of the fullerene cage facilitates coupling between PC61BM molecules irrespective of their relative orientation, whereas for K12 strong electronic coupling is only expected for molecules oriented with their π systems parallel to each other. Comparison between the eutectic compositions of the P3HT:PC61BM and P3HT:K12 shows that the former contains enough fullerene to form a percolation pathway for electrons, whereas the latter contains a sub

  20. Free carrier generation in organic photovoltaic bulk heterojunctions of conjugated polymers with molecular acceptors: planar versus spherical acceptors.

    PubMed

    Nardes, Alexandre M; Ferguson, Andrew J; Wolfer, Pascal; Gui, Kurt; Burn, Paul L; Meredith, Paul; Kopidakis, Nikos

    2014-06-01

    A comparative study of the photophysical performance of the prototypical fullerene derivative PC61BM with a planar small-molecule acceptor in an organic photovoltaic device is presented. The small-molecule planar acceptor is 2-[{7-(9,9-di-n-propyl-9H-fluoren-2-yl)benzo[c][1,2,5]thiadiazol-4-yl}methylene]malononitrile, termed K12. We discuss photoinduced free charge-carrier generation and transport in blends of PC61BM or K12 with poly(3-n-hexylthiophene) (P3HT), surveying literature results for P3HT:PC61BM and presenting new results on P3HT:K12. For both systems we also review previous work on film structure and correlate the structural and photophysical results. In both cases, a disordered mixed phase is formed between P3HT and the acceptor, although the photophysical properties of this mixed phase differ markedly for PC61BM and K12. In the case of PC61BM the mixed phase acts as a free carrier generation region that can efficiently shuttle carriers to the pure polymer and fullerene domains. As a result, the vast majority of excitons quenched in P3HT:PC61BM blends yield free carriers detected by the contactless time-resolved microwave conductivity (TRMC) method. In contrast, approximately 85% of the excitons quenched in P3HT:K12 do not result in free carriers over the nanosecond timescale of the TRMC experiment. We attribute this to poor electron-transport properties in the mixed P3HT:K12 phase. We propose that the observed differences can be traced to the respective shapes of PC61BM and K12: the three-dimensional nature of the fullerene cage facilitates coupling between PC61BM molecules irrespective of their relative orientation, whereas for K12 strong electronic coupling is only expected for molecules oriented with their π systems parallel to each other. Comparison between the eutectic compositions of the P3HT:PC61BM and P3HT:K12 shows that the former contains enough fullerene to form a percolation pathway for electrons, whereas the latter contains a sub

  1. Free carrier generation in organic photovoltaic bulk heterojunctions of conjugated polymers with molecular acceptors: planar versus spherical acceptors.

    PubMed

    Nardes, Alexandre M; Ferguson, Andrew J; Wolfer, Pascal; Gui, Kurt; Burn, Paul L; Meredith, Paul; Kopidakis, Nikos

    2014-06-01

    A comparative study of the photophysical performance of the prototypical fullerene derivative PC61BM with a planar small-molecule acceptor in an organic photovoltaic device is presented. The small-molecule planar acceptor is 2-[{7-(9,9-di-n-propyl-9H-fluoren-2-yl)benzo[c][1,2,5]thiadiazol-4-yl}methylene]malononitrile, termed K12. We discuss photoinduced free charge-carrier generation and transport in blends of PC61BM or K12 with poly(3-n-hexylthiophene) (P3HT), surveying literature results for P3HT:PC61BM and presenting new results on P3HT:K12. For both systems we also review previous work on film structure and correlate the structural and photophysical results. In both cases, a disordered mixed phase is formed between P3HT and the acceptor, although the photophysical properties of this mixed phase differ markedly for PC61BM and K12. In the case of PC61BM the mixed phase acts as a free carrier generation region that can efficiently shuttle carriers to the pure polymer and fullerene domains. As a result, the vast majority of excitons quenched in P3HT:PC61BM blends yield free carriers detected by the contactless time-resolved microwave conductivity (TRMC) method. In contrast, approximately 85% of the excitons quenched in P3HT:K12 do not result in free carriers over the nanosecond timescale of the TRMC experiment. We attribute this to poor electron-transport properties in the mixed P3HT:K12 phase. We propose that the observed differences can be traced to the respective shapes of PC61BM and K12: the three-dimensional nature of the fullerene cage facilitates coupling between PC61BM molecules irrespective of their relative orientation, whereas for K12 strong electronic coupling is only expected for molecules oriented with their π systems parallel to each other. Comparison between the eutectic compositions of the P3HT:PC61BM and P3HT:K12 shows that the former contains enough fullerene to form a percolation pathway for electrons, whereas the latter contains a sub

  2. Quantum confined acceptors and donors in InSe nanosheets

    SciTech Connect

    Mudd, G. W.; Patanè, A. Makarovsky, O.; Eaves, L.; Kudrynskyi, Z. R.; Kovalyuk, Z. D.; Fay, M. W.; Zólyomi, V.; Falko, V.

    2014-12-01

    We report on the radiative recombination of photo-excited carriers bound at native donors and acceptors in exfoliated nanoflakes of nominally undoped rhombohedral γ-polytype InSe. The binding energies of these states are found to increase with the decrease in flake thickness, L. We model their dependence on L using a two-dimensional hydrogenic model for impurities and show that they are strongly sensitive to the position of the impurities within the nanolayer.

  3. 2012 ELECTRON DONOR-ACCEPTOR INTERACTIONS GORDON RESEARCH CONFERENCE, AUGUST 5-10, 2012

    SciTech Connect

    McCusker, James

    2012-08-10

    The upcoming incarnation of the Gordon Research Conference on Electron Donor Acceptor Interactions will feature sessions on classic topics including proton-coupled electron transfer, dye-sensitized solar cells, and biological electron transfer, as well as emerging areas such as quantum coherence effects in donor-acceptor interactions, spintronics, and the application of donor-acceptor interactions in chemical synthesis.

  4. An extended Foerster-Dexter model for correlated donor-acceptor placement in solid state materials

    NASA Astrophysics Data System (ADS)

    Rotman, S. R.; Hartmann, F. X.

    1987-09-01

    The current theory of donor-acceptor interactions in solid-state materials is based on a random distribution of donors and acceptors through the crystal. In this paper, we present a model to calculate the observable transfer rates for the correlated positioning of donors and acceptors in laser materials. Chemical effects leading to such correlations are discussed.

  5. The glucose 6-phosphate shunt around the Calvin-Benson cycle.

    PubMed

    Sharkey, Thomas D; Weise, Sean E

    2016-07-01

    It is just over 60 years since a cycle for the regeneration of the CO2-acceptor used in photosynthesis was proposed. In this opinion paper, we revisit the origins of the Calvin-Benson cycle that occurred at the time that the hexose monophosphate shunt, now called the pentose phosphate pathway, was being worked out. Eventually the pentose phosphate pathway was separated into two branches, an oxidative branch and a non-oxidative branch. It is generally thought that the Calvin-Benson cycle is the reverse of the non-oxidative branch of the pentose phosphate pathway but we describe crucial differences and also propose that some carbon routinely passes through the oxidative branch of the pentose phosphate pathway. This creates a futile cycle but may help to stabilize photosynthesis. If it occurs it could explain a number of enigmas including the lack of complete labelling of the Calvin-Benson cycle intermediates when carbon isotopes are fed to photosynthesizing leaves.

  6. Income-generating activities for family planning acceptors.

    PubMed

    1989-07-01

    The Income Generating Activities program for Family Planning Acceptors was introduced in Indonesia in 1979. Capital input by the Indonesian National Family Planning Coordination Board and the UN Fund for Population Activities was used to set up small businesses by family planning acceptors. In 2 years, when the businesses become self-sufficient, the loans are repaid, and the money is used to set up new family planning acceptors in business. The program strengthens family planning acceptance, improves the status of women, and enhances community self-reliance. The increase in household income generated by the program raises the standards of child nutrition, encourages reliance on the survival of children, and decreases the value of large families. Approximately 18,000 Family Planning-Income Generating Activities groups are now functioning all over Indonesia, with financial assistance from the central and local governments, the World Bank, the US Agency for International Development, the UN Population Fund, the Government of the Netherlands, and the Government of Australia through the Association of South East Asian Nations.

  7. Virtual screening of electron acceptor materials for organic photovoltaic applications

    NASA Astrophysics Data System (ADS)

    Halls, Mathew D.; Djurovich, Peter J.; Giesen, David J.; Goldberg, Alexander; Sommer, Jonathan; McAnally, Eric; Thompson, Mark E.

    2013-10-01

    Virtual screening involves the generation of structure libraries, automated analysis to predict properties related to application performance and subsequent screening to identify lead systems and estimate critical structure-property limits across a targeted chemical design space. This approach holds great promise for informing experimental discovery and development efforts for next-generation materials, such as organic semiconductors. In this work, the virtual screening approach is illustrated for nitrogen-substituted pentacene molecules to identify systems for development as electron acceptor materials for use in organic photovoltaic (OPV) devices. A structure library of tetra-azapentacenes (TAPs) was generated by substituting four nitrogens for CH at 12 sites on the pentacene molecular framework. Molecular properties (e.g. ELUMO, Eg and μ) were computed for each candidate structure using hybrid DFT at the B3LYP/6-311G** level of theory. The resulting TAPs library was then analyzed with respect to intrinsic properties associated with OPV acceptor performance. Marcus reorganization energies for charge transport for the most favorable TAP candidates were then calculated to further determine suitability as OPV electron acceptors. The synthesis, characterization and OPV device testing of TAP materials is underway, guided by these results.

  8. Design directed self-assembly of donor-acceptor polymers.

    PubMed

    Marszalek, Tomasz; Li, Mengmeng; Pisula, Wojciech

    2016-09-21

    Donor-acceptor polymers with an alternating array of donor and acceptor moieties have gained particular attention during recent years as active components of organic electronics. By implementation of suitable subunits within the conjugated backbone, these polymers can be made either electron-deficient or -rich. Additionally, their band gap and light absorption can be precisely tuned for improved light-harvesting in solar cells. On the other hand, the polymer design can also be modified to encode the desired supramolecular self-assembly in the solid-state that is essential for an unhindered transport of charge carriers. This review focuses on three major factors playing a role in the assembly of donor-acceptor polymers on surfaces which are (1) nature, geometry and substitution position of solubilizing alkyl side chains, (2) shape of the conjugated polymer defined by the backbone curvature, and (3) molecular weight which determines the conjugation length of the polymer. These factors adjust the fine balance between attractive and repulsive forces and ensure a close polymer packing important for an efficient charge hopping between neighboring chains. On the microscopic scale, an appropriate domain formation with a low density of structural defects in the solution deposited thin film is crucial for the charge transport. The charge carrier transport through such thin films is characterized by field-effect transistors as basic electronic elements. PMID:27440174

  9. Fullerene derivatives as electron acceptors for organic photovoltaic cells.

    PubMed

    Mi, Dongbo; Kim, Ji-Hoon; Kim, Hee Un; Xu, Fei; Hwang, Do-Hoon

    2014-02-01

    Energy is currently one of the most important problems humankind faces. Depletion of traditional energy sources such as coal and oil results in the need to develop new ways to create, transport, and store electricity. In this regard, the sun, which can be considered as a giant nuclear fusion reactor, represents the most powerful source of energy available in our solar system. For photovoltaic cells to gain widespread acceptance as a source of clean and renewable energy, the cost per watt of solar energy must be decreased. Organic photovoltaic cells, developed in the past two decades, have potential as alternatives to traditional inorganic semiconductor photovoltaic cells, which suffer from high environmental pollution and energy consumption during production. Organic photovoltaic cells are composed of a blended film of a conjugated-polymer donor and a soluble fullerene-derivative acceptor sandwiched between a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)-coated indium tin oxide positive electrode and a low-work-function metal negative electrode. Considerable research efforts aim at designing and synthesizing novel fullerene derivatives as electron acceptors with up-raised lowest unoccupied molecular orbital energy, better light-harvesting properties, higher electron mobility, and better miscibility with the polymer donor for improving the power conversion efficiency of the organic photovoltaic cells. In this paper, we systematically review novel fullerene acceptors synthesized through chemical modification for enhancing the photovoltaic performance by increasing open-circuit voltage, short-circuit current, and fill factor, which determine the performance of organic photovoltaic cells.

  10. Total Phosphate Influences the Rate of Hydrocarbon Degradation but Phosphate Mineralogy Shapes Microbial Community Composition in Cold-Region Calcareous Soils.

    PubMed

    Siciliano, Steven D; Chen, Tingting; Phillips, Courtney; Hamilton, Jordan; Hilger, David; Chartrand, Blaine; Grosskleg, Jay; Bradshaw, Kris; Carlson, Trevor; Peak, Derek

    2016-05-17

    Managing phosphorus bioaccessibility is critical for the bioremediation of hydrocarbons in calcareous soils. This paper explores how soil mineralogy interacts with a novel biostimulatory solution to both control phosphorus bioavailability and influence bioremediation. Two large bore infiltrators (1 m diameter) were installed at a PHC contaminated site and continuously supplied with a solution containing nutrients and an electron acceptor. Soils from eight contaminated sites were prepared and pretreated, analyzed pretrial, spiked with diesel, placed into nylon bags into the infiltrators, and removed after 3 months. From XAS, we learned that three principal phosphate phases had formed: adsorbed phosphate, brushite, and newberyite. All measures of biodegradation in the samples (in situ degradation estimates, mineralization assays, culturable bacteria, catabolic genes) varied depending upon the soil's phosphate speciation. Notably, adsorbed phosphate increased anaerobic phenanthrene degradation and bzdN catabolic gene prevalence. The dominant mineralogical constraints on community composition were the relative amounts of adsorbed phosphate, brushite, and newberyite. Overall, this study finds that total phosphate influences microbial community phenotypes whereas relative percentages of phosphate minerals influences microbial community genotype composition. PMID:27082646

  11. Spectral, thermal and kinetic studies of charge-transfer complexes formed between the highly effective antibiotic drug metronidazole and two types of acceptors: σ- and π-acceptors

    NASA Astrophysics Data System (ADS)

    Refat, Moamen S.; Saad, Hosam A.; Adam, Abdel Majid A.

    2015-04-01

    Understanding the interaction between drugs and small inorganic or organic molecules is critical in being able to interpret the drug-receptor interactions and acting mechanism of these drugs. A combined solution and solid state study was performed to describe the complexation chemistry of drug metronidazole (MZ) which has a broad-spectrum antibacterial activity with two types of acceptors. The acceptors include, σ-acceptor (i.e., iodine) and π-acceptors (i.e., dichlorodicyanobenzoquinone (DDQ), chloranil (CHL) and picric acid (PA)). The molecular structure, spectroscopic characteristics, the binding modes as well as the thermal stability were deduced from IR, UV-vis, 1H NMR and thermal studies. The binding ratio of complexation (MZ: acceptor) was determined to be 1:2 for the iodine acceptor and 1:1 for the DDQ, CHL or PA acceptor, according to the CHN elemental analyses and spectrophotometric titrations. It has been found that the complexation with CHL and PA acceptors increases the values of enthalpy and entropy, while the complexation with DDQ and iodine acceptors decreases the values of these parameters compared with the free MZ donor.

  12. Biological nitrogen removal with enhanced phosphate uptake in a sequencing batch reactor using single sludge system.

    PubMed

    Lee, D S; Jeon, C O; Park, J M

    2001-11-01

    Simultaneous biological phosphorus and nitrogen removal with enhanced anoxic phosphate uptake was investigated in an anaerobic-aerobic-anoxic-aerobic sequencing batch reactor ((AO)2 SBR). Significant amounts of phosphorus-accumulation organisms (PAOs) capable of denitrification could be accumulated in a single sludge system coexisting with nitrifiers. The ratio of the anoxic phosphate uptake to the aerobic phosphate uptake capacity was increased from 11% to 64% by introducing an anoxic phase in an anaerobic aerobic SBR. The (AO)2 SBR system showed stable phosphorus and nitrogen removal performance. Average removal efficiencies of TOC, total nitrogen, and phosphorus were 92%, 88%, and 100%, respectively. It was found that nitrite (up to 10 mg NO2(-)-N/l) was not detrimental to the anoxic phosphate uptake and could serve as an electron acceptor like nitrate. In fact, the phosphate uptake rate was even faster in the presence of nitrite as an electron acceptor compared to the presence of nitrate. It was found that on-line sensor values of pH, ORP, and DO were somehow related with the dynamic behaviours of nutrient concentrations (NH4+, NO3-, and PO4(3-)) in the SBR. These on-line sensor values were used as real-time control parameters to adjust the duration of each operational phase in the (AO)2 SBR. The real-time controlled SBR exhibited better performance in the removal of phosphorus and nitrogen than the SBR with fixed-time operation. PMID:12230180

  13. Magnetite nanoparticles facilitate methane production from ethanol via acting as electron acceptors

    PubMed Central

    Yang, Zhiman; Shi, Xiaoshuang; Wang, Chuanshui; Wang, Lin; Guo, Rongbo

    2015-01-01

    Potential for interspecies hydrogen transfer within paddy soil enrichments obtained via addition of magnetite nanoparticles and ethanol (named as PEM) was investigated. To do this, PEM derived from rice field of Hangzhou (named as PEM-HZ) was employed, because it offered the best methane production performance. Methane production and Fe (III) reduction proceeded in parallel in the presence of magnetite. Inhibition experiments with 2-bromoethane sulfonate (BES) or phosphate showed that interspecies hydrogen transfer and Fe (III) reduction also occurred in methane production from ethanol. 16S rRNA-based Illumina sequencing results showed that Dechloromonas, Thauera, Desulfovibrio and Clostridium were the dominant putative Fe (III) -reducers, and that hydrogenotrophic Methanobacterium accounted for about 88% of the total archaeal community. These results indicated that magnetite nanoparticles that acted as electron acceptor could facilitate rapid oxidation of ethanol by members of the Fe (III) -reducers in PEM-HZ and establishment of the syntrophic relationship of Fe (III) -reducers with Methanobacterium via interspecies hydrogen transfer. Our results could offer a model to understand the microbial interaction with magnetite from a novel angle during methanogenesis. PMID:26559132

  14. Magnetite nanoparticles facilitate methane production from ethanol via acting as electron acceptors

    NASA Astrophysics Data System (ADS)

    Yang, Zhiman; Shi, Xiaoshuang; Wang, Chuanshui; Wang, Lin; Guo, Rongbo

    2015-11-01

    Potential for interspecies hydrogen transfer within paddy soil enrichments obtained via addition of magnetite nanoparticles and ethanol (named as PEM) was investigated. To do this, PEM derived from rice field of Hangzhou (named as PEM-HZ) was employed, because it offered the best methane production performance. Methane production and Fe (III) reduction proceeded in parallel in the presence of magnetite. Inhibition experiments with 2-bromoethane sulfonate (BES) or phosphate showed that interspecies hydrogen transfer and Fe (III) reduction also occurred in methane production from ethanol. 16S rRNA-based Illumina sequencing results showed that Dechloromonas, Thauera, Desulfovibrio and Clostridium were the dominant putative Fe (III) -reducers, and that hydrogenotrophic Methanobacterium accounted for about 88% of the total archaeal community. These results indicated that magnetite nanoparticles that acted as electron acceptor could facilitate rapid oxidation of ethanol by members of the Fe (III) -reducers in PEM-HZ and establishment of the syntrophic relationship of Fe (III) -reducers with Methanobacterium via interspecies hydrogen transfer. Our results could offer a model to understand the microbial interaction with magnetite from a novel angle during methanogenesis.

  15. Magnetite nanoparticles facilitate methane production from ethanol via acting as electron acceptors.

    PubMed

    Yang, Zhiman; Shi, Xiaoshuang; Wang, Chuanshui; Wang, Lin; Guo, Rongbo

    2015-01-01

    Potential for interspecies hydrogen transfer within paddy soil enrichments obtained via addition of magnetite nanoparticles and ethanol (named as PEM) was investigated. To do this, PEM derived from rice field of Hangzhou (named as PEM-HZ) was employed, because it offered the best methane production performance. Methane production and Fe (III) reduction proceeded in parallel in the presence of magnetite. Inhibition experiments with 2-bromoethane sulfonate (BES) or phosphate showed that interspecies hydrogen transfer and Fe (III) reduction also occurred in methane production from ethanol. 16S rRNA-based Illumina sequencing results showed that Dechloromonas, Thauera, Desulfovibrio and Clostridium were the dominant putative Fe (III) -reducers, and that hydrogenotrophic Methanobacterium accounted for about 88% of the total archaeal community. These results indicated that magnetite nanoparticles that acted as electron acceptor could facilitate rapid oxidation of ethanol by members of the Fe (III) -reducers in PEM-HZ and establishment of the syntrophic relationship of Fe (III) -reducers with Methanobacterium via interspecies hydrogen transfer. Our results could offer a model to understand the microbial interaction with magnetite from a novel angle during methanogenesis. PMID:26559132

  16. Magnetite nanoparticles facilitate methane production from ethanol via acting as electron acceptors.

    PubMed

    Yang, Zhiman; Shi, Xiaoshuang; Wang, Chuanshui; Wang, Lin; Guo, Rongbo

    2015-11-12

    Potential for interspecies hydrogen transfer within paddy soil enrichments obtained via addition of magnetite nanoparticles and ethanol (named as PEM) was investigated. To do this, PEM derived from rice field of Hangzhou (named as PEM-HZ) was employed, because it offered the best methane production performance. Methane production and Fe (III) reduction proceeded in parallel in the presence of magnetite. Inhibition experiments with 2-bromoethane sulfonate (BES) or phosphate showed that interspecies hydrogen transfer and Fe (III) reduction also occurred in methane production from ethanol. 16S rRNA-based Illumina sequencing results showed that Dechloromonas, Thauera, Desulfovibrio and Clostridium were the dominant putative Fe (III) -reducers, and that hydrogenotrophic Methanobacterium accounted for about 88% of the total archaeal community. These results indicated that magnetite nanoparticles that acted as electron acceptor could facilitate rapid oxidation of ethanol by members of the Fe (III) -reducers in PEM-HZ and establishment of the syntrophic relationship of Fe (III) -reducers with Methanobacterium via interspecies hydrogen transfer. Our results could offer a model to understand the microbial interaction with magnetite from a novel angle during methanogenesis.

  17. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries.

    PubMed

    Schmidt, Thorsten L; Beliveau, Brian J; Uca, Yavuz O; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M

    2015-11-16

    Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.

  18. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

    PubMed Central

    Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

    2015-01-01

    Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes. PMID:26567534

  19. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

    NASA Astrophysics Data System (ADS)

    Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

    2015-11-01

    Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.

  20. Flexibility of C3h -Symmetrical Linkers in Tris-oligonucleotide-Based Tetrahedral Scaffolds.

    PubMed

    Panagiotidis, Christos; Kath-Schorr, Stephanie; von Kiedrowski, Günter

    2016-02-01

    Flexibility of tris-oligonucleotides is determined by the length of their connecting hydrocarbon chains. Tris-oligonucleotides are branched DNA building blocks with three oligonucleotide arms attached to a C3h -symmetrical linker core at these chains. Four tris-oligonucleotides hybridise into a tetrahedral nanocage by sequence-determined self-assembly. The influence of methylene, ethylene and propylene chains was studied by synthesising sets of tris-oligonucleotides and analysing the relative stability of the hybridisation products against digestion by mung bean nuclease by using gel electrophoresis. Linkers with ethylene chains showed sufficient flexibility, whereas methylene-chain linkers were too rigid. Tris-oligonucleotides based on the latter still formed tetrahedral scaffolds in intermixing experiments with linkers of higher flexibility. Thus, a new generation of versatile isocyanurate-based linkers was established.

  1. Nanoparticle-bridge assay for amplification-free electrical detection of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Teimouri, Manouchehr

    The aim of this research is to investigate a highly sensitive, fast, inexpensive, and field-applicable amplification-free nanoparticle-based oligonucleotide detection method which does not rely on any enzymatic or signal amplification process. In this approach, target oligonucleotide strands are detected through the formation of nanoparticle satellites which make an electrical path between two electrodes. This method enables an extremely sensitive oligonucleotide detection because even a few oligonucleotide strands can form a single nanoparticle satellite which can solely generates an electrical output signal. Results showed that this oligonucleotide detection method can detect oligonucleotide single strands at concentrations as low as 50 femtomolar without any amplification process. This detection method can be implemented in many fields such as biodefense, food safety, clinical research, and forensics.

  2. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore

    NASA Astrophysics Data System (ADS)

    Cao, Chan; Ying, Yi-Lun; Hu, Zheng-Li; Liao, Dong-Fang; Tian, He; Long, Yi-Tao

    2016-08-01

    Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.

  3. Flexibility of C3h -Symmetrical Linkers in Tris-oligonucleotide-Based Tetrahedral Scaffolds.

    PubMed

    Panagiotidis, Christos; Kath-Schorr, Stephanie; von Kiedrowski, Günter

    2016-02-01

    Flexibility of tris-oligonucleotides is determined by the length of their connecting hydrocarbon chains. Tris-oligonucleotides are branched DNA building blocks with three oligonucleotide arms attached to a C3h -symmetrical linker core at these chains. Four tris-oligonucleotides hybridise into a tetrahedral nanocage by sequence-determined self-assembly. The influence of methylene, ethylene and propylene chains was studied by synthesising sets of tris-oligonucleotides and analysing the relative stability of the hybridisation products against digestion by mung bean nuclease by using gel electrophoresis. Linkers with ethylene chains showed sufficient flexibility, whereas methylene-chain linkers were too rigid. Tris-oligonucleotides based on the latter still formed tetrahedral scaffolds in intermixing experiments with linkers of higher flexibility. Thus, a new generation of versatile isocyanurate-based linkers was established. PMID:26593127

  4. Method for producing and regenerating a synthetic CO.sub.2 acceptor

    DOEpatents

    Lancet, Michael S [Pittsburgh, PA; Curran, George P [Pittsburgh, PA; Gorin, Everett [San Rafael, CA

    1982-01-01

    A method for producing a synthetic CO.sub.2 acceptor by feeding a mixture of finely divided silica and at least one finely divided calcium compound selected from the group consisting of calcium oxide and calcium carbonate to a fluidized bed; operating the fluidized bed at suitable conditions to produce pellets of synthetic CO.sub.2 acceptor and recovering the pellets of synthetic CO.sub.2 acceptor from the fluidized bed. Optionally, spent synthetic CO.sub.2 acceptor can be charged to the fluidized bed to produce regenerated pellets of synthetic CO.sub.2 acceptor.

  5. Method for producing and regenerating a synthetic CO[sub 2] acceptor

    DOEpatents

    Lancet, M. S.; Curran, G. P.; Gorin, E.

    1982-05-18

    A method is described for producing a synthetic CO[sub 2] acceptor by feeding a mixture of finely divided silica and at least one finely divided calcium compound selected from the group consisting of calcium oxide and calcium carbonate to a fluidized bed; operating the fluidized bed at suitable conditions to produce pellets of synthetic CO[sub 2] acceptor and recovering the pellets of synthetic CO[sub 2] acceptor from the fluidized bed. Optionally, spent synthetic CO[sub 2] acceptor can be charged to the fluidized bed to produce regenerated pellets of synthetic CO[sub 2] acceptor. 1 fig.

  6. Dynamics of iron-acceptor-pair formation in co-doped silicon

    SciTech Connect

    Bartel, T.; Gibaja, F.; Graf, O.; Gross, D.; Kaes, M.; Heuer, M.; Kirscht, F.; Möller, C.; Lauer, K.

    2013-11-11

    The pairing dynamics of interstitial iron and dopants in silicon co-doped with phosphorous and several acceptor types are presented. The classical picture of iron-acceptor pairing dynamics is expanded to include the thermalization of iron between different dopants. The thermalization is quantitatively described using Boltzmann statistics and different iron-acceptor binding energies. The proper understanding of the pairing dynamics of iron in co-doped silicon will provide additional information on the electronic properties of iron-acceptor pairs and may become an analytical method to quantify and differentiate acceptors in co-doped silicon.

  7. Hemopoiesis-stimulating activity of immobilized oligonucleotides and hyaluronidase during cytostatic-induced myelosuppression.

    PubMed

    Dygai, A M; Skurikhin, E G; Pershina, O V; Zhdanov, V V; Khmelevskaya, A M; Andreeva, T V; Poponina, A M; Zjuzkov, G N; Udut, E V; Khrichkova, T Ju; Simanina, E V; Miroshnichenko, L A; Stavrova, L A; Tchaikovsky, A S; Markova, T S; Gurto, R V; Brjushinina, O S; Slepichev, V A

    2011-03-01

    The hemopoiesis-stimulating effect of combined treatment with immobilized oligonucleotides and hyaluronidase preparations was studied during cytostatic-induced myelosuppression caused by cyclophosphamide administration. Immobilized hyaluronidase was shown to increase the efficiency of correction of changes in the erythroid and granulocytic hemopoietic stems with immobilized oligonucleotides. This potentiation of the effect of immobilized oligonucleotides by immobilized hyaluronidase was related to an increase in functional activity of committed hemopoietic precursors.

  8. MALDI MS analysis of oligonucleotides: desalting by functional magnetite beads using microwave-assisted extraction.

    PubMed

    Chen, Wei-Yu; Chen, Yu-Chie

    2007-11-01

    The presence of alkali cation adductions of oligonucleotides commonly deteriorates matrix-assisted laser desorption/ionization (MALDI) mass spectra. Thus, desalting is required for oligonucleotide samples prior to MALDI MS analysis in order to prevent the mass spectra from developing poor quality. In this paper, we demonstrate a new approach to extract traces of oligonucleotides from aqueous solutions containing high concentrations of salts using microwave-assisted extraction. The C18-presenting magnetite beads, capable of absorbing microwave irradiation, are used as affinity probes for oligonucleotides with the addition of triethylammonium acetate as the counterions. This new microwave-assisted extraction approach using magnetite beads as the trapping agents and as microwave-absorbers has been demonstrated to be very effective in the selective binding of oligonucleotides from aqueous solutions. The extraction of oligonucleotides from solutions onto the C18-presenting magnetite beads takes only 30 s to enrich oligonucleotides in sufficient quantities for MALDI MS analysis. After using this desalting approach, alkali cation adductions of oligonucleotides are dramatically reduced in the MALDI mass spectra. The presence of saturated NaCl (approximately 6 M) in the oligonucleotide sample is tolerated without degrading the mass spectra. The detection limit for d(A)6 is approximately 2.8 fmol. PMID:17902633

  9. 2'-O-[2-(guanidinium)ethyl]-modified oligonucleotides: stabilizing effect on duplex and triplex structures

    SciTech Connect

    Prakash, T.P.; Puschl, A.; Lesnik, E.; Mohan, V.; Tereshko, V.; Egli, M.; Manoharan, M.

    2010-03-08

    Oligonucleotides with a novel 2'-O-[2-(guanidinium)ethyl] (2'-O-GE) modification have been synthesized using a novel protecting group strategy for the guanidinium group. This modification enhances the binding affinity of oligonucleotides to RNA as well as duplex DNA ({Delta}T{sub m} 3.2 C per modification). The 2'-O-GE modified oligonucleotides exhibited exceptional resistance to nuclease degradation. The crystal structure of a palindromic duplex formed by a DNA oligonucleotide with a single 2'-O-GE modification was solved at 1.16 {angstrom} resolution.

  10. Properties of amphiphilic oligonucleotide films at the air/water interface and after film transfer.

    PubMed

    Keller, R; Kwak, M; de Vries, J W; Sawaryn, C; Wang, J; Anaya, M; Müllen, K; Butt, H-J; Herrmann, A; Berger, R

    2013-11-01

    The self-assembly of amphiphilic hybrid materials containing an oligonucleotide sequence at the air/water interface was investigated by means of pressure-molecular area (Π-A) isotherms. In addition, films were transferred onto solid substrates and imaged using scanning force microscopy. We used oligonucleotide molecules with lipid tails, which consisted of a single stranded oligonucleotide 11 mer containing two hydrophobically modified 5-(dodec-1-ynyl)uracil nucleobases (dU11) at the 5'-end of the oligonucleotide sequence. The air/water interface was used as confinement for the self-assembling process of dU11. Scanning force microscopy of films transferred via Langmuir-Blodgett technique revealed mono-, bi- (Π ≥ 2 mN/m) and multilayer formation (Π ≥ 30 mN/m). The first layer was 1.6 ± 0.1 nm thick. It was oriented with the hydrophilic oligonucleotide moiety facing the hydrophilic substrate while the hydrophobic alkyl chains faced air. In the second layer the oligonucleotide moiety was found to face the air. The second layer was found to cover up to 95% of the sample area. Our measurements indicated that the rearrangement of the molecules into bi- and multiple bilayers happened already at the air/water interface. Similar results were obtained with a second type of oligonucleotide amphiphile, an oligonucleotide block copolymer, which was composed of an oligonucleotide 11 mer covalently attached at the terminus to polypropyleneoxide (PPO).

  11. Phosphate Mines, Jordan

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Jordan's leading industry and export commodities are phosphate and potash, ranked in the top three in the world. These are used to make fertilizer. The Jordan Phosphate Mines Company is the sole producer, having started operations in 1935. In addition to mining activities, the company produces phosphoric acid (for fertilizers, detergents, pharmaceuticals), diammonium phosphate (for fertilizer), sulphuric acid (many uses), and aluminum fluoride (a catalyst to make aluminum and magnesium).

    The image covers an area of 27.5 x 49.4 km, was acquired on September 17, 2005, and is located near 30.8 degrees north latitude, 36.1 degrees east longitude.

    The U.S. science team is located at NASA's Jet Propulsion Laboratory, Pasadena, Calif. The Terra mission is part of NASA's Science Mission Directorate.

  12. [Dynamics of charge transfer along an oligonucleotide at finite temperature].

    PubMed

    Lakhno, V D; Fialko, N S

    2004-01-01

    The quantum-statistical approach was used to describe the charge transfer in nucleotide sequences. The results of numerical modeling for hole transfer in the GTTGGG sequence with background temperature noise are given. It was shown that, since guanine has an oxidation potential lower than thymine, the hole created at the G donor in this sequence passes through the thymine barrier into the guanine triplet (acceptor) at a time of approximately 10 ps at a temperature of 37 degrees C.

  13. Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

    PubMed

    Ma, Youlong; Libera, Matthew

    2016-06-28

    Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes. PMID:27253904

  14. Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray.

    PubMed

    Wang, Lih-Chiann; Huang, Dean; Pu, Chang-En; Wang, Ching-Ho

    2014-12-15

    Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions.

  15. Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

    PubMed

    Ma, Youlong; Libera, Matthew

    2016-06-28

    Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes.

  16. Fundamentals of phosphate transfer.

    PubMed

    Kirby, Anthony J; Nome, Faruk

    2015-07-21

    Historically, the chemistry of phosphate transfer-a class of reactions fundamental to the chemistry of Life-has been discussed almost exclusively in terms of the nucleophile and the leaving group. Reactivity always depends significantly on both factors; but recent results for reactions of phosphate triesters have shown that it can also depend strongly on the nature of the nonleaving or "spectator" groups. The extreme stabilities of fully ionised mono- and dialkyl phosphate esters can be seen as extensions of the same effect, with one or two triester OR groups replaced by O(-). Our chosen lead reaction is hydrolysis-phosphate transfer to water: because water is the medium in which biological chemistry takes place; because the half-life of a system in water is an accepted basic index of stability; and because the typical mechanisms of hydrolysis, with solvent H2O providing specific molecules to act as nucleophiles and as general acids or bases, are models for reactions involving better nucleophiles and stronger general species catalysts. Not least those available in enzyme active sites. Alkyl monoester dianions compete with alkyl diester monoanions for the slowest estimated rates of spontaneous hydrolysis. High stability at physiological pH is a vital factor in the biological roles of organic phosphates, but a significant limitation for experimental investigations. Almost all kinetic measurements of phosphate transfer reactions involving mono- and diesters have been followed by UV-visible spectroscopy using activated systems, conveniently compounds with good leaving groups. (A "good leaving group" OR* is electron-withdrawing, and can be displaced to generate an anion R*O(-) in water near pH 7.) Reactivities at normal temperatures of P-O-alkyl derivatives-better models for typical biological substrates-have typically had to be estimated: by extended extrapolation from linear free energy relationships, or from rate measurements at high temperatures. Calculation is free

  17. Species-Level Identification of Orthopoxviruses with an Oligonucleotide Microchip

    PubMed Central

    Lapa, Sergey; Mikheev, Maxim; Shchelkunov, Sergei; Mikhailovich, Vladimir; Sobolev, Alexander; Blinov, Vladimir; Babkin, Igor; Guskov, Alexander; Sokunova, Elena; Zasedatelev, Alexander; Sandakhchiev, Lev; Mirzabekov, Andrei

    2002-01-01

    A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used). PMID:11880388

  18. PCR amplification on microarrays of gel immobilized oligonucleotides

    SciTech Connect

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  19. Oligonucleotide microarray for subtyping of influenza A viruses

    NASA Astrophysics Data System (ADS)

    Klotchenko, S. A.; Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Chervyakova, O. V.; Smirnova, E. A.; Kushnareva, E. V.; Strochkov, V. M.; Taylakova, E. T.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Sansyzbay, A. R.

    2012-02-01

    Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

  20. Chemopreventive Agents from Physalis minima Function as Michael Reaction Acceptors

    PubMed Central

    Men, Ruizhi; Li, Ning; Ding, Chihong; Tang, Yingzhan; Xing, Yachao; Ding, Wanjing; Ma, Zhongjun

    2016-01-01

    Background: The fruits of some varieties of genus Physalis have been used as delicious fruits and functional food in the Northeast of China. Materials and Methods: To reveal the functional material basis, we performed bioactivity-guided phytochemical research and chemopreventive effect assay of the constituents from Physalis minima. Results: It was demonstrated that the ethyl acetate extract of P. minima L. (EEPM) had potential quinone reductase (QR) inducing activity with induction ratio (IR, QR induction activity) value of 1.47 ± 0.24, and glutathione binding property as potential Michael reaction acceptors (with an α, β-unsaturated ketone moiety). Furthermore, bioactivity-guided phytochemical research led eight compounds (1–8), which were elucidated as 3-isopropyl-5-acetoxycyclohexene-2-one-1 (1), isophysalin B (2), physalin G (3), physalin D (4), physalin I (5), physordinose B (6), stigmasterol-3-O-β-D-glucopyranoside (7) and 5α-6β-dihydroxyphysalin R (8) on the basis of nuclear magnetic resonance spectroscopy analyses and HRESIMS. Then, isophysalin B (2) and physordinose B (6) showed significant QR inducing activity with IR value of 2.80 ± 0.19 and 2.38 ± 0.46, respectively. SUMMARY An ultra-performance liquid chromatographic method with glutathione as the substrate was used to detect the Michael reaction acceptors in extracts of Physalis minima (EPM)We investigated the chemical constituents of EPM guided by biological activity methodIsophysalin B (1) and physordinose B (6) showed strong quinone reductase inducing activity with induction ratio values of 2.80 ± 0.19 and 2.38 ± 0.46This study generated useful information for consumers and many encourage researchers to utilize edible fruits from Physalis as a source of phytochemicals Abbreviations used: EPM: Extracts of Physalis minima, EEPM: Ethyl acetate extract of Physalis minima L., GSH: Glutathione, MRAs: Michael reaction acceptors, QR: Quinone reductase. PMID:27279713

  1. Nanostructured donor-acceptor self assembly with improved photoconductivity.

    PubMed

    Saibal, B; Ashar, A Z; Devi, R Nandini; Narayan, K S; Asha, S K

    2014-11-12

    Nanostructured supramolecular donor-acceptor assemblies were formed when an unsymmetrical N-substituted pyridine functionalized perylenebisimide (UPBI-Py) was complexed with oligo(p-phenylenevinylene) (OPVM-OH) complementarily functionalized with hydroxyl unit and polymerizable methacrylamide unit at the two termini. The resulting supramolecular complex [UPBI-Py (OPVM-OH)]1.0 upon polymerization by irradiation in the presence of photoinitiator formed well-defined supramolecular polymeric nanostructures. Self-assembly studies using fluorescence emission from thin film samples showed that subtle structural changes occurred on the OPV donor moiety following polymerization. The 1:1 supramolecular complex showed red-shifted aggregate emission from both OPV (∼500 nm) and PBI (∼640 nm) units, whereas the OPV aggregate emission was replaced by intense monomeric emission (∼430 nm) upon polymerizing the methacrylamide units on the OPVM-OH. The bulk structure was studied using wide-angle X-ray diffraction (WXRD). Complex formation resulted in distinct changes in the cell parameters of OPVM-OH. In contrast, a physical mixture of 1 mol each of OPVM-OH and UPBI-Py prepared by mixing the powdered solid samples together showed only a combination of reflections from both parent molecules. Thin film morphology of the 1:1 molecular complex as well as the supramolecular polymer complex showed uniform lamellar structures in the domain range <10 nm. The donor-acceptor supramolecular complex [UPBI-Py (OPVM-OH)]1.0 exhibited space charge limited current (SCLC) with a bulk mobility estimate of an order of magnitude higher accompanied by a higher photoconductivity yield compared to the pristine UPBI-Py. This is a very versatile method to obtain spatially defined organization of n and p-type semiconductor materials based on suitably functionalized donor and acceptor molecules resulting in improved photocurrent response using self-assembly.

  2. Nanostructured donor-acceptor self assembly with improved photoconductivity.

    PubMed

    Saibal, B; Ashar, A Z; Devi, R Nandini; Narayan, K S; Asha, S K

    2014-11-12

    Nanostructured supramolecular donor-acceptor assemblies were formed when an unsymmetrical N-substituted pyridine functionalized perylenebisimide (UPBI-Py) was complexed with oligo(p-phenylenevinylene) (OPVM-OH) complementarily functionalized with hydroxyl unit and polymerizable methacrylamide unit at the two termini. The resulting supramolecular complex [UPBI-Py (OPVM-OH)]1.0 upon polymerization by irradiation in the presence of photoinitiator formed well-defined supramolecular polymeric nanostructures. Self-assembly studies using fluorescence emission from thin film samples showed that subtle structural changes occurred on the OPV donor moiety following polymerization. The 1:1 supramolecular complex showed red-shifted aggregate emission from both OPV (∼500 nm) and PBI (∼640 nm) units, whereas the OPV aggregate emission was replaced by intense monomeric emission (∼430 nm) upon polymerizing the methacrylamide units on the OPVM-OH. The bulk structure was studied using wide-angle X-ray diffraction (WXRD). Complex formation resulted in distinct changes in the cell parameters of OPVM-OH. In contrast, a physical mixture of 1 mol each of OPVM-OH and UPBI-Py prepared by mixing the powdered solid samples together showed only a combination of reflections from both parent molecules. Thin film morphology of the 1:1 molecular complex as well as the supramolecular polymer complex showed uniform lamellar structures in the domain range <10 nm. The donor-acceptor supramolecular complex [UPBI-Py (OPVM-OH)]1.0 exhibited space charge limited current (SCLC) with a bulk mobility estimate of an order of magnitude higher accompanied by a higher photoconductivity yield compared to the pristine UPBI-Py. This is a very versatile method to obtain spatially defined organization of n and p-type semiconductor materials based on suitably functionalized donor and acceptor molecules resulting in improved photocurrent response using self-assembly. PMID:25283356

  3. Incorporation of Cu Acceptors in ZnO Nanocrystals

    SciTech Connect

    Oo, W.M.H.; Mccluskey, Matthew D.; Huso, Jesse; Morrison, J.; Bergman, Leah; Engelhard, Mark H.; Saraf, Laxmikant V.

    2010-09-16

    Doping of semiconductor nanocrystals is an important problem in nanomaterials research. Using infrared (IR) and x-ray photoelectron spectroscopy (XPS), we have observed Cu acceptor dopants that were intentionally introduced into ZnO nanocrystals. The incorporation of Cu2+ dopants increased as the diameter of the nanocrystals was increased from ~3 to 5 nm. Etching the nanocrystals with acetic acid revealed a core-shell structure, where a 2-nm lightly doped core is surrounded by a heavily doped shell. These observations are consistent with the trapped dopant model, in which dopant atoms stick to the surface of the core and are overgrown by the nanocrystal material.

  4. Ribose-5-phosphate isomerase and ribulose-5-phosphate kinase show apparent specificity for a specific ribulose 5-phosphate species.

    PubMed

    Anderson, L E

    1987-02-01

    Ribose-5-phosphate isomerase and ribulose-5-phosphate kinase appear to show specificity for a particular ribulose 5-phosphate species. The effect of this specificity will be channeling of ribulose 5-phosphate from the isomerase to the kinase during photosynthesis.

  5. Domestic phosphate deposits

    USGS Publications Warehouse

    McKelvey, V.E.; Cathcart, J.B.; Altschuler, Z.S.; Swanson, R.W.; Lutz, Katherine

    1953-01-01

    Most of the worlds phosphate deposits can be grouped into six types: 1) igneous apatite deposits; 2) marine phosphorites; 3) residual phosphorites; 4) river pebble deposits; 5) phosphatized rock; and 6) guano. The igneous apatites and marine phosphorites form deposits measurable in millions or billions of tons; the residual deposits are measurable in thousands or millions; and the other types generally only in thousands of tons. Igneous apatite deposits have been mined on a small scale in New York, New Jersey, and Virginia. Marine phosphorites have been mined in Montana, Idaho, Utah, Wyoming, Arkansas, Tennessee, North Carolina, South Carolina, Georgia, and Florida. Residual phosphorites have been mined in Tennessee, Pennsylvania, and Florida. River pebble has been produced in South Carolina and Florida; phosphatized rock in Tennessee and Florida; and guano in New Mexico and Texas. Present production is limited almost entirely to Florida, Tennessee, Montana, Idaho, and Wyoming. Incomplete but recently partly revised estimates indicate the presence of about 5 billion tons of phosphate deposits in the United States that is minable under present economic conditions. Deposits too lean in quality or thickness to compete with those in the western and southeastern fields probably contain tens of billions of tons.

  6. Prodrugs of phosphonates and phosphates: crossing the membrane barrier

    PubMed Central

    Wiemer, Andrew J.; Wiemer, David F.

    2016-01-01

    A substantial portion of metabolism involves transformation of phosphate esters, including pathways leading to nucleotides and oligonucleotides, carbohydrates, isoprenoids and steroids, and phosphorylated proteins. Because the natural substrates bear one or more negative charges, drugs that target these enzymes generally must be charged as well but small charged molecules can have difficulty traversing the cell membrane other than by endocytosis. The resulting dichotomy has stimulated abundant effort to develop effective prodrugs, compounds that carry little or no charge to enable them to transit biological membranes but then able to release the parent drug once inside the target cell. This chapter will present recent studies on advances in prodrug forms, along with representative examples of their application to marketed and developmental drugs. PMID:25391982

  7. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

    PubMed Central

    Sun, Hongguang; Zhu, Xun; Lu, Patrick Y; Rosato, Roberto R; Tan, Wen; Zu, Youli

    2014-01-01

    Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy. PMID:25093706

  8. In vivo delivery of transcription factors with multifunctional oligonucleotides

    NASA Astrophysics Data System (ADS)

    Lee, Kunwoo; Rafi, Mohammad; Wang, Xiaojian; Aran, Kiana; Feng, Xuli; Lo Sterzo, Carlo; Tang, Richard; Lingampalli, Nithya; Kim, Hyun Jin; Murthy, Niren

    2015-07-01

    Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury.

  9. Molecular dynamics study on DNA oligonucleotide translocation through carbon nanotubes.

    PubMed

    Pei, Q X; Lim, C G; Cheng, Y; Gao, Huajian

    2008-09-28

    Molecular dynamics simulations are performed to study the translocation of a DNA oligonucleotide in a carbon nanotube (CNT) channel consisting of CNTs of two different diameters. A strong gravitational acceleration field is applied to the DNA molecule and water solvent as an external driving force for the translocation. It is observed that both the CNT channel size and the strength of gravitational field have significant influence on the DNA translocation process. It is found that the DNA oligonucleotide is unable to pass through the (8,8) CNT even under strong gravitational fields, which extends previous finding that DNA cannot be self-inserted into a (8,8) CNT. It is shown that the DNA can pass through the (10,10)-(12,12) and (12,12)-(14,14) CNTs with stronger gravitational field resulting in faster translocation. The translocation time tau is found to follow the inverse power law relationship with the gravitational acceleration a as tau approximately a(-1.21). The energetic analysis of the translocation process shows that there is an energy barrier for DNA translocation into the (10,10) tube from the (14,14) tube, which is in contrast to previous report that DNA can be self-inserted into a (10,10) tube from outside the CNT. This difference with previous report shows that the dynamic behavior of DNA translocation inside a CNT channel is quite different from that of DNA translocation into a CNT from outside the CNT.

  10. Dendritic nanoconjugates for intracellular delivery of neutral oligonucleotides

    NASA Astrophysics Data System (ADS)

    Ming, Xin; Wu, Lin; Carver, Kyle; Yuan, Ahu; Min, Yuanzeng

    2015-07-01

    Dendrimer-based gene delivery has been constrained by intrinsic toxicity and suboptimal nanostructure. Conjugation of neutral morpholino oligonucleotides (ONs) with PAMAM dendrimers resulted in neutral, uniform, and ultra-small (~10 nm) nanoconjugates. The nanoconjugates dramatically enhanced cellular delivery of the ONs in cancer cells. After release from the dendrimer in the cytosol, the ONs produced potent functional activity without causing significant cytotoxicity. When carrying an apoptosis-promoting ON, the nanoconjugates produced cancer cell killing directly. Thus, the dendritic nanoconjugates may provide an effective tool for delivering ONs to tumors and other diseased tissues.Dendrimer-based gene delivery has been constrained by intrinsic toxicity and suboptimal nanostructure. Conjugation of neutral morpholino oligonucleotides (ONs) with PAMAM dendrimers resulted in neutral, uniform, and ultra-small (~10 nm) nanoconjugates. The nanoconjugates dramatically enhanced cellular delivery of the ONs in cancer cells. After release from the dendrimer in the cytosol, the ONs produced potent functional activity without causing significant cytotoxicity. When carrying an apoptosis-promoting ON, the nanoconjugates produced cancer cell killing directly. Thus, the dendritic nanoconjugates may provide an effective tool for delivering ONs to tumors and other diseased tissues. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01665g

  11. Gas-phase Dissociation of homo-DNA Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Stucki, Silvan R.; Désiron, Camille; Nyakas, Adrien; Marti, Simon; Leumann, Christian J.; Schürch, Stefan

    2013-12-01

    Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS3 of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series.

  12. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration.

    PubMed

    Richter, Katrin; Schicklberger, Marcus; Gescher, Johannes

    2012-02-01

    An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions.

  13. Functional analysis of a C. elegans trans-splice acceptor.

    PubMed Central

    Conrad, R; Liou, R F; Blumenthal, T

    1993-01-01

    The rol-6 gene is trans-spliced to the 22 nt leader, SL1, 173 nt downstream of the transcription start. We have analyzed splicing in transformants carrying extrachromosomal arrays of rol-6 with mutations in the trans-splice acceptor site. This site is a close match to the consensus, UUUCAG, that is highly conserved in both trans-splice and intron acceptor sites in C. elegans. When the trans-splice site was inactivated by mutating the perfectly-conserved AG, trans-splicing still occurred, but at a cryptic site 20 nt upstream. We tested the frequency with which splicing switched from the normal site to the cryptic site when the pyrimidines at this site were changed to A's. Since most C. elegans 3' splice sites lack an obvious polypyrimidine tract, we hypothesized that these four pyrimidines might play this role, and indeed mutation of these bases caused splicing to switch to the cryptic site. We also demonstrated that a major reason the downstream site is normally favored is because it occurs at a boundary between A+U rich and non-A+U rich RNA. When the RNA between the two splice sites was made less A+U rich, splicing occurred preferentially at the upstream site. Images PMID:8451190

  14. Dissimilatory Reduction of Extracellular Electron Acceptors in Anaerobic Respiration

    PubMed Central

    Richter, Katrin; Schicklberger, Marcus

    2012-01-01

    An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions. PMID:22179232

  15. Donor-acceptor complexation and dehydrogenation chemistry of aminoboranes.

    PubMed

    Malcolm, Adam C; Sabourin, Kyle J; McDonald, Robert; Ferguson, Michael J; Rivard, Eric

    2012-12-01

    A series of formal donor-acceptor adducts of aminoborane (H(2)BNH(2)) and its N-substituted analogues (H(2)BNRR') were prepared: LB-H(2)BNRR'(2)-BH(3) (LB = DMAP, IPr, IPrCH(2) and PCy(3); R and R' = H, Me or tBu; IPr = [(HCNDipp)(2)C:] and Dipp = 2,6-iPr(2)C(6)H(3)). To potentially access complexes of molecular boron nitride, LB-BN-LA (LA = Lewis acid), preliminary dehydrogenation chemistry involving the parent aminoborane adducts LB-H(2)BNH(2)-BH(3) was investigated using [Rh(COD)Cl](2), CuBr, and NiBr(2) as dehydrogenation catalysts. In place of isolating the intended dehydrogenated BN donor-acceptor complexes, the formation of borazine was noted as a major product. Attempts to prepare the fluoroarylborane-capped aminoborane complexes, LB-H(2)BNH(2)-B(C(6)F(5))(3), are also described. PMID:23153209

  16. An investigation of acceptor-doped grain boundaries in ?

    NASA Astrophysics Data System (ADS)

    Ravikumar, V.; Rodrigues, R. P.; Dravid, V. P.

    1996-07-01

    Grain boundary (GB) doped 0022-3727/29/7/014/img2 exhibits interesting electroceramic phenomena including varistor and barrier layer capacitor behaviour. We present here our investigation of GB acceptor-doped 0022-3727/29/7/014/img2 using analytical electron microscopy including electron holography. Mn was diffused into sintered polycrystalline 0022-3727/29/7/014/img2 to attain GBs which are rich in Mn. The presence and spatial extent of Mn at the GBs were analysed using x-ray emission spectroscopy (XES) and parallel electron energy loss spectroscopy (PEELS). The valence state of Mn was determined using PEELS to be predominantly +2. Finally, transmission high-energy electron holography was utilized to directly image and quantify the electrostatic potential and associated space-charge across the GBs directly. The holography results reveal a negatively charged GB with positive space-charge, indicating that Mn with a valence of +2 resides as an acceptor dopant on the Ti site at the GB core. The barrier height and local charge density distribution, including the Debye length, of the double Schottky barrier at the GB are derived from these holography results. This investigation demonstrates the usefulness of electron holography as a bulk-sensitive technique to probe the statics and dynamics of electrostatic field distribution and electrical charge across interfaces in technologically useful materials, and the need to employ diverse analytical techniques for such an investigation.

  17. Spatially Defined Oligonucleotide Arrays. Technical Report for Phase II

    SciTech Connect

    2000-06-15

    The goal of the Human Genome Project is to sequence all 3 billion base pairs of the human genome. Progress in this has been rapid; GenBank{reg_sign} finished 1994 with 286 million bases of sequence and grew by 2470 in the first quarter of 1995. The challenge to the scientific community is to understand the biological relevance of this genetic information. In most cases the sequence being generated for any single region of the genome represents the genotype of a single individual. A complete understanding of the function of specific genes and other regions of the genome and their role in human disease and development will only become apparent when the sequence of many more individuals is known. Access to genetic information is ultimately limited by the ability to screen DNA sequence. Although the pioneering sequencing methods of Sanger et al. (15) and Maxam and Gilbert (11) have become standard in virtually all molecular biology laboratories, the basic protocols remain largely unchanged. The throughput of this sequencing technology is now becoming the rate-limiting step in both large-scale sequencing projects such as the Human Genome Project and the subsequent efforts to understand genetic diversity. This has inspired the development of advanced DNA sequencing technologies (9), Incremental improvements to Sanger sequencing have been made in DNA labeling and detection. High-speed electrophoresis methods using ultrathin gels or capillary arrays are now being more widely employed. However, these methods are throughput-limited by their sequential nature and the speed and resolution of separations. This limitation will become more pronounced as the need to rapidly screen newly discovered genes for biologically relevant polymorphisms increases. An alternative to gel-based sequencing is to use high-density oligonucleotide probe arrays. Oligonucleotide probe arrays display specific oligonucleotide probes at precise locations in a high density, information-rich format (5

  18. Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides.

    PubMed Central

    Jacobsen, J P; Pedersen, J B; Hansen, L F; Wemmer, D E

    1995-01-01

    We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4- (3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolin ium tetraiodide (TOTO), to various double stranded DNA oligonucleotides. TOTO binds strongly to all the oligonucleotides used, but usually more than one complex is observed and exchange between different binding sites broadens the lines in the NMR spectra. Complete precipitation occurs when TOTO is bound to small oligonucleotides. Binding to larger oligonucleotides occurs by bis-intercalation. The 1:1 complex of TOTO with the oligonucleotide d(CCGACTGATGC):d (GCATCAGTCGG) gave only one complex that was shown to be a bis-intercalation in the CTGA:TCAG binding site. The binding to this site was also characterized by studying the TOTO complex with the d(CCGCTGAGC):d(GCTCAGCGG) oligonucleotide. NOE connectivities and molecular modelling were used to characterize the complex. The 1:1 complex of TOTO with the oligonucleotide d(CCGCTAGCG):d(CGCTAGCGG) containing a CTAG:CTAG binding site was similarly characterized by NMR. It was concluded that the binding of TOTO to larger oligonucleotides is site selective with CTAG:CTAG as the preferred binding site. PMID:7708489

  19. A convenient solid-phase method for synthesis of 3'-conjugates of oligonucleotides.

    PubMed

    Stetsenko, D A; Gait, M J

    2001-01-01

    We present a new procedure for the preparation of 3'-conjugates of oligonucleotides through solid-phase synthesis. A suitable universal solid support was readily prepared using a series of peptide-like coupling reactions to incorporate first a spacer and then an L-homoserine branching unit. The N-alpha-position of the homoserine carries an Fmoc protecting group that is removed by treatment with piperidine to liberate an amino group suitable for attachment of the conjugate (e.g., small organic molecule, fluorescent group, cholesterol, biotin, amino acid, etc.) or for assembly of a short peptide. The side-chain hydroxyl group of the homoserine carries a trityl protecting group. After TFA deprotection, the hydroxyl group acts as the site for oligonucleotide assembly. An additional spacer, such as aminohexanoyl, may be incorporated easily between the conjugate molecule and the oligonucleotide. A number of examples of synthesis of 3'-conjugates of oligonucleotides and their analogues are described that involve standard automated oligonucleotide assembly and use of commercially available materials. The linkage between oligonucleotide and 3'-conjugate is chirally pure and is stable to conventional ammonia treatment used for oligonucleotide deprotection and release from the solid support. The homoserine-functionalized solid support system represents a simple and universal route to 3'-conjugates of oligonucleotides and their derivatives.

  20. Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides.

    PubMed

    Jacobsen, J P; Pedersen, J B; Hansen, L F; Wemmer, D E

    1995-03-11

    We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4- (3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolin ium tetraiodide (TOTO), to various double stranded DNA oligonucleotides. TOTO binds strongly to all the oligonucleotides used, but usually more than one complex is observed and exchange between different binding sites broadens the lines in the NMR spectra. Complete precipitation occurs when TOTO is bound to small oligonucleotides. Binding to larger oligonucleotides occurs by bis-intercalation. The 1:1 complex of TOTO with the oligonucleotide d(CCGACTGATGC):d (GCATCAGTCGG) gave only one complex that was shown to be a bis-intercalation in the CTGA:TCAG binding site. The binding to this site was also characterized by studying the TOTO complex with the d(CCGCTGAGC):d(GCTCAGCGG) oligonucleotide. NOE connectivities and molecular modelling were used to characterize the complex. The 1:1 complex of TOTO with the oligonucleotide d(CCGCTAGCG):d(CGCTAGCGG) containing a CTAG:CTAG binding site was similarly characterized by NMR. It was concluded that the binding of TOTO to larger oligonucleotides is site selective with CTAG:CTAG as the preferred binding site.

  1. Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

    DOEpatents

    Golova, Julia B.; Chernov, Boris K.

    2010-04-27

    New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

  2. Oligonucleotide-modified screen-printed gold electrodes for enzyme-amplified sensing of nucleic acids.

    PubMed

    Carpini, Guido; Lucarelli, Fausto; Marrazza, Giovanna; Mascini, Marco

    2004-09-15

    An electrochemical genosensor for the detection of specific sequences of DNA has been developed using disposable screen-printed gold electrodes. Screen-printed gold electrodes were firstly modified with a mixed monolayer of a 25-mer thiol-tethered DNA probe and a spacer thiol, 6-mercapto-1-hexanol (MCH). The DNA probe sequence was internal to the sequence of the 35S promoter, which sequence is inserted in the genome of GMOs regulating the transgene expression. An enzyme-amplified detection scheme, based on the coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the electroinactive alpha-naphthyl phosphate to alpha-naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. The assay was, firstly, characterised using synthetic oligonucleotides. Relevant parameters, such as the probe concentration and the immobilisation time, the use of the MCH and different enzymatic conjugates, were investigated and optimised. The genosensor response was found to be linearly related to the target concentration between 0 and 25 nmol/L; the detection limit was 0.25 nmol/L. The analytical procedure was then applied for the detection of the 35S promoter sequence, which was amplified from the pBI121 plasmid by polymerase chain reaction (PCR). Hybridisation conditions (i.e., hybridisation buffer and hybridisation time) were further optimised. The selectivity of the assay was confirmed using biotinylated non-complementary amplicons and PCR blanks. The results showed that the genosensor enabled sensitive (detection limit: 1 nmol/L) and specific detection of GMO-related sequences, thus providing a useful tool for the screening analysis of bioengineered food samples.

  3. The structure and bonding of iron-acceptor pairs in silicon

    SciTech Connect

    Zhao, S.; Assali, L.V.C.; Kimerling, L.C.

    1995-08-01

    The highly mobile interstitial iron and Group III impurities (B, Al, Ga, In) form iron-acceptor pairs in silicon. Based on the migration kinetics and taking host silicon as a dielectric medium, we have simulated the pairing process in a static silicon lattice. Different from the conventional point charge ionic model, our phenomenological calculations include (1) a correction that takes into account valence electron cloud polarization which adds a short range, attractive interaction in the iron-acceptor pair bonding; and (2) silicon lattice relaxation due to the atomic size difference which causes a local strain field. Our model explains qualitatively (1) trends among the iron-acceptor pairs revealing an increase of the electronic state hole emission energy with increasing principal quantum number of acceptor and decreasing pair separation distance; and (2) the stable and metastable sites and configurational symmetries of the iron-acceptor pairs. The iron-acceptor pairing and bonding mechanism is also discussed.

  4. Binding characteristics of homogeneous molecularly imprinted polymers for acyclovir using an (acceptor-donor-donor)-(donor-acceptor-acceptor) hydrogen-bond strategy, and analytical applications for serum samples.

    PubMed

    Wu, Suqin; Tan, Lei; Wang, Ganquan; Peng, Guiming; Kang, Chengcheng; Tang, Youwen

    2013-04-12

    This paper demonstrates a novel approach to assembling homogeneous molecularly imprinted polymers (MIPs) based on mimicking multiple hydrogen bonds between nucleotide bases by preparing acyclovir (ACV) as a template and using coatings grafted on silica supports. (1)H NMR studies confirmed the AAD-DDA (A for acceptor, D for donor) hydrogen-bond array between template and functional monomer, while the resultant monodisperse molecularly imprinted microspheres (MIMs) were evaluated using a binding experiment, high performance liquid chromatography (HPLC), and solid phase extraction. The Langmuir isothermal model and the Langmuir-Freundlich isothermal model suggest that ACV-MIMs have more homogeneous binding sites than MIPs prepared through normal imprinting. In contrast to previous MIP-HPLC columns, there were no apparent tailings for the ACV peaks, and ACV-MIMs had excellent specific binding properties with a Ka peak of 3.44 × 10(5)M(-1). A complete baseline separation is obtained for ACV and structurally similar compounds. This work also successfully used MIMs as a specific sorbent for capturing ACV from serum samples. The detection limit and mean recovery of ACV was 1.8 ng/mL(-1) and 95.6%, respectively, for molecularly imprinted solid phase extraction coupled with HPLC. To our knowledge, this was the first example of MIPs using AAD-DDA hydrogen bonds.

  5. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

    PubMed

    Soontornworajit, Boonchoy; Zhou, Jing; Snipes, Matthew P; Battig, Mark R; Wang, Yong

    2011-10-01

    Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

  6. Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics.

    PubMed

    Fahy, E; Davis, G R; DiMichele, L J; Ghosh, S S

    1993-04-25

    Polyacrylamide supports, in a range of pore sizes, were investigated as nucleic acid affinity matrices for the detection of target DNA or RNA sequences using a sandwich hybridization format. Bromoacetyl and thiol oligonucleotide derivatives were covalently linked to sulfhydryl- and bromoacetyl-polyacrylamide supports with greater than 95% end-attachment efficiencies. These polyacrylamide-oligonucleotide supports were further derivatized with anionic residues to provide multi-functional supports which show low non-specific binding for non-complementary nucleic acids. While all the polyacrylamide-oligonucleotide supports capture complementary oligonucleotides with high affinity, the pore size was found to be a critical parameter in sandwich hybridization reactions. The superior hybridization characteristics of the Trisacryl support was ascribed to a combination of its macroporous nature, hydrophilicity and the terminal attachment of its capture oligonucleotides.

  7. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.

    PubMed Central

    Guo, Z; Guilfoyle, R A; Thiel, A J; Wang, R; Smith, L M

    1994-01-01

    A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene. Images PMID:7816638

  8. Donor-Acceptor-Type Semiconducting Polymers Consisting of Benzothiadiazole Derivatives as Electron-Acceptor Units for Organic Photovoltaic Cells.

    PubMed

    Kim, Hee Su; Park, Jong Baek; Kim, Ji-Hoon; Hwang, Do-Hoon

    2015-11-01

    We synthesized two fused pentacyclic donor-acceptor structures, where the two different outer electron rich thiophene (DTPBT) and electron poor benzene (ICTh) moieties are covalently bonded to the central electron-deficient benzothiadiazole core by two nitrogen bridges. These new electron-acceptor DTPBT and ICTh building blocks were copolymerized with fluorene, as the electron donor group, via Suzuki coupling polymerization, to produce two new alternating copolymers, PFDTPBT and PFICTh, respectively. The average molecular weights of the synthesized polymers were determined by GPC. The number-average molecular weights of PFDTPBT and PFICTh were 19,000 (PDI = 2.5) and 20,000 (PDI = 4.0), respectively. The optical bandgap energies of the polymers were measured from their absorption onsets to be 2.15 and 2.55 eV, depending on the polymer structure. The HOMO energy levels of the polymers were determined, by measuring the oxidation onsets of the polymer films by cyclic voltammetry. The measured HOMO energy levels of PFDTPBT and PFICTh were -5.10 and -5.57 eV, respectively. When the polymers were blended with PC71BM, as the active layer for bulk-heterojunction photovoltaic devices, power conversion efficiencies were 2.08% and 0.34%, respectively, under AM 1.5 G (100 mW cm(-2)) conditions.

  9. 21 CFR 184.1434 - Magnesium phosphate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Magnesium phosphate. 184.1434 Section 184.1434... Listing of Specific Substances Affirmed as GRAS § 184.1434 Magnesium phosphate. (a) Magnesium phosphate includes both magnesium phosphate, dibasic, and magnesium phosphate, tribasic. Magnesium phosphate,...

  10. 21 CFR 184.1434 - Magnesium phosphate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Magnesium phosphate. 184.1434 Section 184.1434... Listing of Specific Substances Affirmed as GRAS § 184.1434 Magnesium phosphate. (a) Magnesium phosphate includes both magnesium phosphate, dibasic, and magnesium phosphate, tribasic. Magnesium phosphate,...

  11. 21 CFR 184.1434 - Magnesium phosphate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Magnesium phosphate. 184.1434 Section 184.1434... Listing of Specific Substances Affirmed as GRAS § 184.1434 Magnesium phosphate. (a) Magnesium phosphate includes both magnesium phosphate, dibasic, and magnesium phosphate, tribasic. Magnesium phosphate,...

  12. 21 CFR 184.1434 - Magnesium phosphate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Magnesium phosphate. 184.1434 Section 184.1434... GRAS § 184.1434 Magnesium phosphate. (a) Magnesium phosphate includes both magnesium phosphate, dibasic, and magnesium phosphate, tribasic. Magnesium phosphate, dibasic (MgHPO4·3H2O, CAS Reg. No....

  13. 21 CFR 184.1434 - Magnesium phosphate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Magnesium phosphate. 184.1434 Section 184.1434 Food... Specific Substances Affirmed as GRAS § 184.1434 Magnesium phosphate. (a) Magnesium phosphate includes both magnesium phosphate, dibasic, and magnesium phosphate, tribasic. Magnesium phosphate, dibasic...

  14. Preliminary time-of-flight neutron diffraction studies of Escherichia coli ABC transport receptor phosphate-binding protein at the Protein Crystallography Station

    PubMed Central

    Sippel, K. H.; Bacik, J.; Quiocho, F. A.; Fisher, S. Z.

    2014-01-01

    Inorganic phosphate is an essential molecule for all known life. Organisms have developed many mechanisms to ensure an adequate supply, even in low-phosphate conditions. In prokaryotes phosphate transport is instigated by the phosphate-binding protein (PBP), the initial receptor for the ATP-binding cassette (ABC) phosphate transporter. In the crystal structure of the PBP–phosphate complex, the phosphate is completely desolvated and sequestered in a deep cleft and is bound by 13 hydrogen bonds: 12 to protein NH and OH donor groups and one to a carboxylate acceptor group. The carboxylate plays a key recognition role by accepting a phosphate hydrogen. PBP phosphate affinity is relatively consistent across a broad pH range, indicating the capacity to bind monobasic (H2PO4 −) and dibasic (HPO4 2−) phosphate; however, the mechanism by which it might accommodate the second hydrogen of monobasic phosphate is unclear. To answer this question, neutron diffraction studies were initiated. Large single crystals with a volume of 8 mm3 were grown and subjected to hydrogen/deuterium exchange. A 2.5 Å resolution data set was collected on the Protein Crystallography Station at the Los Alamos Neutron Science Center. Initial refinement of the neutron data shows significant nuclear density, and refinement is ongoing. This is the first report of a neutron study from this superfamily. PMID:24915101

  15. Biomediated continuous release phosphate fertilizer

    DOEpatents

    Goldstein, Alan H.; Rogers, Robert D.

    1999-01-01

    A composition is disclosed for providing phosphate fertilizer to the root zone of plants. The composition comprises a microorganism capable of producing and secreting a solubilization agent, a carbon source for providing raw material for the microorganism to convert into the solubilization agent, and rock phosphate ore for providing a source of insoluble phosphate that is solubilized by the solubilization agent and released as soluble phosphate. The composition is provided in a physical form, such as a granule, that retains the microorganism, carbon source, and rock phosphate ore, but permits water and soluble phosphate to diffuse into the soil. A method of using the composition for providing phosphate fertilizer to plants is also disclosed.

  16. Biomediated continuous release phosphate fertilizer

    DOEpatents

    Goldstein, A.H.; Rogers, R.D.

    1999-06-15

    A composition is disclosed for providing phosphate fertilizer to the root zone of plants. The composition comprises a microorganism capable of producing and secreting a solubilization agent, a carbon source for providing raw material for the microorganism to convert into the solubilization agent, and rock phosphate ore for providing a source of insoluble phosphate that is solubilized by the solubilization agent and released as soluble phosphate. The composition is provided in a physical form, such as a granule, that retains the microorganism, carbon source, and rock phosphate ore, but permits water and soluble phosphate to diffuse into the soil. A method of using the composition for providing phosphate fertilizer to plants is also disclosed. 13 figs.

  17. Generation of Nitrogen Acceptors in ZnO using Pulse Thermal Processing

    SciTech Connect

    Xu, Jun; Ott, Ronald D; Sabau, Adrian S; Pan, Zhengwei; Xiu, Faxian; Liu, Jilin; Erie, Jean-Marie; Norton, David P

    2008-01-01

    Bipolar doping in wide bandgap semiconductors is difficult to achieve under equilibrium conditions because of the spontaneous formation of compensating defects and unfavorable energetics for dopant substitution. In this work, we explored the use of rapid pulse thermal processing for activating nitrogen dopants into acceptor states in ZnO. Low-temperature photoluminescence spectra revealed both acceptor-bound exciton (A{sup 0}X) and donor-acceptor pair emissions, which present direct evidence for acceptors generated after pulse thermal processing of nitrogen-doped ZnO. This work suggests that pulse thermal processing is potentially an effective method for p-type doping of ZnO.

  18. Dysregulation of phosphate metabolism and conditions associated with phosphate toxicity

    PubMed Central

    Brown, Ronald B; Razzaque, Mohammed S

    2015-01-01

    Phosphate homeostasis is coordinated and regulated by complex cross-organ talk through delicate hormonal networks. Parathyroid hormone (PTH), secreted in response to low serum calcium, has an important role in maintaining phosphate homeostasis by influencing renal synthesis of 1,25-dihydroxyvitamin D, thereby increasing intestinal phosphate absorption. Moreover, PTH can increase phosphate efflux from bone and contribute to renal phosphate homeostasis through phosphaturic effects. In addition, PTH can induce skeletal synthesis of another potent phosphaturic hormone, fibroblast growth factor 23 (FGF23), which is able to inhibit renal tubular phosphate reabsorption, thereby increasing urinary phosphate excretion. FGF23 can also fine-tune vitamin D homeostasis by suppressing renal expression of 1-alpha hydroxylase (1α(OH)ase). This review briefly discusses how FGF23, by forming a bone–kidney axis, regulates phosphate homeostasis, and how its dysregulation can lead to phosphate toxicity that induces widespread tissue injury. We also provide evidence to explain how phosphate toxicity related to dietary phosphorus overload may facilitate incidence of noncommunicable diseases including kidney disease, cardiovascular disease, cancers and skeletal disorders. PMID:26131357

  19. Dysregulation of phosphate metabolism and conditions associated with phosphate toxicity.

    PubMed

    Brown, Ronald B; Razzaque, Mohammed S

    2015-01-01

    Phosphate homeostasis is coordinated and regulated by complex cross-organ talk through delicate hormonal networks. Parathyroid hormone (PTH), secreted in response to low serum calcium, has an important role in maintaining phosphate homeostasis by influencing renal synthesis of 1,25-dihydroxyvitamin D, thereby increasing intestinal phosphate absorption. Moreover, PTH can increase phosphate efflux from bone and contribute to renal phosphate homeostasis through phosphaturic effects. In addition, PTH can induce skeletal synthesis of another potent phosphaturic hormone, fibroblast growth factor 23 (FGF23), which is able to inhibit renal tubular phosphate reabsorption, thereby increasing urinary phosphate excretion. FGF23 can also fine-tune vitamin D homeostasis by suppressing renal expression of 1-alpha hydroxylase (1α(OH)ase). This review briefly discusses how FGF23, by forming a bone-kidney axis, regulates phosphate homeostasis, and how its dysregulation can lead to phosphate toxicity that induces widespread tissue injury. We also provide evidence to explain how phosphate toxicity related to dietary phosphorus overload may facilitate incidence of noncommunicable diseases including kidney disease, cardiovascular disease, cancers and skeletal disorders. PMID:26131357

  20. Quantum information processing using acceptors in silicon and phonon entanglement

    NASA Astrophysics Data System (ADS)

    Clark, Susan; Reinke, Charles; McGuinness, Hayden; El-Kady, Ihab

    2014-03-01

    Quantum computing with large numbers of qubits remains challenging due to the decoherence and complexity that arise as more qubits are added to a system. Here I propose a new platform for semiconductor quantum computing which may be robust to common sources of decoherence and may not be difficult to fabricate repeatedly. This system consists of a hole bound to an acceptor in silicon which has been implanted in the center of a mechanical cavity (similar to a photonic crystal cavity) and connected to other cavities by a system of waveguides. I will outline a basic entangling gate and calculations showing the promise of this platform as the ideal qubit. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U. S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  1. Insights to primitive replication derived from structures of small oligonucleotides

    NASA Technical Reports Server (NTRS)

    Smith, G. K.; Fox, G. E.

    1995-01-01

    Available information on the structure of small oligonucleotides is surveyed. It is observed that even small oligomers typically exhibit defined structures over a wide range of pH and temperature. These structures rely on a plethora of non-standard base-base interactions in addition to the traditional Watson-Crick pairings. Stable duplexes, though typically antiparallel, can be parallel or staggered and perfect complementarity is not essential. These results imply that primitive template directed reactions do not require high fidelity. Hence, the extensive use of Watson-Crick complementarity in genes rather than being a direct consequence of the primitive condensation process, may instead reflect subsequent selection based on the advantage of accuracy in maintaining the primitive genetic machinery once it arose.

  2. [Research progress of probe design software of oligonucleotide microarrays].

    PubMed

    Chen, Xi; Wu, Zaoquan; Liu, Zhengchun

    2014-02-01

    DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software.

  3. Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

    PubMed

    Steffens, David L; Williams, John G K

    2007-07-01

    We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%. PMID:17595310

  4. Oligonucleotide synthesis catalyzed by the Zn/2+/ ion

    NASA Technical Reports Server (NTRS)

    Sawai, H.; Orgel, L. E.

    1975-01-01

    Results of experiments are reported in which Zn(2+) ion catalyzed the formation of oligonucleotides from nucleoside phosphorimidazolides in aqueous solution, even in the absence of a template. Specifically, the imidazolides (ImpU or ImpA) polymerized to form ImpApA, and pApA, pApApA, and pApApApA, or the analogous uracil compounds. In addition, the expected hydrolysis products of the hydrolysis of ImpA were formed (pA, imidazole). Judging from the ratio of pA(n) over pA (with and without zinc ion), this ion increased the efficiency of phosphodiester-bond formation by up to 10 times. Possible mechanisms for the reaction are tentatively proposed.

  5. A review of statistical methods for preprocessing oligonucleotide microarrays.

    PubMed

    Wu, Zhijin

    2009-12-01

    Microarrays have become an indispensable tool in biomedical research. This powerful technology not only makes it possible to quantify a large number of nucleic acid molecules simultaneously, but also produces data with many sources of noise. A number of preprocessing steps are therefore necessary to convert the raw data, usually in the form of hybridisation images, to measures of biological meaning that can be used in further statistical analysis. Preprocessing of oligonucleotide arrays includes image processing, background adjustment, data normalisation/transformation and sometimes summarisation when multiple probes are used to target one genomic unit. In this article, we review the issues encountered in each preprocessing step and introduce the statistical models and methods in preprocessing.

  6. Aptamer Oligonucleotides: Novel Potential Therapeutic Agents in Autoimmune Disease.

    PubMed

    Li, Weibin; Lan, Xiaopeng

    2015-08-01

    Aptamers are single-stranded deoxyribonucleic acid or ribonucleic acid oligonucleotides generated in vitro based on affinity for certain target molecules by a process known as Systematic Evolution of Ligands by Exponential Enrichment. Aptamers can bind their target molecules with high specificity and selectivity by means of structure compatibility, stacking of aromatic rings, electrostatic and van der Waals interactions, and hydrogen bonding. With several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch to batch variation, flexible modification and stability, lack of toxicity and low immunogenicity, aptamers are becoming promising novel diagnostic and therapeutic agents. This review focuses on the development of aptamers as potential therapeutics for autoimmune diseases, including diabetes mellitus, multiple sclerosis, rheumatoid arthritis, myasthenia gravis, and systemic lupus erythematosus. PMID:25993618

  7. Computer simulation in template-directed oligonucleotide synthesis

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia; Benasconi, Claude F.

    1990-01-01

    It is commonly assumed that template-directed polymerizations have played a key role in prebiotic evolution. A computer simulation that models up to 33 competing reactions was used to investigate the product distribution in a template-directed oligonucleotide synthesis as a function of time and concentration of the reactants. The study focuses on the poly(C)-directed elongation reaction of oligoguanylates, and how it is affected by the competing processes of hydrolysis and dimerization of the activated monomer, which have the potential of severely curtailing the elongation and reducing the size and yield of the synthesized polymers. The simulations show that realistic and probably prebiotically plausible conditions can be found where hydrolysis and dimerization are either negligible or where a high degree of polymerization can be attained even in the face of substantial hydrolysis and/or dimerization.

  8. Beyond fullerenes: design of nonfullerene acceptors for efficient organic photovoltaics.

    PubMed

    Li, Haiyan; Earmme, Taeshik; Ren, Guoqiang; Saeki, Akinori; Yoshikawa, Saya; Murari, Nishit M; Subramaniyan, Selvam; Crane, Matthew J; Seki, Shu; Jenekhe, Samson A

    2014-10-15

    New electron-acceptor materials are long sought to overcome the small photovoltage, high-cost, poor photochemical stability, and other limitations of fullerene-based organic photovoltaics. However, all known nonfullerene acceptors have so far shown inferior photovoltaic properties compared to fullerene benchmark [6,6]-phenyl-C60-butyric acid methyl ester (PC60BM), and there are as yet no established design principles for realizing improved materials. Herein we report a design strategy that has produced a novel multichromophoric, large size, nonplanar three-dimensional (3D) organic molecule, DBFI-T, whose π-conjugated framework occupies space comparable to an aggregate of 9 [C60]-fullerene molecules. Comparative studies of DBFI-T with its planar monomeric analogue (BFI-P2) and PC60BM in bulk heterojunction (BHJ) solar cells, by using a common thiazolothiazole-dithienosilole copolymer donor (PSEHTT), showed that DBFI-T has superior charge photogeneration and photovoltaic properties; PSEHTT:DBFI-T solar cells combined a high short-circuit current (10.14 mA/cm(2)) with a high open-circuit voltage (0.86 V) to give a power conversion efficiency of 5.0%. The external quantum efficiency spectrum of PSEHTT:DBFI-T devices had peaks of 60-65% in the 380-620 nm range, demonstrating that both hole transfer from photoexcited DBFI-T to PSEHTT and electron transfer from photoexcited PSEHTT to DBFI-T contribute substantially to charge photogeneration. The superior charge photogeneration and electron-accepting properties of DBFI-T were further confirmed by independent Xenon-flash time-resolved microwave conductivity measurements, which correctly predict the relative magnitudes of the conversion efficiencies of the BHJ solar cells: PSEHTT:DBFI-T > PSEHTT:PC60BM > PSEHTT:BFI-P2. The results demonstrate that the large size, multichromophoric, nonplanar 3D molecular design is a promising approach to more efficient organic photovoltaic materials.

  9. Recent advances in photoinduced donor/acceptor copolymerization

    NASA Astrophysics Data System (ADS)

    Jönsson, S.; Viswanathan, K.; Hoyle, C. E.; Clark, S. C.; Miller, C.; Morel, F.; Decker, C.

    1999-05-01

    Photoinitiated free radical polymerization of donor (D)/acceptor (A) type monomers has gained considerable interest due to the possibility to efficiently photopolymerize non-acrylate based systems. Furthermore, this photoinduced alternating copolymerization can be accomplished without the presence of a conventional free radical generating photoinitiator. In the past, we have shown that the structural influences in the direct photolysis of N-Alkyl and N-Arylmaleimides as well as their corresponding ground state charge transfer complexes (CTC) with suitable donors have carefully been investigated. For certain combinations of A and D type monomers, a direct photolysis of the ground state complex or the excitation of the acceptor, followed by the formation of an exciplex, has been shown to initiate the copolymerization. Herein, we show that the main route of initiation is based on inter or intra molecular H-abstraction from an excited state maleimide, whereby no exciplex formation takes place. H-abstraction will predominantly take place in systems where easily abstractable hydrogens are present. Our laser flash photolysis investigation, ESR [1] (A. Hiroshi, I. Takasi, T. Nosi, Macromol. Chem. 190 (1989) 2821) and phosphorescence emissions [2,3] (K.S. Chen, T. Foster, J.K.S. Wan, J. Phys. Chem. 84 (1980) 2473; C.J. Seliskar, S.P. McGlynn, J. Chem. Phys. 55 (1971) 4337) studies show that triplet excited states of N-alkyl substituted maleimides (RMI), which are well known strong precursors for direct H-abstractions from aliphatic ethers and secondary alcohols, are formed upon excitation. Rates of copolymerization and degrees of conversion for copolymerization of maleimide/vinyl ether pairs in air and nitrogen have been measured as a function of hydrogen abstractability of the excited triplet state MI as well as the influence of concentration and hydrogen donating effect of the hydrogen donor.

  10. Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

    PubMed Central

    Hanecak, R; Brown-Driver, V; Fox, M C; Azad, R F; Furusako, S; Nozaki, C; Ford, C; Sasmor, H; Anderson, K P

    1996-01-01

    Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and

  11. Guanine modification of inhibitory oligonucleotides potentiates their suppressive function.

    PubMed

    Römmler, Franziska; Jurk, Marion; Uhlmann, Eugen; Hammel, Monika; Waldhuber, Anna; Pfeiffer, Lavinia; Wagner, Hermann; Vollmer, Jörg; Miethke, Thomas

    2013-09-15

    Inhibitory TLR7 and/or TLR9 oligonucleotides (inhibitory oligonucleotide [INH-ODN]) are characterized by a phosphorothioate backbone and a CC(T)XXX₃₋₅GGG motif, respectively. INH-ODN 2088 is a prototypic member of this class of INH-ODN and acts as a TLR7 and TLR9 antagonist. It contains a G quadruple that leads to higher order structures by the formation of G tetrads. These structures are unfavorable for the prediction of their pharmacological and immunological behavior. We show in this study that modification of Gs within the G quadruple by 7-deaza-guanine or 7-deaza-2'-O-methyl-guanine avoids higher order structures and improves their inhibitory potential. Whereas TLR9-induced TNF-α secretion of bone marrow-derived macrophages and conventional dendritic cells was equally inhibited by INH-ODN 2088 and G-modified INH-ODNs such as INH-ODN 24888, TLR7-induced TNF-α release and TLR7- and TLR9-induced IL-12p40 release were significantly more impaired by G-modified INH-ODNs. Similarly, the IL-6 release of B cells from wild-type and autoimmune MRL/Mp-lpr/lpr mice was more efficiently impaired by G-modified INH-ODNs. Surprisingly, INH-ODN 2088 stimulated B cells to proliferate when used in higher doses. Finally, in vivo, in wild-type and autoimmune MRL/Mp-lpr/lpr mice, G-modified INH-ODN 24888 was significantly more efficient than unmodified INH-ODN 2088. In summary, G modification allows the development of INH-ODNs with superior inhibitory potency for inflammatory diseases with high medical need such as systemic lupus erythematosus. PMID:23966630

  12. Ultrafast photoinduced charge transport in Pt(II) donor-acceptor assembly bearing naphthalimide electron acceptor and phenothiazine electron donor.

    PubMed

    Sazanovich, Igor V; Best, Jonathan; Scattergood, Paul A; Towrie, Michael; Tikhomirov, Sergei A; Bouganov, Oleg V; Meijer, Anthony J H M; Weinstein, Julia A

    2014-12-21

    Visible light-induced charge transfer dynamics were investigated in a novel transition metal triad acceptor-chromophore-donor, (NDI-phen)Pt(II)(-C≡C-Ph-CH2-PTZ)2 (1), designed for photoinduced charge separation using a combination of time-resolved infrared (TRIR) and femtosecond electronic transient absorption (TA) spectroscopy. In 1, the electron acceptor is 1,4,5,8-naphthalene diimide (NDI), and the electron donor is phenothiazine (PTZ), and [(phen)Pt(-C≡C-Ph-)], where phen is 1,10-phenanthroline, represents the chromophoric core. The first excited state observed in 1 is a (3)MLCT/LL'CT, with {Pt(II)-acetylide}-to-phen character. Following that, charge transfer from the phen-anion onto the NDI subunit to form NDI(-)-phen-[Pt-(C≡C)2](+)-PTZ2 occurs with a time constant of 2.3 ps. This transition is characterised by appearance of the prominent NDI-anion features in both TRIR and TA spectra. The final step of the charge separation in 1 proceeds with a time constant of ∼15 ps during which the hole migrates from the [Pt-(C≡C)2] subunit to one of the PTZ groups. Charge recombination in 1 then occurs with two distinct time constants of 36 ns and 107 ns, corresponding to the back electron transfer to each of the two donor groups; a rather rare occurrence which manifests that the hole in the final charge-separated state is localised on one of the two donor PTZ groups. The assignment of the nature of the excited states and dynamics in 1 was assisted by TRIR investigations of the analogous previously reported ((COOEt)2bpy)Pt(C≡C-Ph-CH2-PTZ)2 (2), (J. E. McGarrah and R. Eisenberg, Inorg. Chem., 2003, 42, 4355; J. E. McGarrah, J. T. Hupp and S. N. Smirnov, J. Phys. Chem. A, 2009, 113, 6430) as well as (bpy)Pt(C≡C-Ph-C7H15)2, which represent the acceptor-free dyad, and the chromophoric core, respectively. Thus, the step-wise formation of the full charge-separated state on the picosecond time scale and charge recombination via tunnelling have been established; and

  13. Contrasting performance of donor-acceptor copolymer pairs in ternary blend solar cells and two-acceptor copolymers in binary blend solar cells.

    PubMed

    Khlyabich, Petr P; Rudenko, Andrey E; Burkhart, Beate; Thompson, Barry C

    2015-02-01

    Here two contrasting approaches to polymer-fullerene solar cells are compared. In the first approach, two distinct semi-random donor-acceptor copolymers are blended with phenyl-C61-butyric acid methyl ester (PC61BM) to form ternary blend solar cells. The two poly(3-hexylthiophene)-based polymers contain either the acceptor thienopyrroledione (TPD) or diketopyrrolopyrrole (DPP). In the second approach, semi-random donor-acceptor copolymers containing both TPD and DPP acceptors in the same polymer backbone, termed two-acceptor polymers, are blended with PC61BM to give binary blend solar cells. The two approaches result in bulk heterojunction solar cells that have the same molecular active-layer components but differ in the manner in which these molecular components are mixed, either by physical mixing (ternary blend) or chemical "mixing" in the two-acceptor (binary blend) case. Optical properties and photon-to-electron conversion efficiencies of the binary and ternary blends were found to have similar features and were described as a linear combination of the individual components. At the same time, significant differences were observed in the open-circuit voltage (Voc) behaviors of binary and ternary blend solar cells. While in case of two-acceptor polymers, the Voc was found to be in the range of 0.495-0.552 V, ternary blend solar cells showed behavior inherent to organic alloy formation, displaying an intermediate, composition-dependent and tunable Voc in the range from 0.582 to 0.684 V, significantly exceeding the values achieved in the two-acceptor containing binary blend solar cells. Despite the differences between the physical and chemical mixing approaches, both pathways provided solar cells with similar power conversion efficiencies, highlighting the advantages of both pathways toward highly efficient organic solar cells.

  14. Oligonucleotides designed to inhibit TLR9 block Herpes simplex virus type 1 infection at multiple steps.

    PubMed

    Sauter, Monica M; Gauger, Joshua J L; Brandt, Curtis R

    2014-09-01

    Herpes simplex virus type 1 (HSV-1) is an important human pathogen which requires activation of nuclear factor-kappa B (NFκB) during its replication cycle. The persistent nature of HSV-1 infection, and the emergence of drug-resistant strains, highlights the importance of research to develop new antiviral agents. Toll-like receptors (TLRs) play a prominent role during the early antiviral response by recognizing viral nucleic acid and gene products, activating NFκB, and stimulating the production of inflammatory cytokines. We demonstrate a significant effect on HSV-1 replication in ARPE-19 and Vero cells when oligonucleotides designed to inhibit TLR9 are added 2h prior to infection. A greater than 90% reduction in the yield of infectious virus was achieved at oligonucleotide concentrations of 10-20 μM. TLR9 inhibitory oligonucleotides prevented expression of essential immediate early herpes gene products as determined by immunofluorescence microscopy and Western blotting. TLR9 oligonucleotides also interfered with viral attachment and entry. A TLR9 inhibitory oligonucleotide containing five adjacent guanosine residues (G-ODN) exhibited virucidal activity and inhibited HSV-1 replication when added post-infection. The antiviral effect of the TLR9 inhibitory oligonucleotides did not depend on the presence of TLR9 protein, suggesting a mechanism of inhibition that is not TLR9 specific. TLR9 inhibitory oligonucleotides also reduced NFκB activity in nuclear extracts. Studies using these TLR inhibitors in the context of viral infection should be interpreted with caution.

  15. Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

    PubMed Central

    Hoehlig, Kai; Bethge, Lucas; Klussmann, Sven

    2015-01-01

    A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications. PMID:25679211

  16. Enzymatic synthesis of modified oligonucleotides by PEAR using Phusion and KOD DNA polymerases.

    PubMed

    Wang, Xuxiang; Zhang, Jianye; Li, Yingjia; Chen, Gang; Wang, Xiaolong

    2015-02-01

    Antisense synthetic oligonucleotides have been developed as potential gene-targeted therapeutics. We previously reported polymerase-endonuclease amplification reaction (PEAR) for amplification of natural and 5'-O-(1-thiotriphosphate) (S)-modified oligonucleotides. Here, we extended the PEAR technique for enzymatic preparation of 2'-deoxy-2'-fluoro-(2'-F) and 2'-F/S double-modified oligonucleotides. The result showed that KOD and Phusion DNA polymerase could synthesize oligonucleotides with one or two modified nucleotides, and KOD DNA polymerase is more suitable than Phusion DNA polymerase for PEAR amplification of 2'-F and 2'-F/S double modified oligonucleotides. The composition of PEAR products were analyzed by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection and showed that the sequence of the PEAR products are maintained at an extremely high accuracy (>99.9%), and after digestion the area percent of full-length modified oligonucleotides reaches 89.24%. PEAR is suitable for synthesis of modified oligonucleotides efficiently and with high purity. PMID:25517220

  17. Solubilization of insoluble phosphates by thermophilic fungi.

    PubMed

    Singh, C P; Mishra, M M; Yadav, K S

    1980-01-01

    The solubilization of tricalcium phosphate and rock phosphate and assimilation of solubilized P by thermophilic fungi isolated from compost were studied. The solubilization of tricalcium phosphate was greater than that of rock phosphate on inoculation with fungi in liquid medium, but growth of most of the fungi was greater in rock phosphate. Torula thermophila solubilized tricalcium phosphate maximally. There was solubilization of rock phosphate in semi-solid lignocellulose medium by Aspergillus fumigatus.

  18. Phthalimides as exceptionally efficient single electron transfer acceptors in reductive coupling reactions promoted by samarium diiodide.

    PubMed

    Vacas, Tatiana; Alvarez, Eleuterio; Chiara, Jose Luis

    2007-12-20

    Experimental and theoretical evidence shows that phthalimides are highly efficient single electron transfer acceptors in reactions promoted by samarium diiodide, affording ketyl radical anion intermediates, which participate in high-yielding inter- and intramolecular reductive coupling processes with different radicophiles including imides, oxime ethers, nitrones, and Michael acceptors.

  19. Effects and mechanisms of hemopoiesis-stimulating activity of immobilized oligonucleotides under conditions of cytostatic myelosuppression.

    PubMed

    Dygai, A M; Goldberg, V E; Artamonov, A V; Bekarev, A A; Vereschagin, E I; Madonov, P G; Skurikhin, E G; Pershina, O V; Andreeva, T V; Khmelevskaya, E S; Ermakova, N N

    2012-02-01

    Hemopoiesis-stimulating activity of immobilized oligonucleotide preparation was studied on the model of cytostatic myelosuppression induced by injection of cyclophosphamide and 5-fluorouracil. Immobilized oligonucleotides stimulated regeneration of erythro- and granulocytopoiesis in the bone marrow under conditions of cytostatic treatment. The counts of neutrophilic granulocytes and platelets in the peripheral blood increased. The stimulatory effect of the drug was more manifest in animals with active behavior. The mechanism of immobilized oligonucleotide effect was based on stimulation of functional activity of erythroid and granulocytic macrophage precursors.

  20. Method for promoting specific alignment of short oligonucleotides on nucleic acids

    DOEpatents

    Studier, F. William; Kieleczawa, Jan; Dunn, John J.

    1996-01-01

    Disclosed is a method for promoting specific alignment of short oligonucleotides on a nucleic acid polymer. The nucleic acid polymer is incubated in a solution containing a single-stranded DNA-binding protein and a plurality of oligonucleotides which are perfectly complementary to distinct but adjacent regions of a predetermined contiguous nucleotide sequence in the nucleic acid polymer. The plurality of oligonucleotides anneal to the nucleic acid polymer to form a contiguous region of double stranded nucleic acid. Specific application of the methods disclosed include priming DNA synthesis and template-directed ligation.

  1. The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

    PubMed Central

    Richards, J. B.; Hemming, F. W.

    1972-01-01

    When pig liver microsomal preparations were incubated with GDP-[14C]mannose, 10–40% of the 14C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [14C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [14C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [14C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [3H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [14C]mannose from GDP-[14C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an

  2. Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides

    PubMed Central

    Joli, Flore; Bouchemal, Nadia; Laigle, Alain; Hartmann, Brigitte; Hantz, Edith

    2006-01-01

    A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G•U wobble pair, capped by the purine-rich 5′r(GGGAUG)3′ hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G•U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2′-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides. PMID:17041234

  3. The effect of intermolecular donor?acceptor energy transfer on emission anisotropy in uniaxially oriented polymer films

    NASA Astrophysics Data System (ADS)

    Sadownik, M.; Bojarski, Piotr

    2004-10-01

    Excitation energy transport between donors and acceptors is studied for uniaxially stretched and unstretched poly(vinyl) alcohol films. Donor emission anisotropy courses versus acceptor concentration as well as donor-acceptor emission anisotropy spectra occurred quite different in stretched and unstretched films. Upon donor excitation the total emission anisotropy rapidly decreases when passing from the donor to the acceptor fluorescence band in disordered systems. However, such a pronounced effect has not been found in partly ordered films. Donors and acceptors exhibit highly preferential mutual orientation in strongly ordered films resulting in the preservation of acceptor emission anisotropy.

  4. Physiological and electrochemical effects of different electron acceptors on bacterial anode respiration in bioelectrochemical systems.

    PubMed

    Yang, Yonggang; Xiang, Yinbo; Xia, Chunyu; Wu, Wei-Min; Sun, Guoping; Xu, Meiying

    2014-07-01

    To understand the interactions between bacterial electrode respiration and the other ambient bacterial electron acceptor reductions, alternative electron acceptors (nitrate, Fe2O3, fumarate, azo dye MB17) were added singly or multiply into Shewanella decolorationis microbial fuel cells (MFCs). All the added electron acceptors were reduced simultaneously with current generation. Adding nitrate or MB17 resulted in more rapid cell growth, higher flavin concentration and higher biofilm metabolic viability, but lower columbic efficiency (CE) and normalized energy recovery (NER) while the CE and NER were enhanced by Fe2O3 or fumarate. The added electron acceptors also significantly influenced the cyclic voltammetry profile of anode biofilm probably via altering the cytochrome c expression. The highest power density was observed in MFCs added with MB17 due to the electron shuttle role of the naphthols from MB17 reduction. The results provided important information for MFCs applied in practical environments where contains various electron acceptors.

  5. Excitation energy transfer in partly ordered polymer films differing in donor and acceptor transition moments orientation

    NASA Astrophysics Data System (ADS)

    Synak, A.; Bojarski, P.; Sadownik, M.; Kułak, L.; Gryczynski, I.; Grobelna, B.; Rangełowa-Jankowska, S.; Jankowski, D.; Kubicki, A.

    2016-09-01

    Based on spectroscopic measurements selected properties of nonradiative Förster energy transport are studied in uniaxially stretched polyvinyl alcohol thin films for three systems differing in donor and acceptor transition moments orientation relative to the axis of stretching. In particular, donor - acceptor emission anisotropy spectra yield completely different regularities for these systems in uniaxially stretched films, whereas they are similar in unstretched films. In particular it is shown that acceptor fluorescence can be either strongly polarized after nonradiative energy transfer in stretched films or depolarized depending on the angular distribution of acceptor transition moments in the matrix. Donor and acceptor emission anisotropy decays exhibit similar regularities to those of steady-state measurements. The obtained results are analyzed with the help of Monte Carlo simulations.

  6. Ultrasound-modulated fluorescence based on donor-acceptor-labeled microbubbles

    PubMed Central

    Liu, Yuan; Feshitan, Jameel A.; Wei, Ming-Yuan; Borden, Mark A.; Yuan, Baohong

    2015-01-01

    Abstract. A fluorescence resonance energy transfer (FRET)-based microbubble contrast agent system was designed to experimentally demonstrate the concept of ultrasound-modulated fluorescence (UMF). Microbubbles were simultaneously labeled with donor and acceptor fluorophores on the surface to minimize self-quenching and maximize FRET. In response to ultrasound, the quenching efficiency was greatly modulated by changing the distance between the donor and acceptor molecules through microbubble size oscillations. Both donors and acceptors exhibited UMF on individual microbubbles. The UMF strength of the donor was more significant compared to that of the acceptor. Furthermore, the UMF of the donor was observed from a microbubble solution in a turbid media. This study exploits the feasibility of donor–acceptor labeled microbubbles as UMF contrast agents. PMID:25789423

  7. Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

    PubMed

    Ciaccio, P J; Jaiswal, A K; Tew, K D

    1994-06-01

    A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents. PMID:7515059

  8. Crystal structure of 4-sulfamoylanilinium di­hydrogen phosphate

    PubMed Central

    Muthuselvi, C.; Mala, N.; Srinivasan, N.; Pandiarajan, S.; Krishnakumar, R. V.

    2014-01-01

    In the crystal structure of the title mol­ecular salt, C6H9N2O2S+·H2PO4 −, the sulfomylalinium cations and the di­hydrogen phosphate anions form independent [100] chains through Ns—H⋯O (s = sulfamo­yl) and O—H⋯O hydrogen bonds, respectively. The chains are cross-linked by Na—H⋯O (a = amine) hydrogen bonds, generating (010) sheets. Two C—H⋯O hydrogen bonds involving diametrically opposite C atoms in the benzene ring of the cation as donors form chains parallel to [202] in which P=O and P—OH groups are acceptors. Together, these inter­actions lead to a three-dimensional network. PMID:25309301

  9. Carboranyl Nucleosides & Oligonucleotides for Neutron Capture Therapy Final Report

    SciTech Connect

    Schinazi, Raymond F.

    2004-12-01

    This proposal enabled us to synthesize and develop boron-rich nucleosides and oligonucleotide analogues for boron neutron capture therapy (BNCT) and the treatment of various malignancies. First, we determined the relationship between structure, cellular accumulation and tissue distribution of 5-o-carboranyl-2'-deoxyuridine (D-CDU) and its derivatives D-ribo-CU and 5-o-carboranyluracil (CU), to potentially target brain and other solid tumors for neutron capture therapy. Synthesized carborane containing nucleoside derivatives of CDU, D- and L-enantiomers of CDU, D-ribo-CU and CU were used. We measured tissue disposition in xenografted mice bearing 9479 human prostate tumors xenografts and in rats bearing 9L gliosarcoma isografts in their flanks and intracranially. The accumulation of D-CDU, 1-({beta}-L-arabinosyl)-5-o-carboranyluracil, D-ribo-CU, and CU were also studied in LnCap human prostate tumor cells and their retention was measured in male nude mice bearing LnCap and 9479 human prostate tumor xenografts. D-CDU, D-ribo-CU and CU levels were measured after administration in mice bearing 9479 human prostate tumors in their flanks. D-CDU achieved high cellular concentrations in LnCap cells and up to 2.5% of the total cellular compound was recovered in the 5'-monophosphorylated form. D-CDU cellular concentrations were similar in LnCap and 9479 tumor xenografts. Studies in tumor bearing animals indicated that increasing the number of hydroxyl moieties in the sugar constituent of the carboranyl nucleosides lead to increased rate and extent of renal elimination, a decrease in serum half-lives and an increased tissue specificity. Tumor/brain ratios were greatest for CDU and D-ribo-CU, while tumor/prostate ratios were greatest with CU. CDU and D-ribo-CU have potential for BNCT of brain malignancies, while CU may be further developed for prostate cancer. A method was developed for the solid phase synthesis of oligonucleotides containing (ocarboran-1-yl

  10. Comparative study of Trichoderma gene expression in interactions with tomato plants using high-density oligonucleotide microarrays.

    PubMed

    Rubio, M Belén; Domínguez, Sara; Monte, Enrique; Hermosa, Rosa

    2012-01-01

    Trichoderma spp. are widely used as biopesticides and biofertilizers to control diseases and to promote positive physiological responses in plants. In vitro and in vivo assays with Trichoderma harzianum CECT 2413 (T34), Trichoderma virens Gv29-8 (T87) and Trichoderma hamatum IMI 224801 (T7) revealed that these strains affected the growth and development of lateral roots in tomato plants in different ways. The early expression profiles of these Trichoderma strains were studied after 20 h of incubation in the presence of tomato plants, using a high-density oligonucleotide (HDO) microarray, and compared to the profiles in the absence of plants. Out of the total 34 138 Trichoderma probe sets deposited on the microarray, 1077 (3.15 %) showed a significant change of at least 2-fold in expression in the presence of tomato plants. The numbers of probe sets identified in the individual Trichoderma strains were 593 in T. harzianum T34, 336 in T. virens T87 and 94 in T. hamatum T7. Carbohydrate metabolism - the chitin degradation enzymes N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase and chitinase - was the most significantly overrepresented process commonly observed in the three Trichoderma strains in early interactions with tomato plants. Strains T7 and T34, which had similar positive effects on plant development in biological assays, showed a significantly overrepresented hexokinase activity in interaction with tomato. In addition, genes encoding a 40S ribosomal protein and a P23 tumour protein were altered in both these strains.

  11. Potassium acceptor doping of ZnO crystals

    SciTech Connect

    Parmar, Narendra S. Lynn, K. G.; Corolewski, Caleb D.; McCluskey, Matthew D.

    2015-05-15

    ZnO bulk single crystals were doped with potassium by diffusion at 950°C. Positron annihilation spectroscopy confirms the filling of zinc vacancies and a different trapping center for positrons. Secondary ion mass spectroscopy measurements show the diffusion of potassium up to 10 μm with concentration ∼1 × 10{sup 16} cm{sup −3}. IR measurements show a local vibrational mode (LVM) at 3226 cm{sup −1}, at a temperature of 9 K, in a potassium doped sample that was subsequently hydrogenated. The LVM is attributed to an O–H bond-stretching mode adjacent to a potassium acceptor. When deuterium substitutes for hydrogen, a peak is observed at 2378 cm{sup −1}. The O-H peak is much broader than the O-D peak, perhaps due to an unusually low vibrational lifetime. The isotopic frequency ratio is similar to values found in other hydrogen complexes. Potassium doping increases the resistivity up to 3 orders of magnitude at room temperature. The doped sample has a donor level at 0.30 eV.

  12. Molecular insights into the terminal energy acceptor in cyanobacterial phycobilisome.

    PubMed

    Gao, Xiang; Wei, Tian-Di; Zhang, Nan; Xie, Bin-Bin; Su, Hai-Nan; Zhang, Xi-Ying; Chen, Xiu-Lan; Zhou, Bai-Cheng; Wang, Zhi-Xin; Wu, Jia-Wei; Zhang, Yu-Zhong

    2012-09-01

    The linker protein L(CM) (ApcE) is postulated as the major component of the phycobilisome terminal energy acceptor (TEA) transferring excitation energy from the phycobilisome to photosystem II. L(CM) is the only phycobilin-attached linker protein in the cyanobacterial phycobilisome through auto-chromophorylation. However, the underlying mechanism for the auto-chromophorylation of L(CM) and the detailed molecular architecture of TEA is still unclear. Here, we demonstrate that the N-terminal phycobiliprotein-like domain of L(CM) (Pfam00502, LP502) can specifically recognize phycocyanobilin (PCB) by itself. Biochemical assays indicated that PCB binds into the same pocket in LP502 as that in the allophycocyanin α-subunit and that Ser152 and Asp155 play a vital role in LP502 auto-chromophorylation. By carefully conducting computational simulations, we arrived at a rational model of the PCB-LP502 complex structure that was supported by extensive mutational studies. In the PCB-LP502 complex, PCB binds into a deep pocket of LP502 with a distorted conformation, and Ser152 and Asp155 form several hydrogen bonds to PCB fixing the PCB Ring A and Ring D. Finally, based on our results, the dipoles and dipole-dipole interactions in TEA are analysed and a molecular structure for TEA is proposed, which gives new insights into the energy transformation mechanism of cyanobacterial phycobilisome. PMID:22758351

  13. Swift Electrofluorochromism of Donor-Acceptor Conjugated Polytriphenylamines.

    PubMed

    Sun, Jingwei; Liang, Ziqi

    2016-07-20

    Electrofluorochromic (EFC) materials, which exhibit electrochemically controllable fluorescence, hold great promise in optoelectronic devices and biological analysis. Here we design such donor-acceptor (D-A) conjugated polymers-P(TPACO) and P(TCEC)-that contain the same electron-rich and oxidizable polytriphenylamine (PTPA) as π-backbone, yet with different electron-deficient ketone and cyano units as pendant groups, respectively. They both exhibit solvatochromic effects due to intrinsic characteristics of intramolecular charge transfer (ICT). Compared to P(TPACO), P(TCEC) shows stronger ICT, which leads to higher electrochemical oxidation potential and lower ion diffusion coefficient. Moreover, both polymers present simultaneous electrochromic (EC) and EFC behaviors with multistate display and remarkably rapid fluorescence response. The response time of P(TPACO) is as short as 0.19 s, nearly 4-fold faster than that of P(TCEC) (0.92 s). Such rapid response is found to be determined by the ion diffusion coefficient which is associated with the ICT nature. Finally, the EFC display device based on P(TPACO) is successfully demonstrated, which shows green fluorescence ON/OFF switching upon applied potentials. This work has successfully demonstrated that swift EFCs can be achieved by rational modulation of the ICT effect in such D-A conjugated polymers. PMID:27347724

  14. Poly(trifluoromethyl)azulenes: structures and acceptor properties

    SciTech Connect

    Clikeman, Tyler T.; Bukovsky, Eric V.; Kuvychko, Igor V.; San, Long K.; Deng, Shihu; Wang, Xue B.; Chen, Yu-Sheng; Strauss, Steven H.; Boltalina, Olga V.

    2014-07-10

    Azulene is a non-alternant, non-benzenoid aromatic hydrocarbon with an intense blue colour, a dipole moment of 1.0 D,1 positive electron affinity, and an “anomalous” emission from the second excited state in violation of Kasha’s rule.2,3 Azulene’s unique properties have potential uses in molecular switches,4,5 molecular diodes,6 organic photovoltaics,7 and charge transfer complexes.8-12 Introduction of electron-withdrawing groups to the azulenic core, such as CN,8,13,14 halogens,15-19 and CF3,20,21 can enhance certain electrical and photophysical properties. In this work, we report six new trifluoromethyl derivatives of azulene (AZUL), three isomers of AZUL(CF3)3 and three isomers of AZUL(CF3)4, and the first X-ray structure of a π-stacked donor-acceptor complex of a trifluoromethyl azulene with donor pyrene.

  15. Analysis of nonlinear optical properties in donor–acceptor materials

    SciTech Connect

    Day, Paul N.; Pachter, Ruth; Nguyen, Kiet A.

    2014-05-14

    Time-dependent density functional theory has been used to calculate nonlinear optical (NLO) properties, including the first and second hyperpolarizabilities as well as the two-photon absorption cross-section, for the donor-acceptor molecules p-nitroaniline and dimethylamino nitrostilbene, and for respective materials attached to a gold dimer. The CAMB3LYP, B3LYP, PBE0, and PBE exchange-correlation functionals all had fair but variable performance when compared to higher-level theory and to experiment. The CAMB3LYP functional had the best performance on these compounds of the functionals tested. However, our comprehensive analysis has shown that quantitative prediction of hyperpolarizabilities is still a challenge, hampered by inadequate functionals, basis sets, and solvation models, requiring further experimental characterization. Attachment of the Au{sub 2}S group to molecules already known for their relatively large NLO properties was found to further enhance the response. While our calculations show a modest enhancement for the first hyperpolarizability, the enhancement of the second hyperpolarizability is predicted to be more than an order of magnitude.

  16. Nucleoside, nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids.

    PubMed

    Gissot, Arnaud; Camplo, Michel; Grinstaff, Mark W; Barthélémy, Philippe

    2008-04-21

    Amphiphilic molecules based on nucleosides, nucleotides and oligonucleotides are finding more and more biotechnological applications. This Perspective highlights their synthesis, supramolecular organization as well as their applications in the field of biotechnology.

  17. Exploiting protected maleimides to modify oligonucleotides, peptides and peptide nucleic acids.

    PubMed

    Paris, Clément; Brun, Omar; Pedroso, Enrique; Grandas, Anna

    2015-04-10

    This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  18. Photoluminescence and electrochemiluminescence of a Ru(II)(bpy)3-quencher dual-labeled oligonucleotide probe.

    PubMed

    Wilson, Robert; Johansson, Mary Katherine

    2003-11-01

    A molecular beacon oligonucleotide probe covalently labeled with Ru(II)(bpy)3 and Black Hole Quencher-2 is synthesized, and hybridization assays are performed using photoluminescence and electrochemiluminescence methods of excitation.

  19. Use of synthetic oligonucleotides for genomic DNA dot hybridization to split the DQw3 haplotype.

    PubMed Central

    Martell, M; Le Gall, I; Millasseau, P; Dausset, J; Cohen, D

    1988-01-01

    Comparison of two different HLA-DQ beta gene sequences from two DR4 individuals, probably corresponding to DQw3.2 (DQR4) and DQw3.1 (DQR5) specificities, has shown several nucleotide variations. Eight oligonucleotides (24 bases long), derived from these polymorphic areas, have been synthesized. Each oligonucleotide was hybridized to BamHI-digested DNA samples from eight families with HLA-DR4 individuals. Four polymorphic BamHI fragments were detected. Two of eight oligonucleotides gave a single signal (8.9 kilobases) on DQw3.2-positive haplotypes. We used one of these oligonucleotides in a genomic DNA dot hybridization and detected a hybridization signal only in DQw3.2-positive individuals. A very simple test like this allows the screening of a large population sample within a very short period. Images PMID:2895927

  20. A Polyethylenimine-Containing and Transferrin-Conjugated Lipid Nanoparticle System for Antisense Oligonucleotide Delivery to AML

    PubMed Central

    Yuan, Yiming; Zhang, Lijing; Cao, Hua; Yang, Yi; Zheng, Yu; Yang, Xiao-juan

    2016-01-01

    Limited success of antisense oligonucleotides (ASO) in clinical anticancer therapy calls for more effective delivery carriers. The goal of this study was to develop a nanoparticle system for delivery of ASO G3139, which targets mRNA of antiapoptotic protein Bcl-2, to acute myeloid leukemia (AML) cells. The synthesized nanoparticle Tf-LPN-G3139 contained a small molecular weight polyethylenimine and two cationic lipids as condensing agents, with transferrin on its surface for selective binding and enhanced cellular uptake. The optimized nitrogen to phosphate (N/P) ratio was 4 to achieve small particle size and high G3139 entrapment efficiency. The Tf-LPN-G3139 exhibited excellent colloidal stability during storage for at least 12 weeks and remained intact for 4 hours in nuclease-containing serum. The cellular uptake results showed extensive internalization of fluorescence-labelled G3139 in MV4-11 cells through Tf-LPN. Following transfection, Tf-LPN-G3139 at 1 µM ASO level induced 54% Bcl-2 downregulation and >20-fold apoptosis compared to no treatment. When evaluated in mice bearing human xenograft AML tumors, Tf-LPN-G3139 suppressed tumor growth by ~60% at the end of treatment period, accompanied by remarkable pharmacological effect of Bcl-2 inhibition in tumor. In conclusion, Tf-LPN-G3139 is a promising nanoparticle system for ASO G3139 delivery to AML and warrants further investigations. PMID:27034925

  1. Sheath liquid effects in capillary high-performance liquid chromatography-electrospray mass spectrometry of oligonucleotides.

    PubMed

    Huber, C G; Krajete, A

    2000-02-18

    Fused-silica capillary columns of 200 microm inner diameter were packed with micropellicular, octadecylated, 2.3 microm poly(styrene-divinylbenzene) particles and applied to the separation of oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography. Oligonucleotides were eluted at 50 degrees C with gradients of 3-13% acetonitrile in 50 mM triethylammonium bicarbonate. Addition of sheath liquid to the column effluent allowed the detection of oligonucleotides by electrospray ionization mass spectrometry using full-scan data acquisition with a detectability comparable to that obtained with UV detection. The signal-to-noise ratios with different sheath liquids increased in the order isopropanololigonucleotides longer than 20 nucleotide units whereas no significant effect was observed with shorter oligonucleotides. Organic acids and bases in the sheath liquid generally deteriorated the signal-to-noise ratios in the chromatograms and mass spectra mainly because of increased background noise. Only a few charge states were observed in the mass spectra of oligonucleotides because of charge state reduction due to the presence of carbonic acid in the eluent. With triethylammonium hydrogencarbonate as chromatographic eluent and acetonitrile as sheath liquid, very few cation adducts of oligonucleotides were observed in the mass spectra. However, the presence of small amounts of monopotassium adducts enabled the calculation of the charge state of multiply charged ions. With acetonitrile as sheath liquid, 710 amol of a 16-mer oligonucleotide were detected using selected ion monitoring data acquisition with a signal-to-noise ratio of 3:1. Finally, capillary ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was

  2. Complexes of carbon nanotubes with oligonucleotides in thin Langmuir-Blodgett films to detect electrochemically hybridization

    NASA Astrophysics Data System (ADS)

    Egorov, A. S.; Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Orekhovskaya, T. I.; Veligura, A. A.; Govorov, M. I.; Shulitsky, B. G.

    2014-10-01

    Self-assembled complexes consisting of thin multi-walled carbon nanotubes (MWCNTs) and DNA-oligonucleotides which are able to a cooperative binding to complementary oligonucleotides have been investigated. It was establised a high-performance charge transport in nanostructured Langmuir-Blodgett complexes thin MWCNTs/DNA. A method to electrochemically detect DNA hybridization on the self-organized structures has been proposed.

  3. [A prototype of oligonucleotide microarray for detection of pathogens relating to arena- and Filoviridae families].

    PubMed

    Zhirnov, I V; Ryabinin, V A; Sinyakov, A N; Ternovoy, V A; Shikov, A N

    2015-01-01

    A prototype of oligonucleotide microarray for detection of Lassa, Junin, Machupo, Guanarito viruses (Arenaviridae family), Ebola and Marburg viruses (Filoviridae family) was presented. An original approach founded on virus proteins (nucleocapsid protein for Junin, Guanarito, Machupo viruses and RNA-dependent RNA-polymerase for Lassa, Ebola and Marburg viruses) amino acid sequences analysis with subsequent transform of revealed unique peptides into due sets of oligonucleotides was used to design probes for hybridization and primers.

  4. Rates of primary electron transfer reactions in the photosystem I reaction center reconstituted with different quinones as the secondary acceptor

    SciTech Connect

    Kumazaki, Shigeichi; Kandori, Hideki; Yoshihara, Keitaro ); Iwaki, Masayo; Itoh, Shigeru ); Ikegamu, Isamu )

    1994-10-27

    Rates of sequential electron transfer reactions from the primary electron donor chlorophyll dimer (P700) to the electron acceptor chlorophyll a-686 (A[sub 0]) and to the secondary acceptor quinone (Q[sub [phi

  5. AFM and electroanalytical studies of synthetic oligonucleotide hybridization.

    PubMed

    Chiorcea Paquim, A-M; Diculescu, V C; Oretskaya, T S; Oliveira Brett, A M

    2004-11-15

    The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.

  6. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    NASA Astrophysics Data System (ADS)

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao

    2016-05-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

  7. Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides

    PubMed Central

    Wang, Shiyu; Sun, Hong; Tanowitz, Michael; Liang, Xue-hai; Crooke, Stanley T.

    2016-01-01

    Chemically modified antisense oligonucleotides (ASOs) designed to mediate site-specific cleavage of RNA by RNase H1 are used as research tools and as therapeutics. ASOs modified with phosphorothioate (PS) linkages enter cells via endocytotic pathways. The mechanisms by which PS-ASOs are released from membrane-enclosed endocytotic organelles to reach target RNAs remain largely unknown. We recently found that annexin A2 (ANXA2) co-localizes with PS-ASOs in late endosomes (LEs) and enhances ASO activity. Here, we show that co-localization of ANXA2 with PS-ASO is not dependent on their direct interactions or mediated by ANXA2 partner protein S100A10. Instead, ANXA2 accompanies the transport of PS-ASOs to LEs, as ANXA2/PS-ASO co-localization was observed inside LEs. Although ANXA2 appears not to affect levels of PS-ASO internalization, ANXA2 reduction caused significant accumulation of ASOs in early endosomes (EEs) and reduced localization in LEs and decreased PS-ASO activity. Importantly, the kinetics of PS-ASO activity upon free uptake show that target mRNA reduction occurs at least 4 hrs after PS-ASOs exit from EEs and is coincident with release from LEs. Taken together, our results indicate that ANXA2 facilitates PS-ASO trafficking from early to late endosomes where it may also contribute to PS-ASO release. PMID:27378781

  8. Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides.

    PubMed

    Han, Yonghua; Zhang, Tao; Thammapichai, Paradee; Weng, Yiqun; Jiang, Jiming

    2015-07-01

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000-27,000 oligos. These probes spanned 8.3-17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5-3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F1 hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids. PMID:25971668

  9. Dicationic Surfactants with Glycine Counter Ions for Oligonucleotide Transportation.

    PubMed

    Pietralik, Zuzanna; Skrzypczak, Andrzej; Kozak, Maciej

    2016-08-01

    Gemini surfactants are good candidates to bind, protect, and deliver nucleic acids. Herein, the concept of amino acids (namely glycine) as counter ions of gemini surfactants for gene therapy application was explored. This study was conducted on DNA and RNA oligomers and two quaternary bis-imidazolium salts, having 2,5-dioxahexane and 2,8-dioxanonane spacer groups. The toxicity level of surfactants was assessed by an MTT assay, and their ability to bind nucleic acids was tested through electrophoresis. The nucleic acid conformation was established based on circular dichroism and infrared spectroscopic analyses. The structures of the formed complexes were characterized by small-angle scattering of synchrotron radiation. Both studied surfactants appear to be suitable for gene therapy; however, although they vary by only three methylene groups in the spacer, they differ in binding ability and toxicity. The tested oligonucleotides maintained their native conformations upon surfactant addition and the studied lipoplexes formed a variety of structures. In systems based on a 2,5-dioxahexane spacer, a hexagonal phase was observed for DNA-surfactant complexes and a micellar phase was dominant with RNA. For the surfactant with a 2,8-dioxanonane spacer group, the primitive cubic phase prevailed. PMID:27214208

  10. Elucidation of gene function using C-5 propyne antisense oligonucleotides.

    PubMed

    Flanagan, W M; Su, L L; Wagner, R W

    1996-09-01

    Identification of human disease-causing genes continues to be an intense area of research. While cloning of genes may lead to diagnostic tests, development of a cure requires an understanding of the gene's function in both normal and diseased cells. Thus, there exists a need for a reproducible and simple method to elucidate gene function. We evaluate C-5 propyne pyrimidine modified phosphorothioate antisense oligonucleotides (ONs) targeted against two human cell cycle proteins that are aberrantly expressed in breast cancer: p34cdc2 kinase and cyclin B1. Dose-dependent, sequence-specific, and gene-specific inhibition of both proteins was achieved at nanomolar concentrations of ONs in normal and breast cancer cells. Precise binding of the antisense ONs to their target RNA was absolutely required for antisense activity. Four or six base-mismatched ONs eliminated antisense activity confirming the sequence specificity of the antisense ONs. Antisense inhibition of p34cdc2 kinase resulted in a significant accumulation of cells in the Gap2/mitosis phase of the cell cycle in normal cells, but caused little effect on cell cycle progression in breast cancer cells. These data demonstrate the potency, specificity, and utility of C-5 propyne modified antisense ONs as biological tools and illustrate the redundancy of cell cycle protein function that can occur in cancer cells. PMID:9631067

  11. Anti-tumor activity of splice-switching oligonucleotides

    PubMed Central

    Bauman, John A.; Li, Shyh-Dar; Yang, Angela; Huang, Leaf; Kole, Ryszard

    2010-01-01

    Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. The apoptotic regulator Bcl-x is alternatively spliced to express anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL is upregulated in many cancers and is associated with chemoresistance, distinguishing it as an important target for cancer therapy. We previously showed that redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS induced apoptosis in breast and prostate cancer cells. In this study, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. SSOs were delivered in vivo using lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. Our findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing. PMID:20719743

  12. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation.

    PubMed

    Kim, Jinho; Olsen, Timothy R; Zhu, Jing; Hilton, John P; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N; Lin, Qiao

    2016-01-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours. PMID:27217242

  13. Molecular beacon probes of oligonucleotides photodamaged by psoralen.

    PubMed

    Shire, Zahra J; Loppnow, Glen R

    2012-01-01

    Ultraviolet A (UVA)-irradiated 4'-hydroxymethyl-4,5',8-trimethyl psoralen (HMT) in the presence of a poly-dT(17) and dA(7) TTA(8) oligonucleotides produces HMT-dT(17) and HMT-dA(7) TTA(8) adducts in aqueous solution. In this article, we determine whether these HMT-dT(17) and HMT-dA(7) TTA(8) adducts can be detected with a molecular beacon (MB) probe. We measure the degree of damage in dT(17) and dA(7) TTA(8) solutions containing UVA-activated HMT via monitoring the decrease in MB fluorescence. Photoproduct formation is confirmed by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-fight mass spectrometry measurements) and absorption spectroscopy. The MB fluorescence decreases upon UVA irradiation in the presence of HMT with a single-exponential time constants of 114.2 ± 6.5 min for HMT-dT(17) adducts and 677.8 ± 181.8 min for HMT-dA(7) TTA(8) adducts. Our results show that fluorescent MB probes are a selective, robust and accurate tool for detecting UVA-activated HMT-induced DNA damage.

  14. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    PubMed Central

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao

    2016-01-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours. PMID:27217242

  15. EvoOligo: oligonucleotide probe design with multiobjective evolutionary algorithms.

    PubMed

    Shin, Soo-Yong; Lee, In-Hee; Cho, Young-Min; Yang, Kyung-Ae; Zhang, Byoung-Tak

    2009-12-01

    Probe design is one of the most important tasks in successful deoxyribonucleic acid microarray experiments. We propose a multiobjective evolutionary optimization method for oligonucleotide probe design based on the multiobjective nature of the probe design problem. The proposed multiobjective evolutionary approach has several distinguished features, compared with previous methods. First, the evolutionary approach can find better probe sets than existing simple filtering methods with fixed threshold values. Second, the multiobjective approach can easily incorporate the user's custom criteria or change the existing criteria. Third, our approach tries to optimize the combination of probes for the given set of genes, in contrast to other tools that independently search each gene for qualifying probes. Lastly, the multiobjective optimization method provides various sets of probe combinations, among which the user can choose, depending on the target application. The proposed method is implemented as a platform called EvoOligo and is available for service on the web. We test the performance of EvoOligo by designing probe sets for 19 types of Human Papillomavirus and 52 genes in the Arabidopsis Calmodulin multigene family. The design results from EvoOligo are proven to be superior to those from well-known existing probe design tools, such as OligoArray and OligoWiz.

  16. Inositol phosphates in the environment.

    PubMed Central

    Turner, Benjamin L; Papházy, Michael J; Haygarth, Philip M; McKelvie, Ian D

    2002-01-01

    The inositol phosphates are a group of organic phosphorus compounds found widely in the natural environment, but that represent the greatest gap in our understanding of the global phosphorus cycle. They exist as inositols in various states of phosphorylation (bound to between one and six phosphate groups) and isomeric forms (e.g. myo, D-chiro, scyllo, neo), although myo-inositol hexakisphosphate is by far the most prevalent form in nature. In terrestrial environments, inositol phosphates are principally derived from plants and accumulate in soils to become the dominant class of organic phosphorus compounds. Inositol phosphates are also present in large amounts in aquatic environments, where they may contribute to eutrophication. Despite the prevalence of inositol phosphates in the environment, their cycling, mobility and bioavailability are poorly understood. This is largely related to analytical difficulties associated with the extraction, separation and detection of inositol phosphates in environmental samples. This review summarizes the current knowledge of inositol phosphates in the environment and the analytical techniques currently available for their detection in environmental samples. Recent advances in technology, such as the development of suitable chromatographic and capillary electrophoresis separation techniques, should help to elucidate some of the more pertinent questions regarding inositol phosphates in the natural environment. PMID:12028785

  17. Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.

    PubMed

    Dirin, Mehrdad; Urban, Ernst; Noe, Christian R; Winkler, Johannes

    2016-10-01

    Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application. PMID:27236069

  18. New aspects of the fragmentation mechanisms of unmodified and methylphosphonate-modified oligonucleotides.

    PubMed

    Monn, Selina T M; Schürch, Stefan

    2007-06-01

    A set of pentanucleotides was investigated by electrospray tandem mass spectrometry with the focus on the fragmentation mechanism. Results reveal new aspects of the fragmentation mechanism of modified and unmodified oligonucleotides and demonstrate the influence of the nucleobases on the decomposition of oligonucleotides. Adenine-rich oligonucleotides fragment easily resulting in abundant peaks corresponding to the DNA-typical a-B- and w-ions. On the other hand, thymine was found to have a stabilizing effect, which is reflected by the preferred formation of the w(4)-ions and the relatively low abundance of shorter w-ions upon dissociation of pentanucleotides. Data from investigation of the formation of w(4)-ions support a beta-elimination mechanism. Results obtained by investigation of oligonucleotides with an abasic site confirm this mechanism, which is independent of nucleobase loss. Experiments with methylphosphonate oligonucleotides show a remarkable change in the fragmentation pattern due to the modification. It was found that charges are located on the nucleobases and initiate the fragmentation mechanism. The stability of the oligonucleotide is reduced and no a-B-fragment ions are formed wherever there is a methylphosphonate group within the backbone. This fact also demonstrates that fragmentation is locally controlled.

  19. Intracerebroventricular Administration of Mineralocorticoid Receptor Antisense Oligonucleotides Attenuates Salt Appetite in the Rat.

    PubMed

    Ma; Itharat; Fluharty; Sakai

    1997-10-01

    The anterior ventral third ventricle (AV3V) region of the brain contains high concentrations of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) that are important in the maintenance of body fluid and electrolyte balance as well as other physiological processes. Daily intracerebroventricular pulse injections of MR antisense oligonucleotides significantly suppressed deoxycorticosterone acetate (DOCA) induced salt appetite in a dose-related manner. Similar administration of GR antisense or scrambled/sense oligonucleotide into the third ventricle failed to inhibit salt appetite. Salt appetite aroused after adrenalectomy was not suppressed by MR antisense oligonucleotide treatments but was suppressed by an antisense oligonucleotide directed against the angiotensin II AT1 receptor subtype. Receptor binding analysis demonstrated that MR and GR oligonucleotide treatments each reduced their respective receptor subtypes. Finally, although GR antisense oligonucleotide treatment was ineffective in suppressing DOCA-induced salt appetite, this treatment did increase stress induced corticosterone release as well as delayed the recovery of corticosterone to basal levels after stress. PMID:9787254

  20. Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides.

    PubMed

    Rytkönen, Jussi; Arukuusk, Piret; Xu, Wujun; Kurrikoff, Kaido; Langel, Ulo; Lehto, Vesa-Pekka; Närvänen, Ale

    2014-02-01

    The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

  1. Targeted Intracellular Delivery of Antisense Oligonucleotides Via Conjugation With Small Molecule Ligands

    PubMed Central

    Nakagawa, Osamu; Ming, Xin; Huang, Leaf; Juliano, Rudolph L.

    2010-01-01

    Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multi-valent conjugates of oligonucleotides with small molecule ligands. Using the sigma receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid phase DNA synthesis. We generated mono- and tri-valent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The tri-valent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the mono-valent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range. PMID:20550198

  2. New approaches to targeting RNA with oligonucleotides: inhibition of group I intron self-splicing.

    PubMed

    Disney, Matthew D; Childs, Jessica L; Turner, Douglas H

    2004-01-01

    RNA is one class of relatively unexplored drug targets. Since RNAs play a myriad of essential roles, it is likely that new drugs can be developed that target RNA. There are several factors that make targeting RNA particularly attractive. First, the amount of information about the roles of RNA in essential biological processes is currently being expanded. Second, sequence information about targetable RNA is pouring out of genome sequencing efforts at unprecedented levels. Third, designing and screening potential oligonucleotide therapeutics to target RNA is relatively simple. The use of oligonucleotides in cell culture, however, presents several challenges such as oligonucleotide uptake and stability, and selective targeting of genes of interest. Here, we review investigations aimed at targeting RNA with oligonucleotides that can circumvent several of these potential problems. The hallmark of the strategies discussed is the use of short oligonucleotides, which may have the advantage of higher cellular uptake and improved binding selectivity compared to longer oligonucleotides. These strategies have been applied to Group I introns from the mammalian pathogens Pneumocystis carinii and Candida albicans. Both are examples of fungal infections that are increasing in number and prevalence. PMID:14691946

  3. Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection.

    PubMed

    Noor, M Omair; Hrovat, David; Moazami-Goudarzi, Maryam; Espie, George S; Krull, Ulrich J

    2015-07-23

    Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.

  4. Analysis of Shewanella oneidensis Membrane Protein Expression in Response to Electron Acceptor Availability

    SciTech Connect

    Giometti, Carol S.; Khare, Tripti; Verberkmoes, Nathan; O'Loughlin, Ed; Lindberg, Carl; Thompson, Melissa; Hettich, Robert

    2006-04-05

    Shewanella oneidensis MR-1, a gram negative metal-reducing bacterium, can utilize a large number of electron acceptors. In the natural environment, S. oneidensis utilizes insoluble metal oxides as well as soluble terminal electron acceptors. The purpose of this ERSP project is to identify differentially expressed proteins associated with the membranes of S. oneidensis MR-1 cells grown with different electron acceptors, including insoluble metal oxides. We hypothesize that through the use of surface labeling, subcellular fractionation, and a combination of proteome analysis tools, proteins involved in the reduction of different terminal electron acceptors will be elucidated. We are comparing the protein profiles from cells grown with the soluble electron acceptors oxygen and fumarate and with those from cells grown with the insoluble iron oxides goethite, ferrihydrite and lepidocrocite. Comparison of the cell surface proteins isolated from cells grown with oxygen or anaerobically with fumarate revealed an increase in the abundance of over 25 proteins in anaerobic cells, including agglutination protein and flagellin proteins along with the several hypothetical proteins. In addition, the surface protein composition of cells grown with the insoluble iron oxides varies considerably from the protein composition observed with either soluble electron acceptor as well as between the different insoluble acceptors.

  5. Light weight phosphate cements

    DOEpatents

    Wagh, Arun S.; Natarajan, Ramkumar,; Kahn, David

    2010-03-09

    A sealant having a specific gravity in the range of from about 0.7 to about 1.6 for heavy oil and/or coal bed methane fields is disclosed. The sealant has a binder including an oxide or hydroxide of Al or of Fe and a phosphoric acid solution. The binder may have MgO or an oxide of Fe and/or an acid phosphate. The binder is present from about 20 to about 50% by weight of the sealant with a lightweight additive present in the range of from about 1 to about 10% by weight of said sealant, a filler, and water sufficient to provide chemically bound water present in the range of from about 9 to about 36% by weight of the sealant when set. A porous ceramic is also disclosed.

  6. Templated, layered manganese phosphate

    DOEpatents

    Thoma, Steven G.; Bonhomme, Francois R.

    2004-08-17

    A new crystalline maganese phosphate composition having an empirical formula: O). The compound was determined to crystallize in the trigonal space group P-3c1 with a=8.8706(4) .ANG., c=26.1580(2) .ANG., and V (volume)=1783 .ANG..sup.3. The structure consists of sheets of corner sharing Mn(II)O.sub.4 and PO.sub.4 tetrahedra with layers of (H.sub.3 NCH.sub.2 CH.sub.2).sub.3 N and water molecules in-between. The pronated (H.sub.3 NCH.sub.2 CH.sub.2).sub.3 N molecules provide charge balancing for the inorganic sheets. A network of hydrogen bonds between water molecules and the inorganic sheets holds the structure together.

  7. Phosphate nutrition: improving low-phosphate tolerance in crops.

    PubMed

    López-Arredondo, Damar Lizbeth; Leyva-González, Marco Antonio; González-Morales, Sandra Isabel; López-Bucio, José; Herrera-Estrella, Luis

    2014-01-01

    Phosphorus is an essential nutrient that is required for all major developmental processes and reproduction in plants. It is also a major constituent of the fertilizers required to sustain high-yield agriculture. Levels of phosphate--the only form of phosphorus that can be assimilated by plants--are suboptimal in most natural and agricultural ecosystems, and when phosphate is applied as fertilizer in soils, it is rapidly immobilized owing to fixation and microbial activity. Thus, cultivated plants use only approximately 20-30% of the applied phosphate, and the rest is lost, eventually causing water eutrophication. Recent advances in the understanding of mechanisms by which wild and cultivated species adapt to low-phosphate stress and the implementation of alternative bacterial pathways for phosphorus metabolism have started to allow the design of more effective breeding and genetic engineering strategies to produce highly phosphate-efficient crops, optimize fertilizer use, and reach agricultural sustainability with a lower environmental cost. In this review, we outline the current advances in research on the complex network of plant responses to low-phosphorus stress and discuss some strategies used to manipulate genes involved in phosphate uptake, remobilization, and metabolism to develop low-phosphate-tolerant crops, which could help in designing more efficient crops.

  8. Pyrimidone-based series of glucokinase activators with alternative donor-acceptor motif.

    PubMed

    Filipski, Kevin J; Guzman-Perez, Angel; Bian, Jianwei; Perreault, Christian; Aspnes, Gary E; Didiuk, Mary T; Dow, Robert L; Hank, Richard F; Jones, Christopher S; Maguire, Robert J; Tu, Meihua; Zeng, Dongxiang; Liu, Shenping; Knafels, John D; Litchfield, John; Atkinson, Karen; Derksen, David R; Bourbonais, Francis; Gajiwala, Ketan S; Hickey, Michael; Johnson, Theodore O; Humphries, Paul S; Pfefferkorn, Jeffrey A

    2013-08-15

    Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor-acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor-acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor-acceptor motifs may find utility in other glucokinase activator series or beyond.

  9. Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor

    NASA Technical Reports Server (NTRS)

    Myers, Charles R.; Nealson, Kenneth H.

    1988-01-01

    Microbes that couple growth to the reduction of manganese could play an important role in the biogeochemistry of certain anaerobic environments. Such a bacterium, Alteromonas putrefaciens MR-1, couples its growth to the reduction of manganese oxides only under anaerobic conditions. The characteristics of this reduction are consistent with a biological, and not an indirect chemical, reduction of manganese, which suggest that this bacterium uses manganic oxide as a terminal electron acceptor. It can also utilize a large number of other compounds as terminal electron acceptors; this versatility could provide a distinct advantage in environments where electron-acceptor concentrations may vary.

  10. A KAP study on MTP acceptors and their contraceptive practice.

    PubMed

    Roy, M; Lahiri, B C; Ghosh, B N

    1978-01-01

    From the point of view of the family planning program, it is necessary to determine whether a medical termination of pregnancy has a learning effect on a woman's subsequent fertility control behavior. In order to determine the knowledge and attitude of medical termination of pregnancy (MTP) acceptors towards abortion and their pre and post abortion contraceptive practices, a study was conducted during January to December 1975 in a family planning clinic in North Calcutta. 300 consecutive cases admitted to the hospital for abortions were the study subjects - the MTP group. They were subjected to repeated interviews in the ward before and after the abortion and were followed up at home within 6-10 months after the discharge from the hospital. 300 women who attended the antenatal clinic of the same hospital during the same period were selected for comparison by systematic sampling. There is no statistically significant difference in the social characteristics of the 2 groups except that the MTP group was from a comparatively poorer economic class. 13.3% of the MTP group had "good" knowledge of physiology of reproduction in contrast to only 5.0% in the comparison group. Only 52% of the MTP group and 63% in the comparison group failed to have even an "average" knowledge of this area. Nearly 55% of the MTP group and 65% of the comparison group had never practiced family planning. Over 55% of the MTP group had poor knowledge about abortion; the most important soruce of their knowledge was their neighbors. 78.7% of the cases considered MTP to be a sinful act. The primary reason for deciding to have an abortion was socioeconomic. Over 27.6% of the cases were using contraceptives to avoid the present pregnancy. The difference is statistically significant suggesting a positive learning effect of MTP on contraceptive behavior of women seeking abortions.

  11. DNA sequence analysis by hybridization with oligonucleotide microchips : MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides.

    SciTech Connect

    Stomakhin, A. A.; Vasiliskov, V. A.; Timofeev, E.; Schulga, D.; Cotter, R. J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Moscow Inst. of Physics and Technology; Middle Atlantic Mass Spectrometry Lab.; Johns Hopkins Univ. School of Medicine

    2000-01-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA-8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10-15{sup o}C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA-8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.

  12. Enzymatic and regulatory properties of the trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum.

    PubMed

    Gao, Yanyan; Jiang, Ying; Liu, Qiulei; Wang, Ruiming; Liu, Xinli; Liu, Bo

    2014-06-01

    Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60 °C and pH 6, and the enzyme exhibited notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives ethanol, EDTA, urea, DTT, SDS, β-mercaptoethanol, methanol, isopropanol and n-butanol. Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases. PMID:24508535

  13. 2-O-[2-(Methylthio)ethyl]-Modified Oligonucleotide: An Analog of 2-O-[2-(Methoxy)ethyl]-Modified Oligonucleotide with Improved Protein Binding Properties and High Binding Affinity to Target RNA

    SciTech Connect

    Prakash, T.P.; Manoharan, M.; Fraser, A.S.; Kawasaki, A.M.; Lesnik, E.; Sioufi, N.; Leeds, J.M.; Teplova, M.; Egli, M.

    2010-03-08

    A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.

  14. 21 CFR 520.823 - Erythromycin phosphate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... (a) Specifications. Erythromycin phosphate is the phosphate salt of the antibiotic substance produced by the growth of Streptomyces erythreus or the same antibiotic substance produced by any other...

  15. 21 CFR 520.823 - Erythromycin phosphate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... (a) Specifications. Erythromycin phosphate is the phosphate salt of the antibiotic substance produced by the growth of Streptomyces erythreus or the same antibiotic substance produced by any other...

  16. 21 CFR 520.823 - Erythromycin phosphate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... (a) Specifications. Erythromycin phosphate is the phosphate salt of the antibiotic substance produced by the growth of Streptomyces erythreus or the same antibiotic substance produced by any other...

  17. 21 CFR 520.823 - Erythromycin phosphate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... (a) Specifications. Erythromycin phosphate is the phosphate salt of the antibiotic substance produced by the growth of Streptomyces erythreus or the same antibiotic substance produced by any other...

  18. 21 CFR 520.823 - Erythromycin phosphate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... (a) Specifications. Erythromycin phosphate is the phosphate salt of the antibiotic substance produced by the growth of Streptomyces erythreus or the same antibiotic substance produced by any other...

  19. Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using D-glucal as donor substrate.

    PubMed

    Wildberger, Patricia; Brecker, Lothar; Nidetzky, Bernd

    2012-07-15

    Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize D-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α-D-glucose 1-phosphate as the product. When performed in D(2)O, enzymatic phosphorolysis of D-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of D-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered D-glucose next to D-glucal and phosphate, CuCPase produces 2-deoxy-β-D-glucosyl-(1→4)-D-glucose and 2-deoxy-α-D-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-β-D-glucosyl-(1→4)-D-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, D-glucal, phosphate, and D-glucose. Conversion of D-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α-D-glucose 1-phosphate that is difficult to prepare chemically.

  20. Quality control in production of suspensions from solid ammonium phosphates (monoammonium phosphate and diammonium phosphate). [Monoammonium phosphate; diammonium phosphate

    SciTech Connect

    Achorn, F.P.; Balay, H.L.

    1982-01-01

    Suspensions of good quality can be produced from MAP and DAP. Suspension quality depends on the amount of impurities in the ammonium phosphate solids used. Tests have shown that adding ammonium fluoride helps lower viscosity of suspensions containing a considerable amount of impurities. Also, adding polyphosphates (such as 10-34-0, 9-32-0, and 11-37-0) as a source of part of the P/sub 2/O/sub 5/ (6 to 15% polyphosphate in the product) helps to produce a suspension that has excellent storage characteristics. When the polyphosphate content of the product (11-33-0 suspension) is between 10 and 15% it usually will not solidify during cold weather storage. Freight and production costs of granular ammonium phosphates are relatively low compared to other sources of P/sub 2/O/sub 5/ for the fluid fertilizer market; therefore, using MAP and DAP to produce suspensions is expected to continue to grow in popularity. 2 refs., 7 figs., 1 tab.

  1. Single-site copper(II) water oxidation electrocatalysis: rate enhancements with HPO₄²⁻ as a proton acceptor at pH 8.

    PubMed

    Coggins, Michael K; Zhang, Ming-Tian; Chen, Zuofeng; Song, Na; Meyer, Thomas J

    2014-11-01

    The complex Cu(II)(Py3P) (1) is an electrocatalyst for water oxidation to dioxygen in H2PO4(-)/HPO4(2-) buffered aqueous solutions. Controlled potential electrolysis experiments with 1 at pH 8.0 at an applied potential of 1.40 V versus the normal hydrogen electrode resulted in the formation of dioxygen (84% Faradaic yield) through multiple catalyst turnovers with minimal catalyst deactivation. The results of an electrochemical kinetics study point to a single-site mechanism for water oxidation catalysis with involvement of phosphate buffer anions either through atom-proton transfer in a rate-limiting O-O bond-forming step with HPO4(2-) as the acceptor base or by concerted electron-proton transfer with electron transfer to the electrode and proton transfer to the HPO4(2-) base. PMID:25243584

  2. Dynamics of phosphorothioate oligonucleotides in normal and laser photocoagulated retina

    PubMed Central

    Shen, W.; Garrett, K.; da Cruz, L.; Constable, I.; Rakoczy, P.

    1999-01-01

    AIMS—To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat.
METHODS—Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 µl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV).
RESULTS—Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue.
CONCLUSIONS—These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV.

 PMID:10381674

  3. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing. PMID:26553470

  4. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  5. Molecular Cloning of MAPK Gene Family Using Synthetic Oligonucleotide Probe.

    PubMed

    Zhou, Song; Wang, Qin; Chen, Jing; Chen, Jiang-Ye

    1999-01-01

    MAPK(mitogen activated protein kinase) is a kind of Ser/Thr protein kinase. The MAPKs play an important role in several different signal transduction pathways. The MAPKs may also have a role in morphorgenesis of Candida albicans. An oligonucleotide probe was used to screen novel MAPKs in C. albicans. All MAPKs shared high homogeneity in their eleven kinase subdomains, especially subdoman VII and VIII. In subdomain VII, nearly all MAPKs have the same KIDFGLAR sequence, and the two known MAPKs in C. albicans CEK1 and MKC1 have only one different nucleotide in that DNA sequence. This probe was hybridized with C. albicans genomic DNA. Under stringent conditions, the probe could only hybridize with CEK1 and MKC1 gene fragment. But when hybridized at 40 degrees in non-SDS solution, two novel bands appeared. This condition was used to screen SC5314 DNA library, and many positive clones with different hybridization density were obtained. The strongest hybridization clones were identified to contain CEK1 and MKC1 gene. From the stronger positive hybridization clones, two novel genes were identified. The first gene, named CRK1(CDC2-related protein kinase 1), shared high homogeneity to MAPKs, but was not of them. It is closest to SGV1 from S. cerevisiae (with homology 47%) and PITALRE from human (with homology 41%), both of which are CDC2-related protein kinases. The second gene called CEK2(Candida albicans extracelluar signal-regulated kinase 2) is a novel MAPK of Candida albicans, which shares the highest identity with CEK1 and its S. cerevisiae homologs, FUS3 and KSS1, two redundant MAPKs in yeast pheromone response and morphogenesis. PMID:12114967

  6. Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

    PubMed Central

    Jankowsky, E; Schwenzer, B

    1996-01-01

    Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

  7. Application of heteronuclear couplings to conformational analysis of oligonucleotides

    SciTech Connect

    Zhu, G.; Live, D.; Bax, A.

    1994-12-01

    The value of vicinal coupling constants extracted from NMR spectra in deducing torsion angles for conformational analysis is well recognized. Due to the abundance of protons, their couplings have been mostly widely used. In many instances, couplings between protons and other nuclei may be a valuable complement to proton-proton couplings or, in some instances, may be the only coupling available to characterize the torsion angle about a bond. Recently, heteronuclear couplings have been used to great benefit in studies of isotopically enriched proteins, and this general approach has been extended to peptides at natural abundance. The possibility of using this approach to study oligonucleotides is also attractive but has not as yet been widely exploited. With the development of strategies for labeling such molecules, particularly RNAs, this may become an important component in conformational analysis. For DNA, labeling is less accessible, but sufficient quantities of unlabeled material are readily available for measuring these couplings at natural abundance. We chose several DNA systems to explore the usefulness of heteronuclear couplings in addressing the sugar conformation and the glycosidic torsion angle. Intensities of cross peaks in long-range HMQC experiments can be related to the couplings. Crosspeaks involving H1{prime} and C1{prime} atoms have been emphasized because of the superior shift dispersion at these positions between sugar protons and carbon atoms. Results will be shown for the self-complementary Dickerson duplex dodecamer sequence d(CGCGAATTCGCG) and for d(GGTCGG), which dimerizes to form a G-tetrad structure incorporating both syn and anti base orientations. The couplings provide a clear discrimination between presence of C3{prime}-endo and C2{prime}-endo conformations of the sugars and syn and anti bases arrangements.

  8. Fluorescence quenching in an organic donor-acceptor dyad: a first principles study.

    PubMed

    Körzdörfer, T; Tretiak, S; Kümmel, S

    2009-07-21

    Perylene bisimide and triphenyl diamine are prototypical organic dyes frequently used in organic solar cells and light emitting devices. Recent Forster-resonant-energy-transfer experiments on a bridged organic dyad consisting of triphenyl diamine as an energy-donor and perylene bisimide as an energy-acceptor revealed a strong fluorescence quenching on the perylene bisimide. This quenching is absent in a solution of free donors and acceptors and thus attributed to the presence of the saturated CH(2)O(CH(2))(12)-bridge. We investigate the cause of the fluorescence quenching as well as the special role of the covalently bound bridge by means of time dependent density functional theory and molecular dynamics. The conformational dynamics of the bridged system leads to a charge transfer process between donor and acceptor that causes the acceptor fluorescence quenching. PMID:19624200

  9. Interface-induced heavy-hole/light-hole splitting of acceptors in silicon

    SciTech Connect

    Mol, J. A.; Salfi, J.; Simmons, M. Y.; Rogge, S.; Rahman, R.; Hsueh, Y.; Klimeck, G.; Miwa, J. A.

    2015-05-18

    The energy spectrum of spin-orbit coupled states of individual sub-surface boron acceptor dopants in silicon have been investigated using scanning tunneling spectroscopy at cryogenic temperatures. The spatially resolved tunnel spectra show two resonances, which we ascribe to the heavy- and light-hole Kramers doublets. This type of broken degeneracy has recently been argued to be advantageous for the lifetime of acceptor-based qubits [R. Ruskov and C. Tahan, Phys. Rev. B 88, 064308 (2013)]. The depth dependent energy splitting between the heavy- and light-hole Kramers doublets is consistent with tight binding calculations, and is in excess of 1 meV for all acceptors within the experimentally accessible depth range (<2 nm from the surface). These results will aid the development of tunable acceptor-based qubits in silicon with long coherence times and the possibility for electrical manipulation.

  10. Dinitrogen extrusion from enoldiazo compounds under thermal conditions: synthesis of donor-acceptor cyclopropenes.

    PubMed

    Deng, Yongming; Jing, Changcheng; Doyle, Michael P

    2015-08-21

    Donor-acceptor cyclopropenes are formed quantitatively or in high yield from enoldiazoacetates and enoldiazoacetamides under moderate thermal conditions. They are more versatile than their corresponding enoldiazocarbonyl compounds as carbene precursors.

  11. Preparation and spectroscopic studies on charge-transfer complexes of 2-hydroxypyridine with electron acceptors

    NASA Astrophysics Data System (ADS)

    Gaballa, Akmal S.

    2013-07-01

    The CT-interactions of electron acceptors such as iodine (I2), chloranilic acid (H2CA) and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) with 2-hydroxypyridine (HPyO) have been investigated in the defined solvent. The data indicate the formation of CT-complexes with the general formula [(HPyO)(acceptor)]. The 1:1 stoichiometry of the (HPyO)-acceptors were based on elemental analysis, IR spectra and thermogravimetric analysis of the solid CT-complexes along with the photometric titration measurements for the reactions. The formation constants (KCT) for the CT-complexes are shown to be strongly dependent on the type and structure of the electron acceptors. Factors affecting the CT-processes are discussed.

  12. Preparation and spectroscopic studies on charge-transfer complexes of famciclovir drug with different electron acceptors

    NASA Astrophysics Data System (ADS)

    Gaballa, Akmal S.; Teleb, Said M.; Nour, El-Metwally

    2012-09-01

    The CT-interaction of electron acceptors such as chloranilic acid (H2CA), 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and and 7,7',8,8'-tetracyano-p-quinodimethane (TCNQ) with the antiviral drug famciclovir (FCV) have been investigated spectrophotometrically in the defined solvent. The data indicate the formation of CT-complexes with the general formula [(FCV)(acceptor)]. The 1:1 stoichiometry of the (FCV)-acceptors were based on elemental analysis, IR spectra and thermogravimetric analysis of the solid CT-complexes along with the photometric titration measurements for the reactions. The formation constants (KCT) for the CT-complexes are shown to be strongly dependent on the type and structure of the electron acceptor. Factors affecting the CT-processes such as redox potentials and steric hinderance of reactants are discussed.

  13. Computational design of donor-bridge-acceptor systems exhibiting pronounced quantum interference effects.

    PubMed

    Gorczak, Natalie; Renaud, Nicolas; Galan, Elena; Eelkema, Rienk; Siebbeles, Laurens D A; Grozema, Ferdinand C

    2016-03-01

    Quantum interference is a well-known phenomenon that dictates charge transport properties of single molecule junctions. However, reports on quantum interference in donor-bridge-acceptor molecules are scarce. This might be due to the difficulties in meeting the conditions for the presence of quantum interference in a donor-bridge-acceptor system. The electronic coupling between the donor, bridge, and acceptor moieties must be weak in order to ensure localised initial and final states for charge transfer. Yet, it must be strong enough to allow all bridge orbitals to mediate charge transfer. We present the computational route to the design of a donor-bridge-acceptor molecule that features the right balance between these contradicting requirements and exhibits pronounced interference effects.

  14. Curved Oligophenylenes as Donors in Shape-Persistent Donor-Acceptor Macrocycles with Solvatofluorochromic Properties.

    PubMed

    Kuwabara, Takuya; Orii, Jun; Segawa, Yasutomo; Itami, Kenichiro

    2015-08-10

    Many optoelectronic organic materials are based on donor-acceptor (D-A) systems with heteroatom-containing electron donors. Herein, we introduce a new molecular design for all-carbon curved oligoparaphenylenes as donors, which results in the generation of unique shape-persistent D-A macrocycles. Two types of acceptor-inserted cycloparaphenylenes were synthesized. These macrocycles display positive solvatofluorochromic properties owing to their D-A characteristics, which were confirmed by theoretical and electrochemical studies. PMID:26140706

  15. Experiments probing the viability of donor-acceptor norbornenes for (5 + 2)-annulation.

    PubMed

    Walker, Morgan M; Goodman, C Guy; Johnson, Jeffrey S

    2014-10-01

    This Note details experiments that probe the mechanism by which donor-acceptor norbornene systems epimerize. A number of mechanistic studies indicate that epimerization in these systems occurs via a Lewis acid catalyzed retro-Diels-Alder/Diels-Alder sequence, rather than bond rotation in an intimate ion pair. These results suggest that, under the reaction conditions examined, the ring strain present in norbornene is inadequate to induce zwitterion formation analogous to that observed with donor-acceptor cyclopropanes.

  16. New acceptor centers of the background impurities in p-CdZnTe

    SciTech Connect

    Plyatsko, S. V. Rashkovetskyi, L. V.

    2013-07-15

    Low-temperature photoluminescence data are used to study the redistribution of the background impurities and host components of p-CdZnTe single crystals with a resistivity of 1-50 {Omega} cm upon their interaction with infrared laser radiation. The effect of widening of the band gap and the formation of new acceptor centers in response to laser-stimulated changes in the system of intrinsic defects are established. The activation energy of the new acceptor centers is determined.

  17. Process for gasification using a synthetic CO.sub.2 acceptor

    DOEpatents

    Lancet, Michael S.; Curran, George P.

    1980-01-01

    A gasification process is disclosed using a synthetic CO.sub.2 acceptor consisting essentially of at least one compound selected from the group consisting of calcium oxide and calcium carbonate supported in a refractory carrier matrix, the carrier having the general formula Ca.sub.5 (SiO.sub.4).sub.2 CO.sub.3. A method for producing the synthetic CO.sub.2 acceptor is also disclosed.

  18. Fast Carrier Formation from Acceptor Exciton in Low-Gap Organic Photovotalic

    NASA Astrophysics Data System (ADS)

    Yonezawa, Kouhei; Kamioka, Hayato; Yasuda, Takeshi; Han, Liyuan; Moritomo, Yutaka

    2012-04-01

    Bulk heterojunction (BHJ) based on a donor (D) polymer and an acceptor (A) fullerene derivative is a promising organic photovoltaic. Here, we investigated the femtosecond charge dynamics after acceptor excitation in poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b '] dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b] thiophenediyl

  19. Effects of size restriction on donor-acceptor recombination in AgBr

    NASA Astrophysics Data System (ADS)

    Rodney, Paul J.; Marchetti, Alfred P.; Fauchet, Philippe M.

    2000-08-01

    The behavior of donor-acceptor (DA) recombination luminescence in quantum confined AgBr has been investigated. The DA luminescence decay, after pulsed excitation, became longer lived as the nanocluster size decreased. This result differs from theoretical expectations and from observations with AgI nanoclusters. The DA lifetime increase with decreasing size is due to an increase in yield and lifetime of ``free'' excitons that slowly dissociate into ``close'' donor-acceptor pairs, giving rise to an ``exciton dribbling'' effect.

  20. Benzodipyrrole-based Donor-Acceptor-type Boron Complexes as Tunable Near-infrared-Absorbing Materials.

    PubMed

    Nakamura, Tomoya; Furukawa, Shunsuke; Nakamura, Eiichi

    2016-07-20

    Benzodipyrrole-based donor-acceptor boron complexes were designed and synthesized as near-infrared-absorbing materials. The electron-rich organic framework combined with the Lewis acidic boron co-ordination enabled us to tune the LUMO energy level and the HOMO-LUMO gap (i.e.,the absorption wavelength) by changing the organic acceptor units, the number of boron atoms, and the substituents on the boron atoms.

  1. Nanosecond intersystem crossing times in fullerene acceptors: implications for organic photovoltaic diodes.

    PubMed

    Chow, Philip C Y; Albert-Seifried, Sebastian; Gélinas, Simon; Friend, Richard H

    2014-07-23

    Triplet-exciton formation through intersystem crossing of photogenerated singlet excitons in fullerene acceptors can compete with charge generation in organic photovoltaic diodes. This article reports the intersystem crossing timescale (τISC ) of the most commonly used fullerene acceptors, PC60 BM and PC70 BM, in solutions and in spin-coated films. These times are on the nanosecond timescale, and are longer than the characteristic times for charge generation (τd ).

  2. Superiorities of time-correlated single-photon counting against standard fluorimetry in exploiting the potential of fluorochromized oligonucleotide probes for biomedical investigation

    NASA Astrophysics Data System (ADS)

    Lamperti, Marco; Nardo, Luca; Bondani, Maria

    2015-05-01

    Site-specific fluorescence-resonance-energy-transfer donor-acceptor dual-labelled oligonucleotide probes are widely used in state-of-art biotechnological applications. Such applications include their usage as primers in polymerase chain reaction. However, the steady-state fluorescence intensity signal emitted by these molecular tools strongly depends from the specificities of the probe conformation. For this reason, the information which can be reliably inferred by steady-state fluorimetry performed on such samples is forcedly confined to a semi-qualitative level. Namely, fluorescent emission is frequently used as ON/OFF indicator of the probe hybridization state, i.e. detection of fluorescence signals indicates either hybridization to or detachment from the template DNA of the probe. Nonetheless, a fully quantitative analysis of their fluorescence emission properties would disclose other exciting applications of dual-labelled probes in biosensing. Here we show how time-correlated single-photon counting can be applied to get rid of the technical limitations and interpretational ambiguities plaguing the intensity analysis, and to derive information on the template DNA reaching single-base.

  3. Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells

    PubMed Central

    Zhong, Yu; Trinh, M. Tuan; Chen, Rongsheng; Purdum, Geoffrey E.; Khlyabich, Petr P.; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; Zhang, Boyuan; Wang, Wei; Nam, Chang-Yong; Sfeir, Matthew Y.; Black, Charles T.; Steigerwald, Michael L.; Loo, Yueh-Lin; Ng, Fay; Zhu, X.-Y.; Nuckolls, Colin

    2015-01-01

    Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealed both electron and hole transfer processes at the donor−acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. This study describes a new motif for designing highly efficient acceptors for organic solar cells. PMID:26382113

  4. Beyond Fullerenes: Designing Alternative Molecular Electron Acceptors for Solution-Processable Bulk Heterojunction Organic Photovoltaics.

    PubMed

    Sauvé, Geneviève; Fernando, Roshan

    2015-09-17

    Organic photovoltaics (OPVs) are promising candidates for providing a low cost, widespread energy source by converting sunlight into electricity. Solution-processable active layers have predominantly consisted of a conjugated polymer donor blended with a fullerene derivative as the acceptor. Although fullerene derivatives have been the acceptor of choice, they have drawbacks such as weak visible light absorption and poor energy tuning that limit overall efficiencies. This has recently fueled new research to explore alternative acceptors that would overcome those limitations. During this exploration, one question arises: what are the important design principles for developing nonfullerene acceptors? It is generally accepted that acceptors should have high electron affinity, electron mobility, and absorption coefficient in the visible and near-IR region of the spectra. In this Perspective, we argue that alternative molecular acceptors, when blended with a conjugated polymer donor, should also have large nonplanar structures to promote nanoscale phase separation, charge separation and charge transport in blend films. Additionally, new material design should address the low dielectric constant of organic semiconductors that have so far limited their widespread application.

  5. Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells

    DOE PAGES

    Yu M. Zhong; Nam, Chang -Yong; Trinh, M. Tuan; Chen, Rongsheng; Purdum, Geoffrey E.; Khlyabich, Petr P.; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; et al

    2015-09-18

    Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealedmore » both electron and hole transfer processes at the donor–acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. As a result, this study describes a new motif for designing highly efficient acceptors for organic solar cells.« less

  6. Differences in gene expression of human xylosyltransferases and determination of acceptor specificities for various proteoglycans

    SciTech Connect

    Roch, Christina; Kuhn, Joachim; Kleesiek, Knut; Goetting, Christian

    2010-01-01

    The xylosyltransferase (XT) isoforms XT-I and XT-II initiate the posttranslational glycosaminoglycan (GAG) synthesis. Here, we determined the relative expression of both isoforms in 33 human cell lines. The majority of tested cell lines showed dominant XYLT2 gene expression, while only in 23132/87, JAR, NCI-H510A and THP-1 was the XT-I mRNA expression higher. Nearly equal expression levels were detected in six cell lines. Additionally, to shed light on putative differences in acceptor specificities the acceptor properties of potential acceptor sequences were determined. Peptides were expressed as glutathione-S-transferase fusion proteins containing putative or known GAG attachment sites of in vivo proteoglycans. Kinetic analysis showed that K{sub m} and V{sub max} values for XT-I mediated xylosylation were slightly higher than those for XT-II, and that XT-I showed a lesser stringency concerning the acceptor sequence. Mutagenesis of the bikunin peptide sequence in the G-S-G attachment site and flanking regions generated potential acceptor molecules. Here, mutations on the N-terminal side and the attachment site were found to be more susceptible to a loss of acceptor function than mutations in the C-terminus. Altogether the known consensus sequence a-a-a-a-G-S-G-a-a/G-a ('a' representing Asp or Glu) for XT-I mediated xylosylation could be approved and additionally extended to apply to XT-II as well.

  7. A new classification of the amino acid side chains based on doublet acceptor energy levels.

    PubMed Central

    Sneddon, S F; Morgan, R S; Brooks, C L

    1988-01-01

    We describe a new classification of the amino acid side chains based on the potential energy level at which each will accept an extra (doublet) electron. The doublet acceptor energy level, and the doublet acceptor orbital were calculated using semiempirical INDO/2-UHF molecular orbital theory. The results of these calculations show that the side chains fall into four groups. We have termed these groups repulsive, insulating, semiconducting, and attractive in accordance with where each lies on the relative energy scale. We use this classification to examine the role of residues between the donor and acceptor in modulating the rate and mechanism of electron transfer in proteins. With the calculated acceptor levels, we construct a potential barrier for those residues between the donor and acceptor. It is the area beneath this barrier that determines the decay of electronic coupling between donor and acceptor, and thus the transfer rate. We have used this schematic approach to characterize the four electron transfer pathways in myoglobin recently studied by Mayo et al. (Mayo, S.L., W.R. Ellis, R.J. Crutchley, and H.B. Gray. 1986. Science [Wash. DC]. 233:948-952). PMID:3342271

  8. Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells

    SciTech Connect

    Yu M. Zhong; Nam, Chang -Yong; Trinh, M. Tuan; Chen, Rongsheng; Purdum, Geoffrey E.; Khlyabich, Petr P.; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; Zhang, Boyuan; Wang, Wei; Sfeir, Matthew Y.; Black, Charles T.; Steigerwald, Michael L.; Loo, Yueh -Lin; Ng, Fay; Zhu, X. -Y.; Nuckolls, Colin

    2015-09-18

    Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealed both electron and hole transfer processes at the donor–acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. As a result, this study describes a new motif for designing highly efficient acceptors for organic solar cells.

  9. Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells.

    PubMed

    Zhong, Yu; Trinh, M Tuan; Chen, Rongsheng; Purdum, Geoffrey E; Khlyabich, Petr P; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; Zhang, Boyuan; Wang, Wei; Nam, Chang-Yong; Sfeir, Matthew Y; Black, Charles T; Steigerwald, Michael L; Loo, Yueh-Lin; Ng, Fay; Zhu, X-Y; Nuckolls, Colin

    2015-09-18

    Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealed both electron and hole transfer processes at the donor-acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. This study describes a new motif for designing highly efficient acceptors for organic solar cells.

  10. Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells.

    PubMed

    Zhong, Yu; Trinh, M Tuan; Chen, Rongsheng; Purdum, Geoffrey E; Khlyabich, Petr P; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; Zhang, Boyuan; Wang, Wei; Nam, Chang-Yong; Sfeir, Matthew Y; Black, Charles T; Steigerwald, Michael L; Loo, Yueh-Lin; Ng, Fay; Zhu, X-Y; Nuckolls, Colin

    2015-01-01

    Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealed both electron and hole transfer processes at the donor-acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. This study describes a new motif for designing highly efficient acceptors for organic solar cells. PMID:26382113

  11. Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin.

    PubMed Central

    Vijaya, S; Steffen, D L; Robinson, H L

    1986-01-01

    Seven cellular loci with acceptor sites for retroviral integrations have been mapped for the presence of DNase I-hypersensitive sites in chromatin. Integrations in three of these loci, chicken c-erbB, rat c-myc, and a rat locus, dsi-1, had been selected for in retrovirus-induced tumors. Of the remaining four, two, designated dsi-3 and dsi-4, harbored acceptor sites for apparently unselected integrations of Moloney murine leukemia virus in a Moloney murine leukemia virus-induced thymoma, and two, designated C and F, harbored unselected acceptor sites for Moloney murine leukemia virus integrations in a rat fibroblast cell line. Each acceptor site mapped to within 500 base pairs of a DNase I-hypersensitive site. In the analyses of the unselected integrations, six hypersensitive sites were observed in 39 kilobases of DNA. The four acceptor sites in this DNA were localized between 0.05 and 0.43 kilobases of a hypersensitive site. The probability of this close association occurring by chance was calculated to be extremely low. Hypersensitive sites were mapped in cells representing the lineage in which integration had occurred as well as in an unrelated lineage. In six of the seven acceptor loci hypersensitive sites could not be detected in the unrelated lineage. Our results indicate that retroviruses preferentially integrate close to DNase I-hypersensitive sites and that many of these sites are expressed in some but not all cells. Images PMID:3490582

  12. Candidatus Accumulibacter phosphatis clades enriched under cyclic anaerobic and microaerobic conditions simultaneously use different electron acceptors.

    PubMed

    Camejo, Pamela Y; Owen, Brian R; Martirano, Joseph; Ma, Juan; Kapoor, Vikram; Santo Domingo, Jorge; McMahon, Katherine D; Noguera, Daniel R

    2016-10-01

    Lab- and pilot-scale simultaneous nitrification, denitrification and phosphorus removal-sequencing batch reactors were operated under cyclic anaerobic and micro-aerobic conditions. The use of oxygen, nitrite, and nitrate as electron acceptors by Candidatus Accumulibacter phosphatis during the micro-aerobic stage was investigated. A complete clade-level characterization of Accumulibacter in both reactors was performed using newly designed qPCR primers targeting the polyphosphate kinase gene (ppk1). In the lab-scale reactor, limited-oxygen conditions led to an alternated dominance of Clade IID and IC over the other clades. Results from batch tests when Clade IC was dominant (i.e., >92% of Accumulibacter) showed that this clade was capable of using oxygen, nitrite and nitrate as electron acceptors for P uptake. A more heterogeneous distribution of clades was found in the pilot-scale system (Clades IIA, IIB, IIC, IID, IA, and IC), and in this reactor, oxygen, nitrite and nitrate were also used as electron acceptors coupled to phosphorus uptake. However, nitrite was not an efficient electron acceptor in either reactor, and nitrate allowed only partial P removal. The results from the Clade IC dominated reactor indicated that either organisms in this clade can simultaneously use multiple electron acceptors under micro-aerobic conditions, or that the use of multiple electron acceptors by Clade IC is due to significant microdiversity within the Accumulibacter clades defined using the ppk1 gene. PMID:27340814

  13. Enhanced natural attenuation of BTEX in the nitrate-reducing environment by different electron acceptors.

    PubMed

    Zhao, Yongsheng; Qu, Dan; Hou, Zhimin; Zhou, Rui

    2015-01-01

    Enhancing natural attenuation of benzene, toluene, ethylbenzene, and xylene (BTEX) in groundwater is a potential remediation technology. This study focused on selecting appropriate electron acceptors to promote BTEX degradation in a nitrate-reducing environment. Nitrate-reducing soil was obtained from simulated BTEX-contaminated column. Enhancing experiments were conducted in the microcosm with nitrate-reducing material and simulated BTEX-polluted groundwater to investigate the promoting feasibility of adding dissolved oxygen (DO), nitrate, chelated Fe(III), and sulphate as electron acceptors. The concentrations of BTEX, electron acceptors, and their reducing products were measured. The order of promoting BTEX degradation with four electron acceptors was nitrate>sulphate>chelated Fe(III)>DO, and the first-order decay coefficients were 0.0432, 0.0333, 0.0240, and 0.0155, respectively. Nitrate, sulphate, and chelated Fe(III) enhanced attenuation. Nitrate was the most effective electron acceptor under nitrate-reducing conditions. Selecting proper electron acceptor is significant in promoting BTEX degradation according to the biogeochemical characteristics of local underground environment.

  14. The role of anaerobic respiration in the immobilization of uranium through biomineralization of phosphate minerals

    NASA Astrophysics Data System (ADS)

    Salome, Kathleen R.; Green, Stefan J.; Beazley, Melanie J.; Webb, Samuel M.; Kostka, Joel E.; Taillefert, Martial

    2013-04-01

    Although bioreduction of uranyl ions (U(VI)) and biomineralization of U(VI)-phosphate minerals are both able to immobilize uranium in contaminated sediments, the competition between these processes and the role of anaerobic respiration in the biomineralization of U(VI)-phosphate minerals has yet to be investigated. In this study, contaminated sediments incubated anaerobically in static microcosms at pH 5.5 and 7.0 were amended with the organophosphate glycerol-2-phosphate (G2P) as sole phosphorus and external carbon source and iron oxides, sulfate, or nitrate as terminal electron acceptors to determine the most favorable geochemical conditions to these two processes. While sulfate reduction was not observed even in the presence of G2P at both pHs, iron reduction was more significant at circumneutral pH irrespective of the addition of G2P. In turn, nitrate reduction was stimulated by G2P at both pH 5.5 and 7.0, suggesting nitrate-reducing bacteria provided the main source of inorganic phosphate in these sediments. U(VI) was rapidly removed from solution in all treatments but was not reduced as determined by X-ray absorption near edge structure (XANES) spectroscopy. Simultaneously, wet chemical extractions and extended X-ray absorption fine structure (EXAFS) spectroscopy of these sediments indicated the presence of U-P species in reactors amended with G2P at both pHs. The rapid removal of dissolved U(VI), the simultaneous production of inorganic phosphate, and the existence of U-P species in the solid phase indicate that uranium was precipitated as U(VI)-phosphate minerals in sediments amended with G2P. Thus, under reducing conditions and in the presence of G2P, bioreduction of U(VI) was outcompeted by the biomineralization of U(VI)-phosphate minerals and U(VI) sorption at both pHs.

  15. Toxicological review of inorganic phosphates.

    PubMed

    Weiner, M L; Salminen, W F; Larson, P R; Barter, R A; Kranetz, J L; Simon, G S

    2001-08-01

    Inorganic phosphate salts are widely used as food ingredients and in a variety of commercial applications. The United States Food and Drug Administration (FDA) considers inorganic phosphates "Generally Recognized As Safe" (GRAS) (FDA, 1973a, 1979) [FDA: Food and Drug Administration 1973a. GRAS (Generally Recognized as Safe) food ingredients-phosphates. NTIS PB-221-224, FDA, Food and Drug Administration, 1979. Phosphates; Proposed Affirmation of and Deletion From GRAS Status as Direct and Human Food Ingredients. Federal Register 44 (244). 74845-74857, 18 December (1979)] and the European Union (EU) allows inorganic phosphates to be added directly to food (EU Directive 95/2/EC as amended by 98/72/EC). In this review, data on the acute, subchronic and chronic toxicity, genotoxicity, teratogenicity and reproductive toxicity from the published literature and from unpublished studies by the manufacturers are reviewed. Based on the toxicity data and similar chemistry, the inorganic phosphates can be separated into four major classes, consisting of monovalent salts, divalent salts, ammonium salts and aluminum salts. The proposed classification scheme supports the use of toxicity data from one compound to assess the toxicity of another compound in the same class. However, in the case of eye and skin irritation, the proposed classification scheme cannot be used because a wide range of responses exists within each class. Therefore, the eye and skin hazards associated with an individual inorganic phosphate should be assessed on a chemical-by-chemical basis. A large amount of toxicity data exists for all four classes of inorganic phosphates. The large and comprehensive database allows an accurate assessment of the toxicity of each class of inorganic phosphate. Overall, all four classes of inorganic phosphates exhibit low oral, inhalation and dermal toxicities. Based on these data, humans are unlikely to experience adverse effects when the daily phosphorus consumption remains

  16. Role of inorganic phosphate during electron transfer in the redox system NADH+flavin

    NASA Astrophysics Data System (ADS)

    Lozinova, T. A.; Brzhevskaya, O. N.; Kisin, A. V.; Nosova, V. M.; Rykov, S. V.; Tychinskaya, L. Yu.

    2006-05-01

    We have used 31P NMR and EPR to study the effect of inorganic phosphate (Pi) on the process of electron transfer in oxidation of reduced nicotinamide adenine dinucleotide (NADH) by flavin in aqueous solutions. Observation of a significant (up to 40%) drop in the integrated intensity of the 31P NMR signal for the inorganic phosphate over the course of the reaction is interpreted as a manifestation of the effect of chemically induced polarization of the phosphorus nuclei during singlet-triplet conversion in charge-transfer complexes [NADH…Pi], and is evidence for a possible catalytic role for Pi in processes involving transfer of an electron (hydrogen atom) from NADH to appropriate acceptors. The results presented support the possibility of direct involvement of Pi in a chain of one-electron conversions in chemical systems.

  17. Localization of dolichyl phosphate- and pyrophosphate-dependent glycosyl transfer reactions in Saccharomyces cerevisiae.

    PubMed Central

    Marriott, M; Tanner, W

    1979-01-01

    Membranes from Saccharomyces cerevisiae protoplasts were fractionated on a continuous sucrose gradient. Six bands were obtained, which contained altogether about 15% of the total cell protein. From their densitites, their behavior in the presence and absence of Mg2+ ions, and the distribution of marker enzymes, it was possible to identify fractions enriched in rough and smooth endoplasmic reticulum and in mitochondria. All glycosyl transfer reactions investigated where dolichyl phosphates served as glycosyl acceptors or where dolichyl phosphate- and pyrophosphate-activated sugars served as glycosyl donors showed the highest specific activity and up to 75% of the total activity in the endoplasmic reticulum. This was the case for the reactions involved in the formation of O-glycosidic as well as N-glycosidic linkages in yeast glycoprotein biosynthesis. Membrane fractions enriched in plasmalemma contained less than 3% of the corresponding activities. PMID:222737

  18. Effect of biogenic photochromic electron acceptors on chlorophyll fluorescence

    NASA Astrophysics Data System (ADS)

    Lobanov, A. V.; Klimenko, I. V.; Nevrova, O. V.; Zhuravleva, T. S.

    2014-05-01

    It is shown that the photophysical properties of chlorophyll a (Chl) depend on the nature and relative amounts of 2-methyl-1,4-naphthoquinone (MNQ) and nicotinamide adenine dinucleotide phosphate (NADP). Photoinduced charge separation occurs in aqueous ethanol solutions of Chl (1 × 10-5 M) and NADP (5 × 10-6-5 × 10-4 M), resulting in the dynamic quenching of Chl fluorescence. Coordination interaction between Chl and NADP is established at an NADP concentration of ≥5 × 10-4 M. The nonlinear Stern-Volmer dependence in this range is due to the input from static quenching. It is shown that the quenching of Chl fluorescence in an MNQ solution at Chl and MNQ concentrations of 1 × 10-5 M and 6.7 × 10-5-1 × 10-4 M, respectively, is described by a linear dependence in the Stern-Volmer coordinates; no complex formation is observed for Chl and MNQ under these conditions, and electron transfer is of the dynamic type. Static or mixed-type energy transfer from MNQ to Chl dominates at elevated MNQ concentrations.

  19. Novel highly biodegradable biphasic tricalcium phosphates composed of alpha-tricalcium phosphate and beta-tricalcium phosphate.

    PubMed

    Li, Yanbao; Weng, Wenjian; Tam, Kim Chiu

    2007-03-01

    Novel biodegradable biphasic tricalcium phosphates (BTCP) composed of alpha-tricalcium phosphate (alpha-TCP) and beta-tricalcium phosphate (beta-TCP) were successfully synthesized by heating amorphous calcium phosphate precursors with different structures at 800 degrees C for 3 h. The ratio of alpha-TCP and beta-TCP in the calcium phosphate particle can be controlled by aging time and pH value during synthesis of the amorphous precursor.

  20. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    PubMed

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  1. Design and Application of an Easy to Use Oligonucleotide Mass Calculation Program

    NASA Astrophysics Data System (ADS)

    Yang, Jiong; Leopold, Peter; Helmy, Roy; Parish, Craig; Arvary, Becky; Mao, Bing; Meng, Fanyu

    2013-08-01

    With the development of new synthesis procedures, an ever increasing number of chemical modifications can now be incorporated into synthetic oligonucleotides, representing new challenges for analytical chemists to efficiently identify and characterize such molecules. While conventional mass spectrometry (MS) has proven to be a powerful tool to study nucleic acids, new and improved methods and software are now needed to address this emerging challenge. In this report, we describe a simple yet powerful program that affords great flexibility in the calculation of theoretical masses for conventional as well as modified oligonucleotide molecules. This easy to use program can accept input oligonucleotide sequences and then calculate the theoretical mass values for full length products, process impurities, potential metabolites, and gas phase fragments. We intentionally designed this software so that modified nucleotide residues can be incorporated into oligonucleotide sequences, and corresponding mass values can be rapidly calculated. To test the utility of this program, two oligonucleotides that contain a large number of chemical modifications were synthesized. We have analyzed these samples using a Q-TOF mass spectrometer and compared the calculated masses to the observed ones. We found that all of the data matched very well with less than 30 ppm mass errors, well within the expectation for our instrument operated in its current mode. These data confirmed the validity of calculations performed with this new software.

  2. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    PubMed

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  3. Oligonucleotide-induced alternative splicing of serotonin 2C receptor reduces food intake.

    PubMed

    Zhang, Zhaiyi; Shen, Manli; Gresch, Paul J; Ghamari-Langroudi, Masoud; Rabchevsky, Alexander G; Emeson, Ronald B; Stamm, Stefan

    2016-01-01

    The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre-mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full-length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full-length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full-length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro-opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild-type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood-brain barrier and its systemic delivery reduced food intake in wild-type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre-mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader-Willi syndrome. PMID:27406820

  4. A novel, one-step amplification and oligonucleotide ligation procedure for multiplex genetic typing

    SciTech Connect

    Eggerding, F.A.

    1994-09-01

    A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method which combines in one assay DNA amplification by the polymerase chain reaction (PCR) with DNA genotyping by the oligonucleotide ligation assay (OLA). By virtue of a difference in the melting temperatures of PCR primer-target DNA and OLA probe-target DNA hybrids, the method allows preferential amplification of DNA during stage I and oligonucleotide ligation during stage II of the reaction. In stage I target DNA is amplified using high-melting primers in a two-step PCR cycle that employs a 72{degrees}C anneal-elongation step. In stage II genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes located between PCR primers is accomplished by several cycles of denaturation at 94{degrees}C followed by anneal-ligation at 55{degrees}C. Ligation products are fluorochrome-labeled at their 3{prime}-ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used for multiplex detection of 30 cystic fibrosis mutations and for analysis of ras gene point mutations. Because mutation detection occurs concurrently with target amplification, the technique is rapid, highly sensitive and specific, easily automatable, and requires minimal sample processing.

  5. Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy.

    PubMed

    Hammond, Suzan M; Hazell, Gareth; Shabanpoor, Fazel; Saleh, Amer F; Bowerman, Melissa; Sleigh, James N; Meijboom, Katharina E; Zhou, Haiyan; Muntoni, Francesco; Talbot, Kevin; Gait, Michael J; Wood, Matthew J A

    2016-09-27

    The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA. PMID:27621445

  6. High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

    PubMed Central

    Borovkov, Alex Y.; Loskutov, Andrey V.; Robida, Mark D.; Day, Kristen M.; Cano, Jose A.; Le Olson, Tien; Patel, Hetal; Brown, Kevin; Hunter, Preston D.; Sykes, Kathryn F.

    2010-01-01

    To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning. PMID:20693531

  7. Modified nucleotides in T1 RNase oligonucleotides of 18S ribosomal RNA of the Novikoff hepatoma.

    PubMed

    Choi, Y C; Busch, H

    1978-06-27

    The primary structure of 18S rRNA of the Novikoff hepatoma cells was investigated. Regardless of whether the primary sequence of 18S rRNA is finally determined by RNA sequencing methods or DNA sequencing methods, it is important to identify numbers and types of the modified nucleotides and accordingly the present study was designed to localize the modified regions in T1 RNase derived oligonucleotide. Modified nucleotides found in 66 different oligonucleotide sequences included 2 m62A, 1 m6A, 1 m7G, 1m1cap3psi, 7 Cm, 13 Am, 9 Gm, 11 Um, and 38 psi residues. A number of these modified nucleotides are now placed in defined sequences of T1 RNase oligonucleotides which are now being searched for in larger fragments derived from partial T1 RNase digests of 18S rRNA. Improved homochromatography fingerprinting (Choi et al. (1976) Cancer Res. 36, 4301) of T1 RNase derived oligonucleotides provided a distinctive pattern for 18S rRNA of Novikoff hepatoma ascites cells. The 116 spots obtained by homochromatography contain 176 oligonucleotide sequences. PMID:209819

  8. Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

    PubMed Central

    Hazell, Gareth; Shabanpoor, Fazel; Saleh, Amer F.; Bowerman, Melissa; Meijboom, Katharina E.; Zhou, Haiyan; Muntoni, Francesco; Talbot, Kevin; Gait, Michael J.; Wood, Matthew J. A.

    2016-01-01

    The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA. PMID:27621445

  9. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    PubMed Central

    2010-01-01

    Background Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. Results Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis) and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at pilot scale to inform

  10. Detection of genomic deletions in rice using oligonucleotide microarrays

    PubMed Central

    Bruce, Myron; Hess, Ann; Bai, Jianfa; Mauleon, Ramil; Diaz, M Genaleen; Sugiyama, Nobuko; Bordeos, Alicia; Wang, Guo-Liang; Leung, Hei; Leach, Jan E

    2009-01-01

    Background The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL). However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. Results We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip® allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to ~500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations . Conclusion We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a database saturated with deletions across the rice genome. This community resource can continue

  11. Conduction electrons in acceptor-doped GaAs/GaAlAs heterostructures: a review

    NASA Astrophysics Data System (ADS)

    Zawadzki, Wlodek; Raymond, Andre; Kubisa, Maciej

    2016-05-01

    We review magneto-optical and magneto-transport effects in GaAs/GaAlAs heterostructures doped in GaAlAs barriers with donors, providing two-dimensional (2D) electron gas (2DEG) in GaAs quantum wells (QWS), and additionally doped with smaller amounts of acceptors (mostly Be atoms) in the vicinity of 2DEG. One may also deal with residual acceptors (mostly C atoms). The behavior of such systems in the presence of a magnetic field differs appreciably from those doped in the vicinity of 2DEG with donors. Three subjects related to the acceptor-doped heterostructures are considered. First is the problem of bound states of conduction electrons confined to the vicinity of negatively charged acceptors by the joint effect of a QW and an external magnetic field parallel to the growth direction. A variational theory of such states is presented, demonstrating that an electron turning around a repulsive center has discrete energies above the corresponding Landau levels. Experimental evidence for the discrete electron energies comes from the work on interband photo-magneto-luminescence, intraband cyclotron resonance and quantum magneto-transport (the Quantum Hall and Shubnikov-de Haas effects). An electron rain-down effect at weak electric fields and a boil-off effect at strong electric fields are introduced. It is demonstrated, both theoretically and experimentally, that a negatively charged acceptor can localize more than one electron. The second subject describes experiment and theory of asymmetric quantized Hall and Shubnikov-de Haas plateaus in acceptor-doped GaAs/GaAlAs heterostructures. It is shown that the main features of the plateau asymmetry can be attributed to asymmetric density of Landau states in the presence of acceptors. However, at high magnetic fields, the rain-down effect is also at work. The third subject deals with the so-called disorder modes (DMs) in the cyclotron resonance of conduction electrons. The DMs originate from random distributions of negatively

  12. Impact of donor-acceptor geometry and metal chelation on photophysical properties and applications of triarylboranes.

    PubMed

    Hudson, Zachary M; Wang, Suning

    2009-10-20

    Three-coordinate organoboron compounds have recently found a wide range of applications in materials chemistry as nonlinear optical materials, chemical sensors, and emitters for organic light-emitting diodes (OLEDs). These compounds are excellent electron acceptors due to the empty p(pi) orbital on the boron center. When accompanied by electron donors such as amines, these molecules possess large electronic dipoles, which promote donor-acceptor charge-transfer upon excitation with light. Because of this, donor-acceptor triarylboranes are often highly luminescent both in the solid state and in solution. In this Account, we describe our research to develop donor-acceptor triarylboranes as efficient blue emitters for OLEDs. Through the use of hole-transporting donor groups such as 1-napthylphenylamines, we have prepared multifunctional triarylboranes that can act as the emissive, electron transport, or hole transport layers in OLEDs. We have also examined donor-acceptor compounds based on 2,2'-dipyridylamine or 7-azaindolyl donors, several of which have fluorescent quantum efficiencies approaching 100%. We are also investigating the chemistry of metal-containing triarylboranes. Our studies show that the electron-deficient boryl group can greatly facilitate metal-to-ligand charge-transfer transitions and phosphorescence. In addition, electronegative linker groups such as 2,2'-bipyridine can act in synergy with metal chelation to greatly improve the electron-accepting ability and Lewis acidity of triarylboranes. Donor-acceptor triarylboranes developed in our laboratory can also serve as a series of "switch-on" sensors for fluoride ions. When the donor and acceptor are linked by rigid naphthyl or nonrigid silane linkers, donor-acceptor conjugation is disrupted and charge transfer occurs primarily through space. The binding of fluoride ions to the boron center disrupts this charge transfer, activating alternative pi --> pi* transitions in the molecule and changing the

  13. Phosphate transport and sensing in Saccharomyces cerevisiae.

    PubMed Central

    Wykoff, D D; O'Shea, E K

    2001-01-01

    Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Delta cells; however, the pho84Delta strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Delta cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Delta strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters. PMID:11779791

  14. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  15. Immobilization of DNA in polyacrylamide gel for the manufacture of DNA and DNA-oligonucleotide microchips.

    SciTech Connect

    Proudnikov, D.; Timofeev, E.; Mirzabekov, A.; Center for Mechanistic Biology and Biotechnology; Engelhardt Inst. of Molecular Biology

    1998-05-15

    Activated DNA was immobilized in aldehyde-containing polyacrylamide gel for use in manufacturing the MAGIChip (microarrays of gel-immobilized compounds on a chip). First, abasic sites were generated in DNA by partial acidic depurination. Amino groups were then introduced into the abasic sites by reaction with ethylenediamine and reduction of the aldimine bonds formed. It was found that DNA could be fragmented at the site of amino group incorporation or preserved mostly unfragmented. In similar reactions, both amino-DNA and amino-oligonucleotides were attached through their amines to polyacrylamide gel derivatized with aldehyde groups. Single- and double-stranded DNA of 40 to 972 nucleotides or base pairs were immobilized on the gel pads to manufacture a DNA microchip. The microchip was hybridized with fluorescently labeled DNA-specific oligonucleotide probes. This procedure for immobilization of amino compounds was used to manufacture MAGIChips containing both DNA and oligonucleotides.

  16. Conjugation of γ-Fe 2O 3 nanoparticles with single strand oligonucleotides

    NASA Astrophysics Data System (ADS)

    Lee, C. W.; Huang, K. T.; Wei, P. K.; Yao, Y. D.

    2006-09-01

    Following the thermodecomposition synthetic route, water soluble γ-Fe 2O 3 nanoparticles have been successfully prepared, with average size 8.8±1.3 nm. Based on the process we developed, the magnetic particles have carboxyl group on their surfaces. By using 1-ethyl-3-(3-dimrthylaminopropyl)carbodiimide hydrochloride [EDC] as a liker reagent, we successfully modified a protein, streptavidin, on the surface of γ-Fe 2O 3 nanoparticles. With the strong affinity between biotin with streptavidin, we could use the streptavidin-modified magnetic nanoparticles to separate the rare biotin functionalized molecules from the mixed solution. On the other hand, streptavidin functionalized Fe 2O 3 can catch a biotin labeled single strand oligonucleotides through the strong affinity between streptavidin and biotin. The oligonucleotides functionalized magnetic nanoparticle would separate a specific oligonucleotide from a mixture. This design may be used to create a technique to detect diseases in the future.

  17. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study

    SciTech Connect

    Sebastiani, F.; Comez, L.; Sacchetti, F.; Orecchini, A.; Petrillo, C.; Paciaroni, A.; De Francesco, A.; Teixeira, S. C. M.

    2015-07-07

    The dynamics of the human oligonucleotide AG{sub 3}(T{sub 2}AG{sub 3}){sub 3} has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

  18. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

    PubMed

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M

    2002-08-01

    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  19. Quantitative analysis of the interaction between l-methionine derivative and oligonucleotides.

    PubMed

    Mota, Élia; Sousa, Fani; Queiroz, João A; Cruz, Carla

    2015-04-01

    This study explores the use of l-methionine derivative as a potential affinity ligand for nucleic acids purification. The l-methionine derivative is synthesized by activation of the carboxylic acid group with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide follow by immobilization on amine sensor surface, previously activated and treated with ethylenediamine. Their affinity towards oligonucleotides has been determined by surface plasmon resonance biosensor. The highest affinity is found for cytosine and thymine, followed by adenine, whereas the lowest affinity is found for guanine. For hetero-oligonucleotides the affinity order is CCCTTT > CCCAAA ≈ AAATTT > GGGTTT, showing that nucleotides with cytosine have the highest affinity, and the presence of guanine reduces the affinity, corroborating with the results obtained with homo-oligonucleotides.

  20. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

    PubMed

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P

    2013-12-01

    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  1. Myelin Basic Protein and a Multiple Sclerosis-related MBP-peptide Bind to Oligonucleotides

    PubMed Central

    Rozenblum, Guido Tomás; Kaufman, Tomás; Vitullo, Alfredo Daniel

    2014-01-01

    Aptamer ligands for myelin basic protein (MBP) were obtained using the systematic evolution of ligand by exponential enrichment (SELEX) method. Two clones were isolated from a pool of oligonucleotides and tested for MBP targeting. Using purified MBP, we demonstrated the binding activity of the aptamers and we also showed the affinity of MBP for oligonucleotides of specific length. Moreover, one selected aptamer competitively inhibited the binding of an MBP-specific antibody to MBP and the aptamer was found more sensitive than a commercial antibody. In addition, we showed the ability of the aptamer to detect myelin-rich regions in paraffin-embedded mouse brain tissue. Therefore, the MBP-binding activity of the selected oligonucleotide may prove useful as a tool for life science and medical research for myelin detection and might be a good lead for testing it in autoimmune diseases such as multiple sclerosis. PMID:25202925

  2. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids.

    PubMed

    Nåbo, Lina J; Madsen, Charlotte S; Jensen, Knud J; Kongsted, Jacob; Astakhova, Kira

    2015-05-26

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our results by electronic structure calculations.

  3. Dipentafluorophenyl carbonate--a reagent for the synthesis of oligonucleotides and their conjugates.

    PubMed Central

    Efimov, V A; Kalinkina, A L; Chakhmakhcheva, O G

    1993-01-01

    Dipentafluorophenyl carbonate has been successfully used as condensing agent for the internucleotide bond formation in the synthesis of oligonucleotides via H-phosphonate approach. The mechanism of a nucleotide component activation with this reagent has been investigated with the help of 31P NMR spectroscopy. It was shown that preactivation of deoxynucleoside H-phosphonate with dipentafluorophenyl carbonate has no influence on the efficiency of the synthesis. This reagent is highly reactive, nonhygroscopic and stable on storage at room temperature. The effectiveness of dipentafluorophenyl carbonate in the oligonucleotide chemistry has been demonstrated in the solid-phase synthesis of 10-50-mers on 0.2, 1 and 10 mumol scales. The use of this reagent for the derivatisation of polymer supports as well as for the synthesis of oligonucleotide conjugates with polyethylene glycol and a lipid is described. Images PMID:8265346

  4. Effect of molecular crowding and ionic strength on the isothermal hybridization of oligonucleotides.

    PubMed

    Markarian, Marie Z; Schlenoff, Joseph B

    2010-08-19

    The isothermal hybridization of complementary oligonucleotides, 15-mer, 25-mer, 35-mer, and a molecular beacon, was investigated under varying conditions of molecular crowding and ionic strength, using hypochromicity to follow strand pairing and polyethylene glycol as a crowding agent. Thermodynamic analysis of the results revealed the addition of counterions to the oligonucleotide backbones, DeltaPsi, to be dependent on the strand GC content and the molecular crowding. A decrease in DeltaPsi was observed, with both increasing GC% and solution PEG content. In contrast, the number of bound water molecules depended on the activity of Na(+), where two regimes were observed. At a(Na(+)) < 0.05 and increasing molecular crowding, water molecules were released into the DNA solutions, and oligonucleotide pairing was favored with both increasing hydrophobic forces, whereas at a(Na(+)) >or= 0.05, water molecules were bound to the strands, and the extent of double strand formation decreased with increasing PEG wt %.

  5. Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

    PubMed

    Pan'kov, S V; Chechetkin, V R; Somova, O G; Antonova, O V; Moiseeva, O V; Prokopenko, D V; Yurasov, R A; Gryadunov, D A; Chudinov, A V

    2009-10-01

    Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

  6. New protocol for oligonucleotide microarray fabrication using SU-8-coated glass microslides.

    PubMed

    Sethi, D; Kumar, A; Gandhi, R P; Kumar, P; Gupta, K C

    2010-09-15

    Microarray technology has become an important tool for detection and analysis of nucleic acid targets. Immobilization of modified and unmodified oligonucleotides on epoxy-functionalized glass surfaces is often used in microarray fabrication. Here, we demonstrate a protocol that employs coating of SU-8 (glycidyl ether of bisphenol A) onto glass microslides to obtain high density of epoxy functions for efficient immobilization of aminoalkyl-, thiophosphoryl-, and phosphorylated oligonucleotides with uniform spot morphology. The resulting microarrays exhibited high immobilization (∼65%) and hybridization efficiency (30-36%) and were sufficiently stable over a range of temperature and pH conditions. The prominent feature of the protocol is that spots can be visualized distinctly at 0.05 μM probe (a 20-mer oligonucleotide) concentration. The constructed microarrays were subsequently used for detection of base mismatches and bacterial diseases (meningitis and typhoid).

  7. Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

    PubMed Central

    Narihiro, Takashi; Sekiguchi, Yuji

    2011-01-01

    Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

  8. Oligonucleotide probes for Bordetella bronchiseptica based on 16S ribosomal RNA sequences.

    PubMed

    Taneda, A; Futo, S; Mitsuse, S; Seto, Y; Okada, M; Sakano, T

    1994-12-01

    Bordetella bronchiseptica 16S ribosomal RNA (rRNA) gene was cloned and identified. On the basis of information from computer-assisted sequence comparison of the B. bronchiseptica 16S RRNA sequences with that of other bacterial species, we constructed B. bronchiseptica-specific oligonucleotide probes complementary to variable regions in the 16S rRNA molecule. Specificity of these 32P-labeled oligo-nucleotide probes was tested in a RNA/DNA hybridization with B. bronchiseptica strains and other bacterial strains. Probe BB4 was more specific than three other oligonucleotide probes. This probe BB4 was sensitive enough to be able to detect 10(4) bacterial cells. PMID:9133055

  9. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study

    NASA Astrophysics Data System (ADS)

    Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A.

    2015-07-01

    The dynamics of the human oligonucleotide AG3(T2AG3)3 has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

  10. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    PubMed Central

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  11. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

    PubMed

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe

  12. Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

    PubMed Central

    Allawi, H T; Dong, F; Ip, H S; Neri, B P; Lyamichev, V I

    2001-01-01

    A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility. PMID:11233988

  13. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle

    PubMed Central

    Roh, Changhyun; Lee, Ho-Young; Kim, Sang-Eun; Jo, Sung-Kee

    2010-01-01

    Globally, approximately 170 million people (representing approximately 3% of the population worldwide), are infected with hepatitis C virus (HCV) and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs)-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B) on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinker™ glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL−1 to 1 ng mL−1 range with a detection limit of 1 ng mL−1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well. PMID:20517476

  14. Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2'-O-Ribonucleic Acid Modifications

    SciTech Connect

    Egli, Martin; Minasov, George; Tereshko, Valentina; Pallan, Pradeep S.; Teplova, Marianna; Inamati, Gopal B.; Lesnik, Elena A.; Owens, Steve R.; Ross, Bruce S.; Prakash, Thazha P.; Manoharan, Muthiah

    2010-03-05

    The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the

  15. Towards nanowriting on plastics: dip-pen nanolithography of acrylamido-functionalized oligonucleotides on polystyrene.

    PubMed

    Turri, Stefano; Torlaj, Luca; Levi, Marinella

    2010-08-01

    Model high density DNA arrays have been realized by direct deposition with Dip-Pen Nanolithography of acrylamido-functionalized oligonucleotides (23-mer) on spin-coated, flat polystyrene surfaces. A highly specific interaction between the acrylamide end functionality and polystyrene was found. The surface morphology of the model array was studied by atomic force microscopy (AFM). Spots are clearly seen both in topography and demodulation modes. The array withstands the hybridization process with label free, complementary oligonucleotides and the following cleaning procedures. The final AFM characterization showed significant changes especially in demodulation images which may be an indication that molecular recognition between complementary oligos has occurred.

  16. Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5' terminus.

    PubMed Central

    Kremsky, J N; Wooters, J L; Dougherty, J P; Meyers, R E; Collins, M; Brown, E L

    1987-01-01

    A general method for the immobilization of DNA through its 5'-end has been developed. A synthetic oligonucleotide, modified at its 5'-end with an aldehyde or carboxylic acid, was attached to latex microspheres containing hydrazide residues. Using T4 polynucleotide ligase and an oligonucleotide splint, a single stranded 98mer was efficiently joined to the immobilized synthetic fragment. After impregnation of the latex microspheres with the fluorescent dye, Nile Red and attachment of an aldehyde 16mer, 5 X 10(5) bead-DNA conjugates could be detected with a conventional fluorimeter. Images PMID:3562241

  17. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    PubMed

    Verga, Daniela; Welter, Moritz; Marx, Andreas

    2016-02-01

    DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye.

  18. Customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable

    DOEpatents

    Mirzabekov, Andrei; Guschin, Dmitry Y.; Chik, Valentine; Drobyshev, Aleksei; Fotin, Alexander; Yershov, Gennadiy; Lysov, Yuri

    2002-01-01

    This invention relates to using customized oligonucleotide microchips as biosensors for the detection and identification of nucleic acids specific for different genes, organisms and/or individuals in the environment, in food and in biological samples. The microchips are designed to convert multiple bits of genetic information into simpler patterns of signals that are interpreted as a unit. Because of an improved method of hybridizing oligonucleotides from samples to microchips, microchips are reusable and transportable. For field study, portable laser or bar code scanners are suitable.

  19. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    PubMed

    Verga, Daniela; Welter, Moritz; Marx, Andreas

    2016-02-01

    DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye. PMID:26774580

  20. Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit.

    PubMed

    Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

    2013-01-01

    Fluorescent probes for the detection of a double-stranded DNA were prepared by labeling a triplex-forming DNA oligonucleotide with a thiazole orange (TO) dimer unit. They belong to ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes which we have previously reported. The excitonic interaction between the two TO molecules was expected to effectively suppress the background fluorescence of the probes. The applicability of the ECHO probes for the detection of double-stranded DNA was confirmed by examining the thermal stability and photophysical and kinetic properties of the DNA triplexes formed by the ECHO probes.

  1. Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology.

    PubMed

    Broude, Natalia E

    2002-06-01

    The specific structural features of stem-loop (hairpin) DNA constructs provide increased specificity of target recognition. Recently, several robust assays have been developed that exploit the potential of structurally constrained oligonucleotides to hybridize with their cognate targets. Here, I review new diagnostic approaches based on the formation of stem-loop DNA oligonucleotides: molecular beacon methodology, suppression PCR approaches and the use of hairpin probes in DNA microarrays. The advantages of these techniques over existing ones for sequence-specific DNA detection, amplification and manipulation are discussed.

  2. Mixed backbone antisense oligonucleotides: design, biochemical and biological properties of oligonucleotides containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments.

    PubMed Central

    Kandimalla, E R; Manning, A; Zhao, Q; Shaw, D R; Byrn, R A; Sasisekharan, V; Agrawal, S

    1997-01-01

    We have designed and synthesized mixed backbone oligonucleotides (MBOs) containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments. Thermal melting studies of the phosphodiester MBOs (three 2'-5'linkages at each end) with the complementary 3'-5'-DNA and -RNA target strands suggest that 2'-5'-ribonucleoside incorporation into 3'-5'-oligodeoxyribonucleotides reduces binding to the target strands compared with an all 3'-5'-oligodeoxyribonucleotide of the same sequence and length. Increasing the number of 2'-5'linkages (from six to nine) further reduces binding to the DNA target strand more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996)Nucleic Acids Symp. Ser., 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with the DNA target strand more than the duplex with the RNA target strand. Circular dichroism studies indicate that the duplexes of MBOs with the DNA and RNA target strands have spectral characteristics of both A- and B-type conformations. Compared with the control oligonucleotide, MBOs exhibit moderately higher stability against snake venom phosphodiesterase, S1 nuclease and in fetal calf serum. Although 2'-5'modification does not evoke RNase H activity, this modification does not effect the RNase H activation property of the 3'-5'-deoxyribonucleotide segment adjacent to the modification. In vitro studies with MBOs suggest that they have lesser effects on cell proliferation, clotting prolongation and hemolytic complement lysis than do control PS oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide with all 3'-5'linkages. The current results suggest that a limited number of 2'-5'linkages could be used in conjunction with PS oligonucleotides to further modulate the properties of antisense oligonucleotides as therapeutic agents. PMID:9016567

  3. A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

    PubMed

    Nöll, Gilbert; Su, Qiang; Heidel, Björn; Yu, Yaming

    2014-01-01

    The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

  4. Non-fullerene electron acceptors for use in organic solar cells.

    PubMed

    Nielsen, Christian B; Holliday, Sarah; Chen, Hung-Yang; Cryer, Samuel J; McCulloch, Iain

    2015-11-17

    The active layer in a solution processed organic photovoltaic device comprises a light absorbing electron donor semiconductor, typically a polymer, and an electron accepting fullerene acceptor. Although there has been huge effort targeted to optimize the absorbing, energetic, and transport properties of the donor material, fullerenes remain as the exclusive electron acceptor in all high performance devices. Very recently, some new non-fullerene acceptors have been demonstrated to outperform fullerenes in comparative devices. This Account describes this progress, discussing molecular design considerations and the structure-property relationships that are emerging. The motivation to replace fullerene acceptors stems from their synthetic inflexibility, leading to constraints in manipulating frontier energy levels, as well as poor absorption in the solar spectrum range, and an inherent tendency to undergo postfabrication crystallization, resulting in device instability. New acceptors have to address these limitations, providing tunable absorption with high extinction coefficients, thus contributing to device photocurrent. The ability to vary and optimize the lowest unoccupied molecular orbital (LUMO) energy level for a specific donor polymer is also an important requirement, ensuring minimal energy loss on electron transfer and as high an internal voltage as possible. Initially perylene diimide acceptors were evaluated as promising acceptor materials. These electron deficient aromatic molecules can exhibit good electron transport, facilitated by close packed herringbone crystal motifs, and their energy levels can be synthetically tuned. The principal drawback of this class of materials, their tendency to crystallize on too large a length scale for an optimal heterojunction nanostructure, has been shown to be overcome through introduction of conformation twisting through steric effects. This has been primarily achieved by coupling two units together, forming dimers with

  5. Non-fullerene electron acceptors for use in organic solar cells.

    PubMed

    Nielsen, Christian B; Holliday, Sarah; Chen, Hung-Yang; Cryer, Samuel J; McCulloch, Iain

    2015-11-17

    The active layer in a solution processed organic photovoltaic device comprises a light absorbing electron donor semiconductor, typically a polymer, and an electron accepting fullerene acceptor. Although there has been huge effort targeted to optimize the absorbing, energetic, and transport properties of the donor material, fullerenes remain as the exclusive electron acceptor in all high performance devices. Very recently, some new non-fullerene acceptors have been demonstrated to outperform fullerenes in comparative devices. This Account describes this progress, discussing molecular design considerations and the structure-property relationships that are emerging. The motivation to replace fullerene acceptors stems from their synthetic inflexibility, leading to constraints in manipulating frontier energy levels, as well as poor absorption in the solar spectrum range, and an inherent tendency to undergo postfabrication crystallization, resulting in device instability. New acceptors have to address these limitations, providing tunable absorption with high extinction coefficients, thus contributing to device photocurrent. The ability to vary and optimize the lowest unoccupied molecular orbital (LUMO) energy level for a specific donor polymer is also an important requirement, ensuring minimal energy loss on electron transfer and as high an internal voltage as possible. Initially perylene diimide acceptors were evaluated as promising acceptor materials. These electron deficient aromatic molecules can exhibit good electron transport, facilitated by close packed herringbone crystal motifs, and their energy levels can be synthetically tuned. The principal drawback of this class of materials, their tendency to crystallize on too large a length scale for an optimal heterojunction nanostructure, has been shown to be overcome through introduction of conformation twisting through steric effects. This has been primarily achieved by coupling two units together, forming dimers with

  6. Non-Fullerene Electron Acceptors for Use in Organic Solar Cells

    PubMed Central

    2015-01-01

    Conspectus The active layer in a solution processed organic photovoltaic device comprises a light absorbing electron donor semiconductor, typically a polymer, and an electron accepting fullerene acceptor. Although there has been huge effort targeted to optimize the absorbing, energetic, and transport properties of the donor material, fullerenes remain as the exclusive electron acceptor in all high performance devices. Very recently, some new non-fullerene acceptors have been demonstrated to outperform fullerenes in comparative devices. This Account describes this progress, discussing molecular design considerations and the structure–property relationships that are emerging. The motivation to replace fullerene acceptors stems from their synthetic inflexibility, leading to constraints in manipulating frontier energy levels, as well as poor absorption in the solar spectrum range, and an inherent tendency to undergo postfabrication crystallization, resulting in device instability. New acceptors have to address these limitations, providing tunable absorption with high extinction coefficients, thus contributing to device photocurrent. The ability to vary and optimize the lowest unoccupied molecular orbital (LUMO) energy level for a specific donor polymer is also an important requirement, ensuring minimal energy loss on electron transfer and as high an internal voltage as possible. Initially perylene diimide acceptors were evaluated as promising acceptor materials. These electron deficient aromatic molecules can exhibit good electron transport, facilitated by close packed herringbone crystal motifs, and their energy levels can be synthetically tuned. The principal drawback of this class of materials, their tendency to crystallize on too large a length scale for an optimal heterojunction nanostructure, has been shown to be overcome through introduction of conformation twisting through steric effects. This has been primarily achieved by coupling two units together

  7. Insights on the design and electron-acceptor properties of conjugated organophosphorus materials.

    PubMed

    Baumgartner, Thomas

    2014-05-20

    The development of conjugated organic materials has become a rapidly evolving field of research, particularly with a view toward practical applications in so-called organic electronics that encompass a variety of device types, such as OLEDs, OPVs, and OFETs. Almost all of these devices minimally require the presence of electron-donor and -acceptor components that act as p- and n-type semiconductors, respectively. Research over the past two decades has shown that while there is an abundant resource of organic p-type materials, suitable n-type species are few and far between. To overcome this severe bottleneck for the further development of organic electronics, researchers have identified organo-main-group avenues as valuable alternatives toward organic electron-acceptor materials that may ultimately be used as n-type components in practical devices. One particular element of interest in this context is phosphorus, which at first glance may not necessarily suggest such properties. In this Account, I provide detailed insights on the origin of the electron-acceptor properties of organophosphorus-based conjugated materials and include an overview of important molecular species that have been developed by my group and others. To this end, I explain that the electron-acceptor properties of conjugated organophosphorus materials originate from an interaction known as negative hyperconjugation. While this particular interaction creates a simply inductively withdrawing phosphoryl substituent for π-conjugated scaffolds, incorporation of a phosphorus atom as an integral part of a cyclic substructure within a π-conjugated system provides a much more complex, versatile, and consequently highly valuable tool for the tuning of the electron-acceptor properties of the materials. Notably, the degree of negative hyperconjugation can effectively be tailored in various ways via simple substitution at the phosphorus center. This is now well established for phosphole-based molecular

  8. Insights on the design and electron-acceptor properties of conjugated organophosphorus materials.

    PubMed

    Baumgartner, Thomas

    2014-05-20

    The development of conjugated organic materials has become a rapidly evolving field of research, particularly with a view toward practical applications in so-called organic electronics that encompass a variety of device types, such as OLEDs, OPVs, and OFETs. Almost all of these devices minimally require the presence of electron-donor and -acceptor components that act as p- and n-type semiconductors, respectively. Research over the past two decades has shown that while there is an abundant resource of organic p-type materials, suitable n-type species are few and far between. To overcome this severe bottleneck for the further development of organic electronics, researchers have identified organo-main-group avenues as valuable alternatives toward organic electron-acceptor materials that may ultimately be used as n-type components in practical devices. One particular element of interest in this context is phosphorus, which at first glance may not necessarily suggest such properties. In this Account, I provide detailed insights on the origin of the electron-acceptor properties of organophosphorus-based conjugated materials and include an overview of important molecular species that have been developed by my group and others. To this end, I explain that the electron-acceptor properties of conjugated organophosphorus materials originate from an interaction known as negative hyperconjugation. While this particular interaction creates a simply inductively withdrawing phosphoryl substituent for π-conjugated scaffolds, incorporation of a phosphorus atom as an integral part of a cyclic substructure within a π-conjugated system provides a much more complex, versatile, and consequently highly valuable tool for the tuning of the electron-acceptor properties of the materials. Notably, the degree of negative hyperconjugation can effectively be tailored in various ways via simple substitution at the phosphorus center. This is now well established for phosphole-based molecular

  9. Uranium endowments in phosphate rock.

    PubMed

    Ulrich, Andrea E; Schnug, Ewald; Prasser, Horst-Michael; Frossard, Emmanuel

    2014-04-15

    This study seeks to identify and specify the components that make up the prospects of U recovery from phosphate rock. A systems approach is taken. The assessment includes i) reviewing past recovery experience and lessons learned; ii) identifying factors that determine recovery; and iii) establishing a contemporary evaluation of U endowments in phosphate rock reserves, as well as the available and recoverable amounts from phosphate rock and phosphoric acid production. We find that in the past, recovery did not fulfill its potential and that the breakup of the Soviet Union worsened then-favorable recovery market conditions in the 1990s. We find that an estimated 5.7 million tU may be recoverable from phosphate rock reserves. In 2010, the recoverable tU from phosphate rock and phosphoric acid production may have been 15,000 tU and 11,000 tU, respectively. This could have filled the world U supply-demand gap for nuclear energy production. The results suggest that the U.S., Morocco, Tunisia, and Russia would be particularly well-suited to recover U, taking infrastructural considerations into account. We demonstrate future research needs, as well as sustainability orientations. We conclude that in order to promote investment and production, it seems necessary to establish long-term contracts at guaranteed prices, ensuring profitability for phosphoric acid producers. PMID:24556272

  10. Uranium endowments in phosphate rock.

    PubMed

    Ulrich, Andrea E; Schnug, Ewald; Prasser, Horst-Michael; Frossard, Emmanuel

    2014-04-15

    This study seeks to identify and specify the components that make up the prospects of U recovery from phosphate rock. A systems approach is taken. The assessment includes i) reviewing past recovery experience and lessons learned; ii) identifying factors that determine recovery; and iii) establishing a contemporary evaluation of U endowments in phosphate rock reserves, as well as the available and recoverable amounts from phosphate rock and phosphoric acid production. We find that in the past, recovery did not fulfill its potential and that the breakup of the Soviet Union worsened then-favorable recovery market conditions in the 1990s. We find that an estimated 5.7 million tU may be recoverable from phosphate rock reserves. In 2010, the recoverable tU from phosphate rock and phosphoric acid production may have been 15,000 tU and 11,000 tU, respectively. This could have filled the world U supply-demand gap for nuclear energy production. The results suggest that the U.S., Morocco, Tunisia, and Russia would be particularly well-suited to recover U, taking infrastructural considerations into account. We demonstrate future research needs, as well as sustainability orientations. We conclude that in order to promote investment and production, it seems necessary to establish long-term contracts at guaranteed prices, ensuring profitability for phosphoric acid producers.

  11. Dissolution of phosphate matrices based on the thorium phosphate diphosphate

    NASA Astrophysics Data System (ADS)

    Dacheux, N.; Thomas, A. C.; Brandel, V.; Genet, M.

    2000-07-01

    Several authors have reported the use of phosphate matrices like apatites, monazites or NZP for the immobilization of actinides coming from an advanced reprocessing or for the final disposal of the excess plutonium from dismantled nuclear weapons. The thorium phosphate diphosphate Th4(PO4)4P2O7 (namely TPD) was also proposed for this purpose. Indeed, its structure allows the replacement of large amounts of tetravalent actinides like uranium, neptunium or plutonium leading to the obtention of solid solutions. The maximum weight loading was estimated to be equal to about 48% for uranium, 33% for neptunium and 26% for plutonium.

  12. Receptor-mediated hepatic uptake of M6P-BSA-conjugated triplex-forming oligonucleotides in rats.

    PubMed

    Ye, Zhaoyang; Cheng, Kun; Guntaka, Ramareddy V; Mahato, Ram I

    2006-01-01

    Excessive production of extracellular matrix, predominantly type I collagen, results in liver fibrosis. Earlier we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) and conjugated to the type I collagen specific triplex-forming oligonucleotide (TFO) for its enhanced delivery to hepatic stellate cells (HSCs), which is the principal liver fibrogenic cell. In this report, we demonstrate a time-dependent cellular uptake of M6P-BSA-33P-TFO by HSC-T6 cells. Both cellular uptake and nuclear deposition of M6P-BSA-33P-TFO were significantly higher than those of 33P-TFO, leading to enhanced inhibition of type I collagen transcription. Following systemic administration into rats, hepatic accumulation of M6P-BSA-33P-TFO increased from 55% to 68% with the number of M6P per BSA from 14 to 27. Unlike 33P-TFO, there was no significant decrease in the hepatic uptake of (M6P)20-BSA-33P-TFO in fibrotic rats. Prior administration of excess M6P-BSA decreased the hepatic uptake of (M6P)20-BSA-33P-TFO from 66% to 40% in normal rats, and from 60% to 15% in fibrotic rats, suggesting M6P/insulin-like growth factor II (M6P/IGF II) receptor-mediated endocytosis of M6P-BSA-33P-TFO by HSCs. Almost 82% of the total liver uptake in fibrotic rats was contributed by HSCs. In conclusion, by conjugation with M6P-BSA, the TFO could be potentially used for the treatment of liver fibrosis.

  13. Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors.

    PubMed Central

    Frébortová, Jitka; Fraaije, Marco W; Galuszka, Petr; Sebela, Marek; Pec, Pavel; Hrbác, Jan; Novák, Ondrej; Bilyeu, Kristin D; English, James T; Frébort, Ivo

    2004-01-01

    The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. 150 s(-1) could be obtained. This suggests that the natural electron acceptor of the enzyme is quite probably a p-quinone or similar compound. By using the stopped-flow technique, it was found that the enzyme is rapidly reduced by N6-(2-isopentenyl)adenine (k(red)=950 s(-1)). Re-oxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming its classification as a dehydrogenase. Furthermore, it was established for the first time that the enzyme is capable of degrading aromatic cytokinins, although at low reaction rates. As a result, the enzyme displays a dual catalytic mode for oxidative degradation of cytokinins: a low-rate and low-substrate specificity reaction with oxygen as the electron acceptor, and high activity and strict specificity for isopentenyladenine and analogous cytokinins with some specific electron acceptors. PMID:14965342

  14. Nature of the attractive interaction between proton acceptors and organic ring systems.

    PubMed

    Arras, Emmanuel; Seitsonen, Ari Paavo; Klappenberger, Florian; Barth, Johannes V

    2012-12-14

    Systematic ab initio calculations are combined with a deconvolution of electrostatic contributions to analyze the interplay between potential hydrogen bond acceptors and organic rings with C(sp(2))-H groups (benzene, pyridine and cyclopentadiene). A distinct anisotropic interaction between the ring systems and the electron lone pairs of cyanide, water and other acceptor species is revealed that favors the in-plane orientation of the proton acceptor group. In the attractive regime this interaction carries a pronounced electrostatic signature. By decomposing the electrostatic contribution into parts attributed to different subunits of the ring systems we demonstrate that a major proportion of the interaction energy gain is originating from the non-adjacent moieties, that are not in close contact with. This behavior holds equally for homocyclic, heterocyclic and non-aromatic rings but contrasts that of the ethyne molecule, taken as reference for a weak hydrogen bond donor clearly exhibiting the expected localized character. The ring interaction requires the presence of π-electron clouds and typically results in an interaction energy gain of 40 to 80 meV. Our findings suggest the proton acceptor-ring interaction as a new category of intermolecular non-covalent interactions.

  15. Experimental investigation of donor-acceptor electron transfer and back transfer in solid solutions

    SciTech Connect

    Dorfman, R.C.; Lin, Y.; Fayer, M.D. )

    1989-08-24

    Electron transfer from an optically excited donor (rubrene) to randomly distributed acceptors (duroquinone) followed by electron back transfer in a rigid solution (sucrose octaacetate) has been studied experimentally. The forward electron-transfer process was observed by time-dependent fluorescence quenching measurements, while the electron back transfer from the radical anion to the radical cation was monitored by use of the picosecond transient grating technique. A statistical mechanics theory is used to describe the time-dependent dynamics of the system and to extract the forward- and back-transfer parameters from the data. The theory includes donor-acceptor and acceptor-acceptor excluded volume. It is found that the inclusion of excluded volume is necessary to obtain accurate transfer parameters. These parameters enable a detailed description of the electron transfer and recombination dynamics to be given. The agreement between theory and experiment is excellent. A variety of time-dependent properties of the system are calculated. These include the time-dependent ion populations and the probability that the ith acceptor is an ion as a function of time and distance. In addition, (R(t)) and ({tau}(t)), which are the average ion separation as a function of time and the average ion existence time as a function of ion separation, respectively, are calculated.

  16. Phosphate-a poison for humans?

    PubMed

    Komaba, Hirotaka; Fukagawa, Masafumi

    2016-10-01

    Maintenance of phosphate balance is essential for life, and mammals have developed a sophisticated system to regulate phosphate homeostasis over the course of evolution. However, due to the dependence of phosphate elimination on the kidney, humans with decreased kidney function are likely to be in a positive phosphate balance. Phosphate excess has been well recognized as a critical factor in the pathogenesis of mineral and bone disorders associated with chronic kidney disease, but recent investigations have also uncovered toxic effects of phosphate on the cardiovascular system and the aging process. Compelling evidence also suggests that increased fibroblastic growth factor 23 and parathyroid hormone levels in response to a positive phosphate balance contribute to adverse clinical outcomes. These insights support the current practice of managing serum phosphate in patients with advanced chronic kidney disease, although definitive evidence of these effects is lacking. Given the potential toxicity of excess phosphate, the general population may also be viewed as a target for phosphate management. However, the widespread implementation of dietary phosphate intervention in the general population may not be warranted due to the limited impact of increased phosphate intake on mineral metabolism and clinical outcomes. Nonetheless, the increasing incidence of kidney disease or injury in our aging society emphasizes the potential importance of this issue. Further work is needed to more completely characterize phosphate toxicity and to establish the optimal therapeutic strategy for managing phosphate in patients with chronic kidney disease and in the general population. PMID:27282935

  17. Process for producing granular diammonium phosphate

    SciTech Connect

    Fairchild, W.D.

    1988-05-17

    A process for the production of solid granular diammonium phosphate is described comprising: reacting anhydrous ammonia with phosphoric acid in a reactor to form a partially reacted slurry of monoammonium phosphate and diammonium phosphate; pumping the slurry to a granulator-reactor and further reacting the slurry with anhydrous ammonia to form a solid granular diammonium phosphate mixture having a particle range size consisting of undersize, oversize and product; drying the solid granular diammonium phosphate mixture in a dryer; dividing the dried solid granular diammonium phosphate mixture being discharged from the dryer into a first portion and a second portion; diverting and feeding the first portion of the dried granular diammonium phosphate mixture back to the granulator-reactor; feeding the second portion of dried granular diammonium phosphate mixture to a classifying means consisting of a set of screens including an oversize screen and a product screen set to a narrow size separation to separate the mixture of the solid granular diammonium phosphate into undersize, oversize and product solid granular diammonium phosphate; milling the oversize granular diammonium phosphate; recycling to the granular-reactor the milled oversized granular diammonium phosphate and the undersized granular particles obtained during the classifying of the solid granular diammonium phosphate mixture; and collecting the desired product granular particle thereby enhancing the production of a narrow range of granular diammonium phosphate particle size distribution within a broad range of particle size distribution.

  18. Effect of acceptor (Mg) concentration on the electrical resistance at room and high (200 deg. C) temperatures of acceptor (Mg)-doped BaTiO{sub 3} ceramics

    SciTech Connect

    Yoon, Seok-Hyun; Hong, Min-Hee; Hong, Jeong-Oh; Kim, Young-Tae; Hur, Kang-Heon

    2007-09-01

    The behaviors of the electrical resistance at room and high (200 deg. C) temperatures of acceptor (Mg)-doped BaTiO{sub 3} ceramics with the increase of acceptor concentration were investigated. A series of coarse-grained specimens with different acceptor concentrations that were sintered at various oxygen partial pressures was prepared. The critical acceptor concentrations, beyond which the room temperature resistance increases abruptly, were experimentally evaluated and they were found to increase with a decrease in oxygen partial pressure during sintering. Each defect and electron concentrations at sintering and room temperature as a function of acceptor concentration were theoretically calculated. The results calculated could explain the experimentally observed behavior of the resistance versus acceptor concentration. The high (200 deg. C) temperature resistances under high electric field showed resistance degradation critically depending on acceptor concentration in the very small concentration range below {approx}0.1 mol %, which occurred easily with the increase of acceptor concentration. It was explained that such behavior was caused by the variation of potential barrier of grain boundaries for the migration of oxygen vacancies as a function of acceptor concentration.

  19. Hydrogen bonding mediated by key orbital interactions determines hydration enthalpy differences of phosphate water clusters.

    PubMed

    Ruben, Eliza A; Chapman, Michael S; Evanseck, Jeffrey D

    2007-10-25

    Electronic structure calculations have been carried out to provide a molecular interpretation for dihydrogen phosphate stability in water relative to that of metaphosphate. Specifically, hydration enthalpies of biologically important metaphosphate and dihydrogen phosphate with one to three waters have been computed with second-order Møller-Plesset perturbation and density functional theory (B3LYP) with up to the aug-cc-pvtz basis set and compared to experiment. The inclusion of basis set superposition error corrections and supplemental diffuse functions are necessary to predict hydration enthalpies within experimental uncertainty. Natural bond orbital analysis is used to rationalize underlying hydrogen bond configurations and key orbital interactions responsible for the experimentally reported difference in hydration enthalpies between metaphosphate and dihydrogen phosphate. In general, dihydrogen phosphate forms stronger hydrogen bonds compared to metaphosphate due to a greater charge transfer or enhanced orbital overlap between the phosphoryl oxygen lone pairs, n(O), and the antibonding O-H bond of water. Intramolecular distal lone pair repulsion with the donor n(O) orbital of dihydrogen phosphate distorts symmetric conformations, which improves n(O) and sigma*(O-H) overlap and ultimately the hydrogen bond strength. Unlike metaphosphate, water complexed to dihydrogen phosphate can serve as both a hydrogen bond donor and a hydrogen bond acceptor, which results in cooperative charge transfer and a reduction of the energy gap between n(O) and sigma*(O-H), leading to stronger hydrogen bonds. This study offers insight into how orbital interactions mediate hydrogen bond strengths with potential implications on the understanding of the kinetics and mechanism in enzymatic phosphoryl transfer reactions.

  20. Phosphoryl transfer from α-d-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types.

    PubMed

    Wildberger, Patricia; Pfeiffer, Martin; Brecker, Lothar; Rechberger, Gerald N; Birner-Gruenberger, Ruth; Nidetzky, Bernd

    2015-03-01

    The Cori ester α-d-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s(-1)) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys(189) and Cys(195). Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 (18)O solvent proceeded with complete (18)O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P. PMID:25527541

  1. Detergent phosphate bans and eutrophication

    SciTech Connect

    Lee, G.F.; Jones, R.A.

    1986-04-01

    The Vollenweider-OECD eutrophication model has been expanded to approximately 400 lakes. It is possible to make a quantitative prediction of the effects of a detergent phosphate ban and thereby to ascertain the potential benefits of such a ban. In order to assess the effect of a detergent phosphate ban on water quality it is necessary to know the percentage of phosphorus in the domestic waste water that enters the water body, either directly or indirectly, and the percentage of the total phosphorus load that is derived from domestic wastewater. Although detergent phosphate bans generally will not result in an overall improvement to water quality, there may be some situations in which eutrophication-related water quality would be improved by a ban. 8 references, 1 figure, 1 table.

  2. Proficiency of acceptor-donor-acceptor organic dye with spiro-MeOTAD HTM on the photovoltaic performance of dye sensitized solar cell

    NASA Astrophysics Data System (ADS)

    Ramavenkateswari, K.; Venkatachalam, P.

    2016-08-01

    This work investigates the proficiency of acceptor-donor-acceptor (A-D-A) organic dye Diisopropyl azodicarboxylate (DIAC) as photosensitizer on the photovoltaic parameters of silver (Ag) doped TiO2 photoanode dye-sensitized solar cells (DSSCs) with quasi-solid state electrolyte/hole transport material (HTM) spiro-MeOTAD. TNSs (TiO2 nanosticks) photoanodes are prepared through sol-gel method and hydrothermal technique. X-ray powder diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM) and BET measurement were used to characterize the structure and morphology of TiO2 nanostructures. The Diisopropyl azodicarboxylate organic dye with TNPs-Ag@TNSs composite photoanode structure and spiro-MeOTAD HTM exhibited better power conversion efficiency (PCE). [Figure not available: see fulltext.

  3. Proficiency of acceptor-donor-acceptor organic dye with spiro-MeOTAD HTM on the photovoltaic performance of dye sensitized solar cell

    NASA Astrophysics Data System (ADS)

    Ramavenkateswari, K.; Venkatachalam, P.

    2016-09-01

    This work investigates the proficiency of acceptor-donor-acceptor (A-D-A) organic dye Diisopropyl azodicarboxylate (DIAC) as photosensitizer on the photovoltaic parameters of silver (Ag) doped TiO2 photoanode dye-sensitized solar cells (DSSCs) with quasi-solid state electrolyte/hole transport material (HTM) spiro-MeOTAD. TNSs (TiO2 nanosticks) photoanodes are prepared through sol-gel method and hydrothermal technique. X-ray powder diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM) and BET measurement were used to characterize the structure and morphology of TiO2 nanostructures. The Diisopropyl azodicarboxylate organic dye with TNPs-Ag@TNSs composite photoanode structure and spiro-MeOTAD HTM exhibited better power conversion efficiency (PCE).

  4. An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

    SciTech Connect

    Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B. )

    1989-12-05

    UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ((beta-35S)UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with (beta-35S). UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue.

  5. [Phosphate metabolism and iron deficiency].

    PubMed

    Yokoyama, Keitaro

    2016-02-01

    Autosomal dominant hypophosphatemic rickets(ADHR)is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage. Fibroblast growth factor 23(FGF23)is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. Low iron status plays a role in the pathophysiology of ADHR. Iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. It was reported that FGF23 elevation in patients with CKD, who are often iron deficient. In patients with nondialysis-dependent CKD, treatment with ferric citrate hydrate resulted in significant reductions in serum phosphate and FGF23.

  6. Growth of strain SES-3 with arsenate and other diverse electron acceptors

    USGS Publications Warehouse

    Laverman, A.M.; Blum, J.S.; Schaefer, J.K.; Phillips, E.J.P.; Lovley, D.R.; Oremland, R.S.

    1995-01-01

    The selenate-respiring bacterial strain SES-3 was able to use a variety of inorganic electron acceptors to sustain growth. SES-3 grew with the reduction of arsenate to arsenite, Fe(III) to Fe(II), or thiosulfate to sulfide. It also grew in medium in which elemental sulfur, Mn(IV), nitrite, trimethylamine N-oxide, or fumarate was provided as an electron acceptor. Growth on oxygen was microaerophilic. There was no growth with arsenite or chromate. Washed suspensions of cells grown on selenate or nitrate had a constitutive ability to reduce arsenate but were unable to reduce arsenite. These results suggest that strain SES-3 may occupy a niche as an environmental opportunist by being able to take advantage of a diversity of electron acceptors.

  7. Effect of cathode electron acceptors on simultaneous anaerobic sulfide and nitrate removal in microbial fuel cell.

    PubMed

    Cai, Jing; Zheng, Ping; Mahmood, Qaisar

    2016-01-01

    The current investigation reports the effect of cathode electron acceptors on simultaneous sulfide and nitrate removal in two-chamber microbial fuel cells (MFCs). Potassium permanganate and potassium ferricyanide were common cathode electron acceptors and evaluated for substrate removal and electricity generation. The abiotic MFCs produced electricity through spontaneous electrochemical oxidation of sulfide. In comparison with abiotic MFC, the biotic MFC showed better ability for simultaneous nitrate and sulfide removal along with electricity generation. Keeping external resistance of 1,000 Ω, both MFCs showed good capacities for substrate removal where nitrogen and sulfate were the main end products. The steady voltage with potassium permanganate electrodes was nearly twice that of with potassium ferricyanide. Cyclic voltammetry curves confirmed that the potassium permanganate had higher catalytic activity than potassium ferricyanide. The potassium permanganate may be a suitable choice as cathode electron acceptor for enhanced electricity generation during simultaneous treatment of sulfide and nitrate in MFCs. PMID:26901739

  8. Förster Resonance Energy Transfer between Quantum Dot Donors and Quantum Dot Acceptors

    PubMed Central

    Chou, Kenny F.; Dennis, Allison M.

    2015-01-01

    Förster (or fluorescence) resonance energy transfer amongst semiconductor quantum dots (QDs) is reviewed, with particular interest in biosensing applications. The unique optical properties of QDs provide certain advantages and also specific challenges with regards to sensor design, compared to other FRET systems. The brightness and photostability of QDs make them attractive for highly sensitive sensing and long-term, repetitive imaging applications, respectively, but the overlapping donor and acceptor excitation signals that arise when QDs serve as both the donor and acceptor lead to high background signals from direct excitation of the acceptor. The fundamentals of FRET within a nominally homogeneous QD population as well as energy transfer between two distinct colors of QDs are discussed. Examples of successful sensors are highlighted, as is cascading FRET, which can be used for solar harvesting. PMID:26057041

  9. Photovoltaic enhancement of organic solar cells by a bridged donor-acceptor block copolymer approach

    NASA Astrophysics Data System (ADS)

    Sun, Sam-Shajing; Zhang, Cheng; Ledbetter, Abram; Choi, Soobum; Seo, Kang; Bonner, Carl E.; Drees, Martin; Sariciftci, Niyazi Serdar

    2007-01-01

    The authors show that a photovoltaic device composed of a -donor-bridge-acceptor-bridge- type block copolymer thin film exhibits a significant performance improvement over its corresponding donor/acceptor blend (Voc increased from 0.14to1.10V and Jsc increased from 0.017 to 0.058mA/cm2) under identical conditions, where donor is an alkyl derivatized poly-p-phenylenevinylene (PPV) conjugated block, acceptor is a sulfone-alkyl derivatized PPV conjugated block, and bridge is a nonconjugated and flexible unit. The authors attribute such improvement to the block copolymer intrinsic nanophase separation and molecular self-assembly that results in the reduction of the exciton and carrier losses.

  10. Mg acceptors in GaN: Dependence of the /g-anisotropy on the doping concentration

    NASA Astrophysics Data System (ADS)

    Hofmann, Detlev M.; Burkhardt, Wolfgang; Leiter, Frank; Walter von Förster; Alves, Helder; Hofstaetter, Albrecht; Meyer, Bruno K.; Romanov, Nikolai G.; Amano, Hiroshi; Akasaki, Isamu

    1999-12-01

    Mg acceptors in GaN epitaxial layers grown by metal-organic vapour-phase epitaxy were investigated by optically detected magnetic resonance (ODMR) spectroscopy. The magnetic resonances were detected on the magnetic circular dichroism (MCD) of the acceptor bound exciton (Mg0X) in the near bandgap region, and in the infrared spectral range on the MCD of the hole ionisation transition Mg0+hν→Mg-+hVB. The observed g-values of the Mg0 acceptors range for g|| from 2.102 to 2.065 and for g⊥ from 1.94 to 2.00, respectively. These variations depend on the Mg doping concentration.

  11. Rational design of two-dimensional molecular donor-acceptor nanostructure arrays

    NASA Astrophysics Data System (ADS)

    Zhang, Jia Lin; Zhong, Shu; Zhong, Jian Qiang; Niu, Tian Chao; Hu, Wen Ping; Wee, Andrew Thye Shen; Chen, Wei

    2015-02-01

    The construction of long-range ordered organic donor-acceptor nanostructure arrays over microscopic areas supported on solid substrates is one of the most challenging tasks towards the realization of molecular nanodevices. They can also be used as ideal model systems to understand light induced charge transfer, charge separation and energy conversion processes and mechanisms at the nanometer scale. The aim of this paper is to highlight recent advances and progress in this topic. Special attention is given to two different strategies for the construction of organic donor-acceptor nanostructure arrays, namely (i) molecular self-assembly on artificially patterned or pre-defined molecular surface nanotemplates and (ii) molecular nanostructure formation steered via directional and selective intermolecular interactions. The interfacial charge transfer and the energy level alignment of these donor-acceptor nanostructures are also discussed.

  12. Spectroscopic studies of charge transfer complexes between colchicine and some π acceptors

    NASA Astrophysics Data System (ADS)

    Arslan, Mustafa; Duymus, Hulya

    2007-07-01

    Charge transfer complexes between colchicine as donor and π acceptors such as tetracyanoethylene (TCNE), 2,3-dichloro-5,6-dicyano- p-benzoquinone (DDQ), p-chloranil ( p-CHL) have been studied spectrophotometrically in dichloromethane at 21 °C. The stoichiometry of the complexes was found to be 1:1 ratio by the Job method between donor and acceptors with the maximum absorption band at a wavelength of 535, 585 and 515 nm. The equilibrium constant and thermodynamic parameters of the complexes were determined by Benesi-Hildebrand and van't Hoff equations. Colchicine in pure form and in dosage form was applied in this study. The formation constants for the complexes were shown to be dependent on the structure of the electron acceptors used.

  13. Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis.

    PubMed

    Krishnan, Shyam; Miller, Rand M; Tian, Boxue; Mullins, R Dyche; Jacobson, Matthew P; Taunton, Jack

    2014-09-10

    Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.

  14. Donor-Acceptor Heterojunction Configurations Based on DNA-Multichromophore Arrays.

    PubMed

    Nakamura, Mitsunobu; Tsuto, Koji; Jomura, Ayumi; Takada, Tadao; Yamana, Kazushige

    2015-08-10

    Multichromophore arrays of bis(2-thienyl)diketopyrrolopyrrole (DPP) and naphthalenediimide (NDI) with two Zn(II) -cyclens were constructed using thymidine DNA as a scaffold through the binding of the Zn(II) -cyclens with thymine bases. We demonstrate photocurrent generation in a donor-acceptor heterojunction configuration consisting of the DPP (donor) and NDI (acceptor) arrays co-immobilized on an Au electrode. The co-immobilized electrode exhibited good photocurrent responses because of the efficient charge separation between the DPP and NDI arrays. In contrast, an immobilized electrode consisting of randomly assembled DPP-NDI arrays generated no photocurrent response because DPP formed ground-state charge-transfer complexes with NDI in the randomly assembled arrays. Therefore, our approach to generate donor-acceptor heterojunctions based on DNA-multichromophore arrays is a useful method to efficiently generate photocurrent. PMID:26179473

  15. Enhanced biodegradation of cyclotetramethylenetetranitramine (HMX) under mixed electron-acceptor condition.

    PubMed

    Boopathy, R

    2001-02-01

    The biodegradation of cyclotetramethylenetetranitramine, commonly known as 'high melting explosive' (HMX), under various electron-acceptor conditions was investigated using enrichment cultures developed from the anaerobic digester sludge of Thibodaux sewage treatment plant. The results indicated that the HMX was biodegraded under sulfate reducing, nitrate reducing, fermenting, methanogenic, and mixed electron accepting conditions. However, the rates of degradation varied among the various conditions studied. The fastest removal of HMX (from 22 ppm on day 0 to < 0.05 ppm on day 11) was observed under mixed electron-acceptor conditions, followed in order by sulfate reducing, fermenting, methanogenic, and nitrate reducing conditions. Under aerobic conditions, HMX was not biodegraded, which indicated that HMX degradation takes place under anaerobic conditions via reduction. HMX was converted to methanol and chloroform under mixed electron-acceptor conditions. This study showed evidence for HMX degradation under anaerobic conditions in a mixed microbial population system similar to any contaminated field sites, where a heterogeneous population exists.

  16. Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

    PubMed

    Kreuzinger, N; Podeu, R; Gruber, F; Göbl, F; Kubicek, C P

    1996-09-01

    Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction. PMID:8795234

  17. Pyridoxal-5'-phosphate-dependent catalytic antibodies.

    PubMed

    Gramatikova, Svetlana; Mouratou, Barbara; Stetefeld, Jörg; Mehta, Perdeep K; Christen, Philipp

    2002-11-01

    Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5'-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten N(alpha)-(5'-phosphopyridoxyl)-L-lysine, a stable analog of the covalent coenzyme-substrate adducts were screened by a competition ELISA for binding of the PLP-amino acid Schiff base adduct. The Schiff base with its C4'-N alpha double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent alpha,beta-elimination reaction of beta-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k(cat)'=0.42 min(-1) with D-alanine at 25 degrees C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme-amino acid adduct. (2) The bond at C alpha to be broken lies together with the C alpha-N bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The alpha-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme-substrate adduct assumes a cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10(4) times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9 k(cat (Ab x PLP))'/k(cat (PLP))'=5 x 10(3)). The analogies of antibody 15A9 with

  18. Hybrid coconut seedlings, scholarships, and discount cards for family planning acceptors.

    PubMed

    Sumarsono

    1989-10-01

    Having learned from failed family planning (FP) incentive schemes in other countries, Indonesia implemented a reward system designed to popularize FP in the community. In order to overcome cultural opposition to FP, many countries in the 1970s opted to give incentives--money, materials, etc.--to new contraceptive acceptors and the FP workers who successfully recruited them. These countries, which oftentimes spent up to 1/4 of their program budget on incentives, saw rapid increases in the number of new acceptors. The results, however, only reflected a superficial acceptance of FP. When the incentives stopped, the number of acceptors dropped considerably. Recognizing this, the Indonesian government set out to increase FP acceptance by making the small family the norm in the community. And one of the approaches for doing so was a reward system. The goals of the reward program were: 1) to raise awareness of the recognition given to individuals or groups that have accepted FP; 2) to create pride among FP workers and new acceptors; and 3) to generate leadership in the community. Villages with high FP acceptance receive rewards such as deep-wells that provide clean water or income generating projects. Individuals also receive rewards that sometimes include hybrid coconut seedlings which, after 3 years, can yield up to 700 coconuts, which can provide a family with a significant supplemental income. The government also gives scholarships to children of FP acceptors. Also, the president of Indonesia publicly recognized family planning acceptors. In 1989, over 800,000 couples received awards for practicing contraception over the past 5-16 years. PMID:12315968

  19. Molecular origin of photovoltaic performance in donor-block-acceptor all-conjugated block copolymers

    DOE PAGES

    Smith, Kendall A.; Lin, Yen -Hao; Mok, Jorge W.; Yager, Kevin G.; Strzalka, Joseph; Nie, Wanyi; Mohite, Aditya D.; Verduzco, Rafael

    2015-11-03

    All-conjugated block copolymers may be an effective route to self-assembled photovoltaic devices, but we lack basic information on the relationship between molecular characteristics and photovoltaic performance. Here, we synthesize a library of poly(3-hexylthiophene) (P3HT) block poly((9,9-dialkylfluorene)-2,7-diyl-alt-[4,7-bis(alkylthiophen-5-yl)-2,1,3-benzothiadiazole]-2',2''-diyl) (PFTBT) donor-block-acceptor all-conjugated block copolymers and carry out a comprehensive study of processing conditions, crystallinity, domain sizes, and side-chain structure on photovoltaic device performance. We find that all block copolymers studied exhibit an out-of-plane crystal orientation after deposition, and on thermal annealing at high temperatures the crystal orientation flips to an in-plane orientation. By varying processing conditions on polymer photovoltaic devices, we show thatmore » the crystal orientation has only a modest effect (15-20%) on photovoltaic performance. The addition of side-chains to the PFTBT block is found to decrease photovoltaic power conversion efficiencies by at least an order of magnitude. Through grazing-incidence X-ray measurements we find that the addition of side-chains to the PFTBT acceptor block results in weak segregation and small (< 10 nm) block copolymer self-assembled donor and acceptor domains. This work is the most comprehensive to date on all-conjugated block copolymer systems and suggests that photovoltaic performance of block copolymers depends strongly on the miscibility of donor and acceptor blocks, which impacts donor and acceptor domain sizes and purity. Lastly, strategies for improving the device performance of block copolymer photovoltaics should seek to increase segregation between donor and acceptor polymer domains.« less

  20. Hybrid coconut seedlings, scholarships, and discount cards for family planning acceptors.

    PubMed

    Sumarsono

    1989-10-01

    Having learned from failed family planning (FP) incentive schemes in other countries, Indonesia implemented a reward system designed to popularize FP in the community. In order to overcome cultural opposition to FP, many countries in the 1970s opted to give incentives--money, materials, etc.--to new contraceptive acceptors and the FP workers who successfully recruited them. These countries, which oftentimes spent up to 1/4 of their program budget on incentives, saw rapid increases in the number of new acceptors. The results, however, only reflected a superficial acceptance of FP. When the incentives stopped, the number of acceptors dropped considerably. Recognizing this, the Indonesian government set out to increase FP acceptance by making the small family the norm in the community. And one of the approaches for doing so was a reward system. The goals of the reward program were: 1) to raise awareness of the recognition given to individuals or groups that have accepted FP; 2) to create pride among FP workers and new acceptors; and 3) to generate leadership in the community. Villages with high FP acceptance receive rewards such as deep-wells that provide clean water or income generating projects. Individuals also receive rewards that sometimes include hybrid coconut seedlings which, after 3 years, can yield up to 700 coconuts, which can provide a family with a significant supplemental income. The government also gives scholarships to children of FP acceptors. Also, the president of Indonesia publicly recognized family planning acceptors. In 1989, over 800,000 couples received awards for practicing contraception over the past 5-16 years.