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Sample records for hairpin ribozyme catalysis

  1. Structural studies on an internal loop from a hairpin ribozyme

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Z.; SantaLucia, J. Jr.; Tinoco, I. Jr. [Univ. of California, Berkeley, CA (United States)

    1994-12-01

    Ribozymes, RNA enzymes, catalyze site-specific RNA cleavage and ligation reactions. We are studying the three-dimensional structure of a hairpin ribozyme derived from the minus strand of tobacco ring spot virus satellite RNA ((-)sTRSV), which has been engineering to specifically cleave the HIV-1 RNA. The minimum structure for the catalytic reaction involves a 50-nucleotide ribozyme and a 14-nucleotide substrate. The proposed secondary structure of the ribozyme-substrate complex consists of four short helices separated by two internal loops. The relatively large size (64-nucleotide) of the ribozyme-substrate complex presents formidable problems in solving the structure using NMR. Therefore we are studying smaller structural subunits of the complex. We are determining the high resolution structure of the symmetric internal loop involving the cleavage site and the flanking helices. One strand of the internal loop was selectively {sup 13}C-labeled at C8 of each purine and C6 of each pyrimidine. By using {sup 13}C-edited two-dimensional NMR, the proton NOESY spectrum was greatly simplified. This allowed unambiguous sequential proton resonance assignments along each strand. Three-dimensional {sup 1}-{sup 13}C HMQC-NOESY was used to further facilitate resonance assignments. We are also enzymatically synthesizing the entire 50-nucleotide ribozyme and will combine it with the {sup 13}C-labeled substrate. Through comparison of the NOE connectivities of the labeled nucleotides from the internal loop alone with those from the entire complex, the differences between the two structures can be elucidated.

  2. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

    Science.gov (United States)

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske

    2016-07-12

    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism.

  3. A conformational switch controls hepatitis delta virus ribozyme catalysis.

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    Ke, Ailong; Zhou, Kaihong; Ding, Fang; Cate, Jamie H D; Doudna, Jennifer A

    2004-05-13

    Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.

  4. Chemical syntheses of inhibitory substrates of the RNA-RNA ligation reaction catalyzed by the hairpin ribozyme.

    Science.gov (United States)

    Massey, Archna P; Sigurdsson, Snorri Th

    2004-01-01

    The chemical syntheses of RNA oligomers containing modifications on the 5'-carbon of the 5'-terminal nucleoside for crystallographic and mechanistic studies of the hairpin ribozyme are reported. Phosphoramidites 4 and 8 were prepared and used in solid phase syntheses of RNA oligomers containing the sequence 5'-N'UCCUCUCC, where N' indicates either 5'-chloro-5'-deoxyguanosine or 5'-amino-5'-deoxyguanosine, respectively. A ribozyme ligation assay with the 5'-chloro- and 5'-amino-modified RNA oligomers demonstrated their inhibition of the hairpin-catalyzed RNA-RNA ligation reaction.

  5. Single-atom imino substitutions at A9 and A10 reveal distinct effects on the fold and function of the hairpin ribozyme catalytic core.

    Science.gov (United States)

    Spitale, Robert C; Volpini, Rosaria; Mungillo, Michael V; Krucinska, Jolanta; Cristalli, Gloria; Wedekind, Joseph E

    2009-08-25

    The hairpin ribozyme cleaves a phosphodiester bond within a cognate substrate. Structural and biochemical data indicate the conserved A9 and A10 bases reside close to the scissile bond but make distinct contributions to catalysis. To investigate these residues, we replaced the imino moiety of each base with N1-deazaadenosine. This single-atom change resulted in an 8-fold loss in k(obs) for A9 and displacement of the base from the active site; no effects were observed for A10. We propose that the imino moiety of A9 promotes a key water-mediated contact that favors transition-state formation, which suggests an enhanced chemical repertoire for RNA.

  6. A transition-state interaction shifts nucleobase ionization toward neutrality to facilitate small ribozyme catalysis.

    Science.gov (United States)

    Liberman, Joseph A; Guo, Man; Jenkins, Jermaine L; Krucinska, Jolanta; Chen, Yuanyuan; Carey, Paul R; Wedekind, Joseph E

    2012-10-17

    One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.

  7. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

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    Robert J Scarborough

    2014-01-01

    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  8. Metal ions: supporting actors in the playbook of small ribozymes.

    Science.gov (United States)

    Johnson-Buck, Alexander E; McDowell, Sarah E; Walter, Nils G

    2011-01-01

    Since the 1980s, several small RNA motifs capable of chemical catalysis have been discovered. These small ribozymes, composed of between approximately 40 and 200 nucleotides, have been found to play vital roles in the replication of subviral and viral pathogens, as well as in gene regulation in prokaryotes, and have recently been discovered in noncoding eukaryotic RNAs. All of the known natural small ribozymes - the hairpin, hammerhead, hepatitis delta virus, Varkud satellite, and glmS ribozymes--catalyze the same self-cleavage reaction as RNase A, resulting in two products, one bearing a 2'-3' cyclic phosphate and the other a 5'-hydroxyl group. Although originally thought to be obligate metalloenzymes like the group I and II self-splicing introns, the small ribozymes are now known to support catalysis in a wide variety of cations that appear to be only indirectly involved in catalysis. Nevertheless, under physiologic conditions, metal ions are essential for the proper folding and function of the small ribozymes, the most effective of these being magnesium. Metal ions contribute to catalysis in the small ribozymes primarily by stabilizing the catalytically active conformation, but in some cases also by activating RNA functional groups for catalysis, directly participating in catalytic acid-base chemistry, and perhaps by neutralizing the developing negative charge of the transition state. Although interactions between the small ribozymes and cations are relatively nonspecific, ribozyme activity is quite sensitive to the types and concentrations of metal ions present in solution, suggesting a close evolutionary relationship between cellular metal ion homeostasis and cation requirements of catalytic RNAs, and perhaps RNA in general.

  9. Mechanisms of RNA catalysis.

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    Lilley, David M J

    2011-10-27

    Ribozymes are RNA molecules that act as chemical catalysts. In contemporary cells, most known ribozymes carry out phosphoryl transfer reactions. The nucleolytic ribozymes comprise a class of five structurally-distinct species that bring about site-specific cleavage by nucleophilic attack of the 2'-O on the adjacent 3'-P to form a cyclic 2',3'-phosphate. In general, they will also catalyse the reverse reaction. As a class, all these ribozymes appear to use general acid-base catalysis to accelerate these reactions by about a million-fold. In the Varkud satellite ribozyme, we have shown that the cleavage reaction is catalysed by guanine and adenine nucleobases acting as general base and acid, respectively. The hairpin ribozyme most probably uses a closely similar mechanism. Guanine nucleobases appear to be a common choice of general base, but the general acid is more variable. By contrast, the larger ribozymes such as the self-splicing introns and RNase P act as metalloenzymes.

  10. Capturing hammerhead ribozyme structures in action by modulating general base catalysis.

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    Young-In Chi

    2008-09-01

    Full Text Available We have obtained precatalytic (enzyme-substrate complex and postcatalytic (enzyme-product complex crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.

  11. EXPLORATION OF SCHISTOSOMA MANSONI HAMMERHEAD RIBOZYME CATALYSIS AND STRUCTURE: TOWARDS DIRECT OBSERVATION OF CLEAVAGE AND LIGATION, AND A 1.55 Å FULL-LENGTH MG2+-BOUND CRYSTAL STRUCTURE

    OpenAIRE

    Anderson, Michael

    2012-01-01

    As a relatively simple and well-studied molecule, the hammerhead ribozyme is an ideal system to study RNA catalysis and structure. A deeper understanding of the hammerhead catalytic mechanism and the role of divalent ions in catalysis lends support to the exploration of more complex RNA machinery such as the ribosome, and ultimately may assist in the design of new medical therapies. Kinetic study of the hammerhead ribozyme to date has largely derived from bulk assays monitoring millions of ...

  12. Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

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    Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

    2014-09-01

    The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

  13. Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis.

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    Gaffarogullari, Ece Cazibe; Krause, André; Balbo, Jessica; Herten, Dirk-Peter; Jäschke, Andres

    2013-12-01

    The analysis of binding interactions between small molecules and biopolymers is important for understanding biological processes. While fluorescence correlation spectroscopy (FCS) requires fluorescence labeling on the small molecule, which often interferes with binding, in microscale thermophoresis (MST) the label can be placed on the biopolymer. Ribozymes have not been analyzed by MST so far. The Diels-Alderase ribozyme (DAse) is a true catalyst, facilitating the Diels-Alder reaction between two free small substrates, anthracene dienes, and maleimide dienophiles. Despite high efforts, the determination of the dissociation constant (KD) of maleimide dienophiles to the DAse by FCS has been unsuccessful. Here, we determined the binding interactions of the DAse to its substrates and the Diels-Alder product using MST. The results supported a positive cooperativity for substrate binding to the DAse. By varying the temperature, we furthermore studied the thermodynamics of dienophile dissociation. The entropic contribution was found to be the energetic driving force for the binding of the dienophile to the DAse.

  14. Enantioselective Catalysis by Using Short, Structurally Defined DNA Hairpins as Scaffold for Hybrid Catalysts.

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    Marek, Jasmin J; Singh, Raghvendra P; Heuer, Andreas; Hennecke, Ulrich

    2017-05-02

    A new type of DNA metal complex hybrid catalyst, which is based on single-stranded DNA oligonucleotides, is described. It was shown that oligonucleotides as short as 14 nucleotides that fold into hairpin structures are suitable as nucleic acid components for DNA hybrid catalysts. With these catalysts, excellent enantioinduction in asymmetric Diels-Alder reactions with selectivity values as high as 96 % enantiomeric excess (ee) can be achieved. Molecular dynamics simulations indicate that a rather flexible loop combined with a rigid stem region provides DNA scaffolds with these high selectivity values. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Investigating the position of the hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis and inhibitor binding.

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    Aitha, Mahesh; Moller, Abraham J; Sahu, Indra D; Horitani, Masaki; Tierney, David L; Crowder, Michael W

    2016-03-01

    In an effort to examine the relative position of a hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis, rapid freeze quench double electron electron resonance (RFQ-DEER) spectroscopy was used. A doubly-labeled mutant of NDM-1, which had one spin label on the invariant loop at position 69 and another label at position 235, was prepared and characterized. The reaction of the doubly spin labeled mutant with chromacef was freeze quenched at 500μs and 10ms. DEER results showed that the average distance between labels decreased by 4Å in the 500μs quenched sample and by 2Å in the 10ms quenched sample, as compared to the distance in the unreacted enzyme, although the peaks corresponding to distance distributions were very broad. DEER spectra with the doubly spin labeled enzyme with two inhibitors showed that the distance between the loop residue at position 69 and the spin label at position 235 does not change upon inhibitor binding. This study suggests that the hairpin loop in NDM-1 moves over the metal ion during the catalysis and then moves back to its original position after hydrolysis, which is consistent with a previous hypothesis based on NMR solution studies on a related metallo-β-lactamase. This study also demonstrates that this loop motion occurs in the millisecond time domain.

  16. Identification of an imino group indispensable for cleavage by a small ribozyme.

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    Spitale, Robert C; Volpini, Rosaria; Heller, Moriah G; Krucinska, Jolanta; Cristalli, Gloria; Wedekind, Joseph E

    2009-05-06

    The hairpin ribozyme is a small, noncoding RNA (ncRNA) that catalyzes a site-specific phosphodiester bond cleavage reaction. Prior biochemical and structural analyses pinpointed the amidine moiety of base Ade38 as a key functional group in catalysis, but base changes designed to probe function resulted in localized misfolding of the active site. To define the requirements for chemical activity using a conservative modification, we synthesized and incorporated N1-deazaadenosine into the full-length ribozyme construct. This single-atom variant severely impairs activity, although the active-site fold remains intact in the accompanying crystal structures. The results demonstrate the essentiality of the imino moiety as well as the importance of its interaction with the substrate in the precatalytic and transition-state conformations. This work demonstrates the efficacy of single-atom approaches in the analysis of ncRNA structure-function relationships.

  17. Thirty-five years of research into ribozymes and nucleic acid catalysis: where do we stand today? [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sabine Müller

    2016-06-01

    Full Text Available Since the discovery of the first catalytic RNA in 1981, the field of ribozyme research has developed from the discovery of catalytic RNA motifs in nature and the elucidation of their structures and catalytic mechanisms, into a field of engineering and design towards application in diagnostics, molecular biology and medicine. Owing to the development of powerful protocols for selection of nucleic acid catalysts with a desired functionality from random libraries, the spectrum of nucleic acid supported reactions has greatly enlarged, and importantly, ribozymes have been accompanied by DNAzymes. Current areas of research are the engineering of allosteric ribozymes for artificial regulation of gene expression, the design of ribozymes and DNAzymes for medicinal and environmental diagnostics, and the demonstration of RNA world relevant ribozyme activities. In addition, new catalytic motifs or novel genomic locations of known motifs continue to be discovered in all branches of life by the help of high-throughput bioinformatic approaches. Understanding the biological role of the catalytic RNA motifs widely distributed in diverse genetic contexts belongs to the big challenges of future RNA research.

  18. Crystallographic analysis of small ribozymes and riboswitches.

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    Lippa, Geoffrey M; Liberman, Joseph A; Jenkins, Jermaine L; Krucinska, Jolanta; Salim, Mohammad; Wedekind, Joseph E

    2012-01-01

    Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ(1) riboswitch, and variants of a larger class II preQ(1) riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, "first choice" RNA crystal screen compiled from our prior successes with commercially available screens.

  19. Group I intron ribozymes

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2012-01-01

    Group I intron ribozymes constitute one of the main classes of ribozymes and have been a particularly important model in the discovery of key concepts in RNA biology as well as in the development of new methods. Compared to other ribozyme classes, group I intron ribozymes display considerable......, the intronic products of these pathways have the potential to integrate into targets and to form various types of circular RNA molecules. Thus, group I intron ribozymes and associated elements found within group I introns is a rich source of biological phenomena. This chapter provides a strategy and protocols...... for initial characterization of new group I intron ribozymes....

  20. Biochemical analysis of hatchet self-cleaving ribozymes.

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    Li, Sanshu; Lünse, Christina E; Harris, Kimberly A; Breaker, Ronald R

    2015-11-01

    Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg(2+) to promote RNA strand scission with a maximum rate constant of ∼4 min(-1). As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2'-oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.

  1. Chemistry and biology of self-cleaving ribozymes

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    Jimenez, Randi M.; Polanco, Julio A.; Lupták, Andrej

    2015-01-01

    Self-cleaving ribozymes were discovered thirty years ago, but their biological distribution and catalytic mechanisms are only beginning to be defined. Each ribozyme family is defined by a distinct structure with unique active sites accelerating the same transesterification reaction across the families. Biochemical studies show that general acid-base catalysis is the most common mechanism of self-cleavage, but metal ions and metabolites can be employed as cofactors. Ribozymes have been discovered in highly diverse genomic contexts throughout nature, from viroids to vertebrates. Their biological roles include self-scission during rolling-circle replication of RNA genomes, co-transcriptional processing of retrotransposons, and metabolite-dependent gene expression regulation in bacteria. Other examples, including highly conserved mammalian ribozymes, suggest that many new biological roles are yet to be discovered. PMID:26481500

  2. A ribozyme transcribed by a ribozyme

    DEFF Research Database (Denmark)

    Bentin, Thomas

    2011-01-01

    Prominent current ideas on how life emerged on Earth include an RNA world hypothesis in which RNA performed informational as well as catalytic functions in the absence of both DNA and protein. Demonstration of a self-replicative system based on ribonucleic acid polymers as both information carriers...... and catalysts would lend support to such a scenario. A pivotal component of this system would be an RNA dependent RNA polymerase ribozyme capable of replicating its own RNA gene. Recent work from the Holliger group at the Laboratory for Molecular Biology in Cambridge has provided synthetic ribozymes1 that just...

  3. Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes.

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    Tanaka, T; Inui, O; Dohi, N; Okada, N; Okada, H; Kikuchi, Y

    2001-07-01

    Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.

  4. RNA reprogramming of alpha-mannosidase mRNA sequences in vitro by myxomycete group IC1 and IE ribozymes.

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    Fiskaa, Tonje; Lundblad, Eirik W; Henriksen, Jørn R; Johansen, Steinar D; Einvik, Christer

    2006-06-01

    Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated alpha-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant alpha-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated alpha-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4-P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing.

  5. Isolation of novel ribozymes that ligate AMP-activated RNA substrates

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    Hager, A. J.; Szostak, J. W.

    1997-01-01

    BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

  6. Crystal Structure of the Catalytic Core of an RNA-Polymerase Ribozyme

    Energy Technology Data Exchange (ETDEWEB)

    Shechner, David M.; Grant, Robert A.; Bagby, Sarah C.; Koldobskaya, Yelena; Piccirilli, Joseph A.; Bartel, David P.; (MIT); (HHMI); (UC)

    2010-09-02

    Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.

  7. Structure of ribonuclease P--a universal ribozyme.

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    Torres-Larios, Alfredo; Swinger, Kerren K; Pan, Tao; Mondragón, Alfonso

    2006-06-01

    Ribonuclease P (RNase P) is one of only two known universal ribozymes and was one of the first ribozymes to be discovered. It is involved in RNA processing, in particular the 5' maturation of tRNA. Unlike most other natural ribozymes, it recognizes and cleaves its substrate in trans. RNase P is a ribonucleoprotein complex containing one RNA subunit and as few as one protein subunit. It has been shown that, in bacteria and in some archaea, the RNA subunit alone can support catalysis. The structure and function of bacterial RNase P RNA have been studied extensively, but the detailed catalytic mechanism is not yet fully understood. Recently, structures of one of the structural domains and of the entire RNA component of RNase P from two different bacteria have been described. These structures provide the first atomic-level information on the structural assembly of the RNA component, and the regions involved in substrate recognition and catalysis. Comparison of these structures reveals a highly conserved core that comprises two universally conserved structural modules. Interestingly, the same structural core can be found in the context of different scaffolds.

  8. Energy landscapes, folding mechanisms, and kinetics of RNA tetraloop hairpins.

    Science.gov (United States)

    Chakraborty, Debayan; Collepardo-Guevara, Rosana; Wales, David J

    2014-12-31

    RNA hairpins play a pivotal role in a diverse range of cellular functions, and are integral components of ribozymes, mRNA, and riboswitches. However, the mechanistic and kinetic details of RNA hairpin folding, which are key determinants of most of its biological functions, are poorly understood. In this work, we use the discrete path sampling (DPS) approach to explore the energy landscapes of two RNA tetraloop hairpins, and provide insights into their folding mechanisms and kinetics in atomistic detail. Our results show that the potential energy landscapes have a distinct funnel-like bias toward the folded hairpin state, consistent with efficient structure-seeking properties. Mechanistic and kinetic information is analyzed in terms of kinetic transition networks. We find microsecond folding times, consistent with temperature jump experiments, for hairpin folding initiated from relatively compact unfolded states. This process is essentially driven by an initial collapse, followed by rapid zippering of the helix stem in the final phase. Much lower folding rates are predicted when the folding is initiated from extended chains, which undergo longer excursions on the energy landscape before nucleation events can occur. Our work therefore explains recent experiments and coarse-grained simulations, where the folding kinetics exhibit precisely this dependency on the initial conditions.

  9. Mechanical unfolding of RNA: From hairpins to structures with internal multiloops

    CERN Document Server

    Hyeon, Changbong

    2007-01-01

    Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer (LOT) experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force ($f$), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer (SOP) model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying complexity. Our work shows (i) the response of RNA to force is largely determined by the native structure; (ii) only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored.

  10. Flanking sequences with an essential role in hydrolysis of a self-cleaving group I-like ribozyme

    Science.gov (United States)

    Einvik, Christer; Nielsen, Henrik; Nour, Reza; Johansen, Steinar

    2000-01-01

    DiGIR1 is a group I-like ribozyme derived from the mobile twin ribozyme group I intron DiSSU1 in the nuclear ribosomal DNA of the myxomycete Didymium iridis. This ribozyme is responsible for intron RNA processing in vitro and in vivo at two internal sites close to the 5′-end of the intron endonuclease open reading frame and is a unique example of a group I ribozyme with an evolved biological function. DiGIR1 is the smallest functional group I ribozyme known from nature and has an unusual core organization including the 6 bp P15 pseudoknot. Here we report results of functional and structural analyses that identify RNA elements critical for hydrolysis outside the DiGIR1 ribozyme core moiety. Results from deletion analysis, disruption/compensation mutagenesis and RNA structure probing analysis all support the existence of two new segments, named P2 and P2.1, involved in the hydrolysis of DiGIR1. Significant decreases in the hydrolysis rate, kobs, were observed in disruption mutants involving both segments. These effects were restored by compensatory base pairing mutants. The possible role of P2 is to tether the ribozyme core, whereas P2.1 appears to be more directly involved in catalysis. PMID:10773091

  11. Ribozymes and their medical implications

    Energy Technology Data Exchange (ETDEWEB)

    Cech, T.R.

    1988-11-25

    Certain RNA molecules can mediate their own cleavage or splicing or act as enzymes to promote reactions on substrate RNA molecules. Thus, RNA is not restricted to being a passive carrier of genetic information but can have an active role in directing cellular biochemistry. These findings suggest the possibility that other cellular RNA's, including the RNA components of small nuclear ribonucleoproteins, of the ribosome, and of various ribonucleoprotein enzymes, are catalysts. RNA enzymes (ribozymes) can be used as sequence-specific RNA cleavage agents in vitro, providing useful tools for biochemical studies of RNA. On a more speculative note, ribozymes directed against viral RNAs have the potential of serving as therapeutic agents. Finally, some infectious agents including hepatitis delta virus and perhaps poliovirus and rhinoviruses, are themselves ribozymes, providing potential targets for pharmaceuticals.

  12. Crystal structure of Pistol, a class of self-cleaving ribozyme

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Laura A.; Wang, Jimin; Steitz, Thomas A. (Yale)

    2017-01-17

    Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.

  13. Ribozyme uses in retinal gene therapy.

    Science.gov (United States)

    Hauswirth, W W; Lewin, A S

    2000-11-01

    In this chapter we discuss the design, delivery and preclinical testing of mutation-specific ribozymes for the treatment of dominantly inherited retinal disease. We focus particular attention on the initial screening of ribozymes in vitro, because the activity of RNA enzymes in cell-free systems can be used to predict their suitability for animal experiments. Current techniques for delivering genes of interest to cells of the retina using viral vectors are then briefly surveyed emphasizing vector properties that best match to the needs of a ribozyme-based therapy. Using these considerations, analysis of ribozyme gene therapy for an autosomal dominant RP-like disease in a rodent model is outlined emphasizing the desirability of combining biochemical, morphological and electrophysiological measures of therapy. Finally, we describe alternative, perhaps more general, ribozyme approaches that have yet to be tested in the context of retinal disease.

  14. Ribozyme modulation of lipopolysaccharide-induced tumor necrosis factor-alpha production by peritoneal cells in vitro and in vivo.

    Science.gov (United States)

    Sioud, M

    1996-05-01

    We have utilized synthetic ribozymes to modulate the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha) by peritoneal cells. Two hammerhead ribozymes (mRz1 and mRz2) were prepared by transcription in vitro and their activities in vitro and in vivo were investigated. Both ribozymes cleaved their RNA target with an apparent turnover number (kcat) of 2 min(-1), and inhibited TNF-alpha gene expression in vitro by 50% and 70%, respectively. When mRz1 and mRz2, entrapped in liposomes, were delivered into mice by intraperitoneal injection, they inhibited LPS-induced TNF-alpha gene expression in vivo with mRz2 being the most effective. This enhanced activity could result from the facilitation of catalysis by cellular endogenous proteins, since they specifically bind to mRz2 as compared to mRz1. Furthermore, a significant mRz2 activity can be recovered from peritoneal cells 2 days post-administration in vivo. The anti-TNF-alpha ribozyme treatment in vivo resulted in a more significant reduction of LPS-induced IFN-gamma protein secretion compared to IL-10. In contrast to this pleiotropic effect, the anti-TNF-alpha ribozyme treatment did not affect the heterogenous expression of Fas ligand by peritoneal cells, indicating the specificity of the treatment. Taken together, the present data indicate that the biological effects of TNF-alpha can be modulated by ribozymes. In addition, the data suggest that ribozymes can be administered in a drug-like manner, and therefore indicate their potential in clinical applications.

  15. Electronic Structure Rearrangements in Hybrid Ribozyme/Protein Catalysis

    Science.gov (United States)

    Kang, Jiyoung; Kino, Hiori; Field, Martin J.; Tateno, Masaru

    2017-04-01

    We analyzed the electronic structural changes that occur in the reaction cycle of a biological catalyst composed of RNA and protein, and elucidated the dynamical rearrangements of the electronic structure that was obtained from our previous study in which ab initio quantum mechanics/molecular mechanics molecular dynamics simulations were performed. Notable results that we obtained include the generation of a reactive HOMO that is responsible for bond formation in the initial stages of the reaction, and the appearance of a reactive LUMO that is involved in the bond rupture that leads to products. We denote these changes as dynamical induction of the reactive HOMO (DIRH) and LUMO (DIRL), respectively. Interestingly, we also find that the induction of the reactive HOMO is enhanced by the formation of a low-barrier hydrogen bond (LBHB), which, to the best of our knowledge, represents a novel role for LBHBs in enzymatic systems.

  16. Heterogeneous Catalysis.

    Science.gov (United States)

    Vannice, M. A.

    1979-01-01

    Described is a graduate course in catalysis offered at Penn State University. A detailed course outline with 30 lecture topics is presented. A list of 42 references on catalysis used in place of a textbook is provided. (BT)

  17. An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

    Science.gov (United States)

    Szewczak, A A; Ortoleva-Donnelly, L; Zivarts, M V; Oyelere, A K; Kazantsev, A V; Strobel, S A

    1999-09-28

    Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

  18. Direct Raman measurement of an elevated base pKa in the active site of a small ribozyme in a precatalytic conformation.

    Science.gov (United States)

    Guo, Man; Spitale, Robert C; Volpini, Rosaria; Krucinska, Jolanta; Cristalli, Gloria; Carey, Paul R; Wedekind, Joseph E

    2009-09-16

    Catalytic RNA molecules can achieve rate acceleration by shifting base pK(a) values toward neutrality. Prior evidence has suggested that base A38 of the hairpin ribozyme plays an important role in phosphoryl transfer, possibly functioning as a general acid, or by orienting a specific water molecule for proton transfer. To address the role of A38, we used Raman spectroscopy to measure directly the pK(a) of the N1-imino moiety in the context of hairpin ribozyme crystals representative of a "precatalytic" conformation. The results revealed that the pK(a) of A38 is shifted to 5.46 +/- 0.05 relative to 3.68 +/- 0.06 derived from a reference solution of the nucleotide AMP. The elevated pK(a) correlates well with the first titration point of the macroscopic pH-rate profile of the hairpin ribozyme in solution and strongly supports A38 as a general acid catalyst in bond scission. The results confirm that A38 is protonated before the transition state, which would promote phosphorane development. Overall, the results establish a cogent structure-function paradigm that expands our understanding of how RNA structure can enhance nucleobase reactivity to catalyze biological reactions.

  19. A Self-Replicating Ligase Ribozyme

    Science.gov (United States)

    Paul, Natasha; Joyce, Gerald F.

    2002-01-01

    A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of Itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 per min, whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(exp -11) M per min. Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

  20. Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world.

    Science.gov (United States)

    Liberman, Joseph A; Wedekind, Joseph E

    2011-06-01

    Genome sequencing has produced thousands of nonprotein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base pK(a) shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ(1) and S-adenosyl-(l)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.

  1. A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jui-Hui; Yajima, Rieko; Chadalavada, Durga M.; Chase, Elaine; Bevilacqua, Philip C.; Golden, Barbara L. (Purdue); (Penn)

    2010-11-01

    The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2{prime},3{prime}-cyclic phosphate and 5{prime}-hydroxyl termini. The active site nucleotide C75 has a pK{sub a} shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg{sup 2+} ion that helps activate the 2{prime}-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg{sup 2+} ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg{sup 2+} ion is also found within the active site where it interacts with a newly resolved G {center_dot} U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-RP oxygen of the scissile phosphate and the 2{prime}-hydroxyl nucleophile are inner-sphere ligands to the active site Mg{sup 2+} ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

  2. A Comparison of Vanadate to a 2'-5' Linkage at the Active Site of a Small Ribozyme Suggests a Role for Water in Transition-State Stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Torelli, A.T.; Krucinska, J.; Wedekind, J.E.

    2009-06-04

    The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 A resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2',5'-phosphodiester linkage at the site of cleavage was also solved at 2.35 A resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2',5' structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2'-O nucleophile with the scissile G+1 phosphorus. The 2',5'-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2',5' linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds.

  3. A new RNA branching activity: the GIR1 ribozyme

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Johansen, Steinar D

    2006-01-01

    ',5'-phosphodiester bonds in biology. We recently described a new ribozyme, the GIR1 branching ribozyme, which catalyzes the formation of a tiny lariat that caps an mRNA. This new example together with work on artificial branching ribozymes and deoxyribozymes shows that branching is facile and points...

  4. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    Directory of Open Access Journals (Sweden)

    Gayani N. P. Dedduwa-Mudalige

    2015-09-01

    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

  5. Hammerhead ribozyme activity and oligonucleotide duplex stability in mixed solutions of water and organic compounds

    Directory of Open Access Journals (Sweden)

    Shu-ichi Nakano

    2014-01-01

    Full Text Available Nucleic acids are useful for biomedical targeting and sensing applications in which the molecular environment is different from that of a dilute aqueous solution. In this study, the influence of various types of mixed solutions of water and water-soluble organic compounds on RNA was investigated by measuring the catalytic activity of the hammerhead ribozyme and the thermodynamic stability of an oligonucleotide duplex. The compounds with a net neutral charge, such as poly(ethylene glycol, small primary alcohols, amide compounds, and aprotic solvent molecules, added at high concentrations changed the ribozyme-catalyzed RNA cleavage rate, with the magnitude of the effect dependent on the NaCl concentration. These compounds also changed the thermodynamic stability of RNA base pairs of an oligonucleotide duplex and its dependence on the NaCl concentration. Specific interactions with RNA molecules and reduced water activity could account for the inhibiting effects on the ribozyme catalysis and destabilizing effects on the duplex stability. The salt concentration dependence data correlated with the dielectric constant, but not with water activity, viscosity, and the size of organic compounds. This observation suggests the significance of the dielectric constant effects on the RNA reactions under molecular crowding conditions created by organic compounds.

  6. Ribozymes:an anti-viral agent

    Institute of Scientific and Technical Information of China (English)

    Asad U.Khan; Shahper N.Khan

    2008-01-01

    The discovery that RNA can act as an enzyme led Thomas Cech to win the Nobel Prize in Chemistry and led immediately to the next wave of attempts to find an effective RNA-based therapy.The tantalizing idea that RNA enzymes called trans-cleaving ribozymes enables them to act as potential antiviral and powerful tool for functional genomic studies.The efficacy of ribozyme function in a complex intracellular environment is depend-ent on the intracellular fate of the RNA that is being targeted.Recently,ribozymes have been used successfully to inhibit gene expression in a variety of biological systems in vitro and in vivo.Ribozyme has also been used successfully to combat many cases of viral infection,as clinical trial.Despite it needs to be investigated and explored as far as its structural and functional aspects are concern.In view of the significance of ribozyme in modern medicine,we reviewed the recent literature on general approach to control viral infection.

  7. General combinatorics of RNA hairpins and cloverleaves.

    Science.gov (United States)

    Liao, Bo; Wang, Tian-ming

    2003-01-01

    For an abstract single-strand RNA, a combinatorial analysis is given for two important structures, hairpins and cloverleaves. The total number of hairpins and cloverleaves of a given length with minimal hairpin loop length m(m > 0) and with minimal stack length l(l > 0) is computed, under the assumption that all base pairs can occur.

  8. Schistosome satellite DNA encodes active hammerhead ribozymes.

    Science.gov (United States)

    Ferbeyre, G; Smith, J M; Cedergren, R

    1998-07-01

    Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smalpha repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smalpha ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

  9. A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme.

    Science.gov (United States)

    Bouchard, Patricia; Legault, Pascale

    2014-09-01

    Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme. © 2014 Bouchard and Legault; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. Chemical fidelity of an RNA polymerase ribozyme

    DEFF Research Database (Denmark)

    Attwater, J.; Tagami, S.; Kimoto, M.

    2013-01-01

    The emergence of catalytically active RNA enzymes (ribozymes) is widely believed to have been an important transition in the origin of life. In the context of a likely heterogeneous chemical environment, substrate specificity and selectivity of these primordial enzymes would have been critical...... for function. Here we have explored the chemical fidelity, i.e. substrate selectivity and specificity for both single and multiple catalytic steps of the Z RNA polymerase ribozyme-a modern day analogue of the primordial RNA replicase. Using a wide range of nucleotide analogues and ionic conditions, we observe...

  11. Ribozymes: applications to functional analysis and gene discovery.

    Science.gov (United States)

    Shiota, Maki; Sano, Masayuki; Miyagishi, Makoto; Taira, Kazunari

    2004-08-01

    Ribozymes are catalytic RNA molecules that cleave RNAs with high specificity. Since the discovery of these non-protein enzymes, the rapidly developing field of ribozymes has been of particular interest because of the potential utility of ribozymes as tools for reversed genetics. However, despite extensive efforts, the activity of ribozymes in vivo has not usually been high enough to achieve the desirable biological effects. Now, by the use of RNA polymerase III (pol III) promoters, the ribozyme activity in cells has been successfully improved by developing efficient transport systems for the transcripts to the cytoplasm. In addition, it is possible to cleave a specific target RNA in cells by using an allosterically controllable ribozyme or an RNA-protein hybrid ribozyme. These ribozymes are potentially applicable to molecular gene therapy and efficient gene discovery systems. Furthermore, the developed pol III expression system is applicable to the expression of small interfering RNAs (siRNAs). The advantage of such ribozymes over siRNAs is the high specificity of the ribozyme that would not cause interferon responses.

  12. Generation and Growth of Single Hairpin Vortices

    Science.gov (United States)

    Haji-Haidari, Ahmad

    The behavior of selectively generated single hairpin vortices are examined within a laminar boundary layer environment over a range of Reynolds numbers, the hairpin vortices are experimentally generated by means of controlled fluid injection from a streamwise slot. Flow visualization using both dye and hydrogen bubble wire is employed in conjunction with hot film anemometry to investigate the growth characteristics and evolution of these single hairpin vortices. Qualitatively, it is established that hairpin vortices form by local destabilization at the interface between the low-speed fluid introduced through the slot and the higher speed boundary layer flow. Kinematical considerations of the hairpin vortex are established. It is observed that a hairpin vortex generally displays visualization and velocity signatures characteristic of those observed for a turbulent boundary layer. Hydrogen-bubble wire visualization results specifically indicate that hairpin vortices generate two purely turbulent-like flow patterns. The first is a low-speed streak pattern developing immediately adjacent to the surface due to surface interaction by the counter -rotating legs of the hairpin vortex; the second pattern is a turbulent pocket-like pattern farther removed from the surface. It is determined from the visualization data that hairpin vortices manifest the necessary flow characteristics which give rise to the regenerative and sustained process required for maintenance of turbulence. The regeneration and the growth process takes place through the formation of similar hairpin-like vortices by one of two means. The first is an inviscid lateral propagation of the initial disturbance which gives rise to outboard (subsidiary), vortices which cause the lateral spreading of the structure. A more complicated and eruptive process occurs by means of viscous-inviscid interactions which give rise to trailing vortices (secondary), which cause the streamwise elongation of the disturbance. A

  13. Exploring ribozyme conformational changes with X-ray crystallography.

    Science.gov (United States)

    Spitale, Robert C; Wedekind, Joseph E

    2009-10-01

    Relating three-dimensional fold to function is a central challenge in RNA structural biology. Toward this goal, X-ray crystallography has long been considered the "gold standard" for structure determinations at atomic resolution, although NMR spectroscopy has become a powerhouse in this arena as well. In the area of dynamics, NMR remains the dominant technique to probe the magnitude and timescales of molecular motion. Although the latter area remains largely unassailable by conventional crystallographic methods, inroads have been made on proteins using Laue radiation on timescales of ms to ns. Proposed 'fourth generation' radiation sources, such as free-electron X-ray lasers, promise ps- to fs-timescale resolution, and credible evidence is emerging that supports the feasibility of single molecule imaging. At present however, the preponderance of RNA structural information has been derived from timescale and motion insensitive crystallographic techniques. Importantly, developments in computing, automation and high-flux synchrotron sources have propelled the rapidity of 'conventional' RNA crystal structure determinations to timeframes of hours once a suitable set of phases is obtained. With a sufficient number of crystal structures, it is possible to create a structural ensemble that can provide insight into global and local molecular motion characteristics that are relevant to biological function. Here we describe techniques to explore conformational changes in the hairpin ribozyme, a representative non-protein-coding RNA catalyst. The approaches discussed include: (i) construct choice and design using prior knowledge to improve X-ray diffraction; (ii) recognition of long-range conformational changes and (iii) use of single-base or single-atom changes to create ensembles. The methods are broadly applicable to other RNA systems.

  14. Ribozymes that can be regulated by external stimuli.

    Science.gov (United States)

    Frommer, Jennifer; Appel, Bettina; Müller, Sabine

    2015-02-01

    Ribozymes have been known for about 30 years, and nowadays are understood well enough to be turned into useful tools for a number of applications in vitro and in vivo. Allosteric ribozymes switch on and off their activity in response to a specific chemical (ligand) or physical (temperature, light) signal. The possibility of controlling ribozyme activity by external stimuli is of particular relevance for applications in different fields, such as environmental and medicinal diagnostics, molecular computing, control of gene expression and others. Herein, we review recent advances and describe selected examples of addressable ribozymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Enantioconvergent catalysis

    Directory of Open Access Journals (Sweden)

    Justin T. Mohr

    2016-09-01

    Full Text Available An enantioconvergent catalytic process has the potential to convert a racemic starting material to a single highly enantioenriched product with a maximum yield of 100%. Three mechanistically distinct approaches to effecting enantioconvergent catalysis are identified, and recent examples of each are highlighted. These processes are compared to related, non-enantioconvergent methods.

  16. The Hammerhead Ribozyme: A Long History for a Short RNA.

    Science.gov (United States)

    de la Peña, Marcos; García-Robles, Inmaculada; Cervera, Amelia

    2017-01-04

    Small nucleolytic ribozymes are a family of naturally occurring RNA motifs that catalyse a self-transesterification reaction in a highly sequence-specific manner. The hammerhead ribozyme was the first reported and the most extensively studied member of this family. However, and despite intense biochemical and structural research for three decades since its discovery, the history of this model ribozyme seems to be far from finished. The hammerhead ribozyme has been regarded as a biological oddity typical of small circular RNA pathogens of plants. More recently, numerous and new variations of this ribozyme have been found to inhabit the genomes of organisms from all life kingdoms, although their precise biological functions are not yet well understood.

  17. The Hammerhead Ribozyme: A Long History for a Short RNA

    Directory of Open Access Journals (Sweden)

    Marcos de la Peña

    2017-01-01

    Full Text Available Small nucleolytic ribozymes are a family of naturally occurring RNA motifs that catalyse a self-transesterification reaction in a highly sequence-specific manner. The hammerhead ribozyme was the first reported and the most extensively studied member of this family. However, and despite intense biochemical and structural research for three decades since its discovery, the history of this model ribozyme seems to be far from finished. The hammerhead ribozyme has been regarded as a biological oddity typical of small circular RNA pathogens of plants. More recently, numerous and new variations of this ribozyme have been found to inhabit the genomes of organisms from all life kingdoms, although their precise biological functions are not yet well understood.

  18. Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes

    DEFF Research Database (Denmark)

    Beckert, Bertrand Dominique; Nielsen, Henrik; Einvik, Christer

    2008-01-01

    related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto...

  19. Zeolites and Catalysis

    Science.gov (United States)

    1999-12-15

    Handbook of Heterogeneous Catalysis ,Vol. als: State of the Art 1994, Studies in Surface Science and 5, Wiley-VCH, Weinheim, 1997, p. 2329. Catalysis, Vol...Weitkamp (Eds.), in Zeolite and Microporous Materials, Studies in Surface Handbook of Heterogeneous Catalysis , Vol. 4, Wiley-VCH, Science and Catalysis

  20. Twister ribozymes as highly versatile expression platforms for artificial riboswitches

    Science.gov (United States)

    Felletti, Michele; Stifel, Julia; Wurmthaler, Lena A.; Geiger, Sophie; Hartig, Jörg S.

    2016-01-01

    The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class. PMID:27670347

  1. Electroanalytical determination of d(GCGAAGC) hairpin.

    Science.gov (United States)

    Trnková, Libuse; Postbieglová, Irena; Holik, Miroslav

    2004-06-01

    Hairpins (or hairpin-like structures) may play a major role in expansion events of triplet repeat expansion diseases (X syndrome, Huntington's disease, Friedreich's ataxia). The d(GCGAAGC) fragment has been found in the replication origins of phage phiX 174 and herpes simplex virus, in a promoter region of an Escherichia coli heat-shock gene, and in rRNA genes. The paper deals with the application of electrochemical methods to the determination of the DNA heptamer-d(GCGAAGC) which forms very stable hairpin structure in aqueous solutions. On mercury electrodes, this hairpin provides voltammetric reduction signals of adenine and cytosine, and oxidation signals of guanine. Both signals have been studied by cyclic voltammetry (CV), linear sweep voltammetry (LSV), and elimination voltammetry with linear scan (EVLS) in dependence on pH, accumulation time, scan rate, and loop sequences. The EVLS in combination with the adsorptive stripping was employed to the determination of the detection limit (LD) of this mini-hairpin (2 nM). Multidimensional voltammetric data were worked up by Fourier Transform (FT) and for the first coefficient a confidence ellipse was calculated in order to drop out some outlier data. The same method was used also for detection limit determinations. The values of LD obtained by two approaches were compared.

  2. Excited singlet states of "hairpin" polyenes

    OpenAIRE

    Froelich, Wolfgang; Dewey, Harry J.; Deger, Hans; Dick, Bernhard; Klingensmith, Kenneth A.; Puettmann, Wilhelm; Vogel, Emanuel; Hohlneicher, Georg; Michl, Josef

    1983-01-01

    The synthesis and UV-visible, polarized-fluorescence and MCD spectra of 6 U-shaped hairpin polyenes (e.g., I) are reported. Qual. arguments and results of p-electron calcns. permit the identification of 4 excited singlet states and their assignment to mixts. of singly and doubly excited configurations. The hairpin polyenes represent a link between the all-trans-polyenes on the 1 hand and the annulenes and acenes on the other; they have the topol. of the former and a geometry near that of the ...

  3. Molecular characterization of a new member of the lariat capping twin-ribozyme introns

    DEFF Research Database (Denmark)

    Tang, Yunjia; Nielsen, Henrik; Masquida, Benoît

    2014-01-01

    in Naegleria amoeboflagellates and the myxomycete Didymium iridis. RESULTS: We characterize structural organization, catalytic properties and molecular evolution of a new twin-ribozyme intron in Allovahlkampfia (Heterolobosea). The intron contains two ribozyme domains with different functions in ribosomal RNA...

  4. Ultrafast Unzipping of a Beta-Hairpin Peptide

    NARCIS (Netherlands)

    Zinth, W.; Schrader, T.E.; Schreier, W.J.; Koller, F.O.; Cordes, T.; Babitzki, G.; Denschlag, R.; Tavan, P.; Löweneck, M.; Dong, Shou-Liang; Moroder, L.; Renner, C.; Corkum, Paul; Jonas, David M.; Miller, R.J. Dwayne; Weiner, Andrew M.

    2007-01-01

    Light induced switching of a beta-hairpin structure is investigated by femtosecond IR spectroscopy. While the unzipping process comprises ultrafast kinetics and is finished within 1 ns, the folding into the hairpin structure is a much slower process.

  5. Catalysis of Photochemical Reactions.

    Science.gov (United States)

    Albini, A.

    1986-01-01

    Offers a classification system of catalytic effects in photochemical reactions, contrasting characteristic properties of photochemical and thermal reactions. Discusses catalysis and sensitization, examples of catalyzed reactions of excepted states, complexing ground state substrates, and catalysis of primary photoproducts. (JM)

  6. Molecular characterization of a new member of the lariat capping twin-ribozyme introns

    Science.gov (United States)

    2014-01-01

    Background Twin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA in Naegleria amoeboflagellates and the myxomycete Didymium iridis. Results We characterize structural organization, catalytic properties and molecular evolution of a new twin-ribozyme intron in Allovahlkampfia (Heterolobosea). The intron contains two ribozyme domains with different functions in ribosomal RNA splicing and homing endonuclease mRNA maturation. We found Allovahlkampfia GIR2 to be a typical group IC1 splicing ribozyme responsible for addition of the exogenous guanosine cofactor (exoG), exon ligation and circularization of intron RNA. The Allovahlkampfia LC ribozyme, by contrast, represents an efficient self-cleaving ribozyme that generates a small 2′,5′ lariat cap at the 5′ end of the homing endonuclease mRNA, and thus contributes to intron mobility. Conclusions The discovery of a twin-ribozyme intron in a member of Heterolobosea expands the distribution pattern of LC ribozymes. We identify a putative regulatory RNA element (AP2.1) in the Allovahlkampfia LC ribozyme that involves homing endonuclease mRNA coding sequences as an important structural component. PMID:25342998

  7. Biochemical analysis of pistol self-cleaving ribozymes.

    Science.gov (United States)

    Harris, Kimberly A; Lünse, Christina E; Li, Sanshu; Brewer, Kenneth I; Breaker, Ronald R

    2015-11-01

    Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min(-1) under physiological Mg(2+) and pH conditions. The reaction proceeds via the nucleophilic attack of a 2'-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.

  8. Destabilization of the TAR hairpin leads to extension of the polyA hairpin and inhibition of HIV-1 polyadenylation

    NARCIS (Netherlands)

    Vrolijk, M.M.; Harwig, A.; Berkhout, B.; Das, A.T.

    2009-01-01

    ABSTRACT: BACKGROUND: Two hairpin structures that are present at both the 5' and 3' end of the HIV-1 RNA genome have important functions in the viral life cycle. The TAR hairpin binds the viral Tat protein and is essential for Tat-mediated activation of transcription. The adjacent polyA hairpin

  9. Advances in catalysis

    CERN Document Server

    Gates, Bruce C

    2012-01-01

    Advances in Catalysis fills the gap between the journal papers and the textbooks across the diverse areas of catalysis research. For more than 60 years Advances in Catalysis has been dedicated to recording progress in the field of catalysis and providing the scientific community with comprehensive and authoritative reviews. This series in invaluable to chemical engineers, physical chemists, biochemists, researchers and industrial chemists working in the fields of catalysis and materials chemistry. * In-depth, critical, state-of-the-art reviews * Comprehensive, covers of all as

  10. Advances in catalysis

    CERN Document Server

    Jentoft, Friederike C

    2014-01-01

    Advances in Catalysis fills the gap between the journal papers and the textbooks across the diverse areas of catalysis research. For more than 60 years Advances in Catalysis has been dedicated to recording progress in the field of catalysis and providing the scientific community with comprehensive and authoritative reviews. This series is invaluable to chemical engineers and chemists working in the field of catalysis in academia or industry. Authoritative reviews written by experts in the field. Topics selected to reflect progress of the field. Insightful and critical articles, fully edite

  11. Kinetic partitioning mechanism of HDV ribozyme folding

    Science.gov (United States)

    Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2014-01-01

    RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.

  12. Kinetic partitioning mechanism of HDV ribozyme folding

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing, E-mail: wbzhang@whu.edu.cn [Department of Physics, Wuhan University, Wuhan, Hubei 430072 (China)

    2014-01-14

    RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.

  13. The Structural Basis of Ribozyme-Catalyzed RNA Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, M.P.; Scott, W.G.; /UC, Santa Cruz

    2007-07-12

    Life originated, according to the RNA World hypothesis, from self-replicating ribozymes that catalyzed ligation of RNA fragments. We have solved the 2.6 angstrom crystal structure of a ligase ribozyme that catalyzes regiospecific formation of a 5' to 3' phosphodiester bond between the 5'-triphosphate and the 3'-hydroxyl termini of two RNA fragments. Invariant residues form tertiary contacts that stabilize a flexible stem of the ribozyme at the ligation site, where an essential magnesium ion coordinates three phosphates. The structure of the active site permits us to suggest how transition-state stabilization and a general base may catalyze the ligation reaction required for prebiotic RNA assembly.

  14. Synthesis of a bifunctional cytidine derivative and its conjugation to RNA for in vitro selection of a cytidine deaminase ribozyme.

    Science.gov (United States)

    Rublack, Nico; Müller, Sabine

    2014-01-01

    Over the past 20 years, the generation of functional RNAs by in vitro selection has become a standard technique. Apart from aptamers for simple binding of defined ligands, also RNAs for catalysis of chemical reactions have been selected. In the latter case, a key step often is the conjugation of one of the two reactants to the library, requiring suitable strategies for terminal or internal RNA functionalization. With the aim of selecting a ribozyme for deamination of cytidine, we have set up a selection scheme involving the attachment of the cytidine acting as deamination substrate to the 3'-terminus of the RNAs in the library, and library immobilization. Here, we report the synthesis of a bifunctional cytidine derivative suitable for conjugation to RNA and linkage of the conjugated library to a streptavidine-coated surface. Successful conjugation of the cytidine derivative to the 3'-terminus of a model RNA is demonstrated.

  15. Synthesis of a bifunctional cytidine derivative and its conjugation to RNA for in vitro selection of a cytidine deaminase ribozyme

    Directory of Open Access Journals (Sweden)

    Nico Rublack

    2014-08-01

    Full Text Available Over the past 20 years, the generation of functional RNAs by in vitro selection has become a standard technique. Apart from aptamers for simple binding of defined ligands, also RNAs for catalysis of chemical reactions have been selected. In the latter case, a key step often is the conjugation of one of the two reactants to the library, requiring suitable strategies for terminal or internal RNA functionalization. With the aim of selecting a ribozyme for deamination of cytidine, we have set up a selection scheme involving the attachment of the cytidine acting as deamination substrate to the 3'-terminus of the RNAs in the library, and library immobilization. Here, we report the synthesis of a bifunctional cytidine derivative suitable for conjugation to RNA and linkage of the conjugated library to a streptavidine-coated surface. Successful conjugation of the cytidine derivative to the 3'-terminus of a model RNA is demonstrated.

  16. Activity of HDV ribozymes to trans-cleave HCV RNA

    Institute of Scientific and Technical Information of China (English)

    Yue-Cheng Yu; Qing Mao; Chang-Hai Gu; Qi-Fen Li; Yu-Ming Wang

    2002-01-01

    AIM: To explore whether HDV ribozymes have the abilityto trans-cleave HCVRNA.METHODS: Three HDV genomic ribozymes weredesigned and named RzC1, RzC2 and RzC3. Thesubstrate RNA contained HCVRNA 5'-noncoding regionand 5'-fragment of C region (5'-NCR-C). All theribozymes and HCV RNA 5'-NCR-C were obtained bytranscription in vibo from their DNA templates, and HCVRNA 5'-NCR-C was radiolabelled at its 5'-end Undercertain pH, temperature, appropriate concentration ofMg2+ and deionized formamide, these ribozymes wererespectively or simultaneously mixed with HCVRNA 5'-NCR-C and reacted for a certain time. The trans-cleavage reaction was stopped at different time points,and the products were separated with polyacrylamidegel electrophoresis (PAGE), displayed byautoradiography. Percentage of trans-deaved productswas measured to indicate the activity of HDV ribozymes.RESULTS: RzC1 and RzC2 could trans-cleave 26 % and21.8 % of HCV RNA 5'-NCR-C under our reactionconditions with 2.5 mol. L-1 deionized formamiderespectively. The percentage of HCV RNA 5'-NCR-Ctrans-cleaved by RzC1, RzC2 or combined usage of thethree ribozymes increased with time, up to 24.9 %, 20.3 %and 37.3 % respectively at 90 min point. Almost noproduct from RzC3 was observed.CONCLUSION: HDV ribozymes are able to trans-cleavespecifically HCV RNA at certain sites under appropriateconditions, and combination of several ribozymesaiming at different target sites can trans-cleave thesubstrate more efficiently than using only one of them.

  17. Speciation of a group I intron into a lariat capping ribozyme

    DEFF Research Database (Denmark)

    Meyer, Mélanie; Nielsen, Henrik; Oliéric, Vincent

    2014-01-01

    The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes...... for a conventional mRNA cap in the downstream pre-mRNA encoding a homing endonuclease. We describe the crystal structures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inherited from group I ribozymes have undergone speciation due to profound changes in molecular selection...... pressure, ultimately giving rise to an original branching ribozyme family. The structures elucidate the role of key elements that regulate the activity of the LC ribozyme by conformational switching and suggest a mechanism by which the signal for branching is transmitted to the catalytic core...

  18. Ultrasensitive monitoring of ribozyme cleavage product using molecular-beacon-ligation system

    Institute of Scientific and Technical Information of China (English)

    MENG XiangXian; TANG ZhiWen; WANG KeMin; TAN WeiHong; YANG XiaoHai; LI Jun; GUO QiuPing

    2007-01-01

    This paper reports a new approach to detect ribozyme cleavage product based on the molecular- beacon-ligation system. The molecular beacon, designed in such a way that one-half of its loop is complementary to ribozyme cleavage product, is used to monitor ligation process of RNA/DNA complex in a homogeneous solution and to convert directly cleavage product information into fluorescence signal. The method need not label ribozyme and ribozyme substrate, which is fast, simple and ultrasensitive for detection of cleavage product. Detection limit of the assay is 0.05 nmol/L. The cleavage product of hammerhead ribozyme against hepatitis C virus RNA (HCV-RNA) was detected perfectly based on this assay. Owing to its ultrasensitivity, excellent specificity, convenience and fidelity, this method might hold out great promise in ribozyme reaction and ribozyme gene therapy.

  19. RNA folding and catalysis mediated by iron (II.

    Directory of Open Access Journals (Sweden)

    Shreyas S Athavale

    Full Text Available Mg²⁺ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²⁺ in the absence of free oxygen as a replacement for Mg²⁺ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²⁺ in RNA folding and function can indeed be served by Fe²⁺. The results of quantum mechanical calculations show that the geometry of coordination of Fe²⁺ by RNA phosphates is similar to that of Mg²⁺. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²⁺ or Mg²⁺. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²⁺, and the hammerhead ribozyme are enhanced in the presence of Fe²⁺ compared to Mg²⁺. All chemical footprinting and ribozyme assays in the presence of Fe²⁺ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²⁺. The combined biochemical and paleogeological data are consistent with a role for Fe²⁺ in an RNA World. RNA and Fe²⁺ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²⁺ alone.

  20. Errors and alternatives in prebiotic replication and catalysis.

    Science.gov (United States)

    Ninio, Jacques

    2007-04-01

    The work on nonenzymatic nucleic acid replication performed by Leslie Orgel and co-workers over the last four decades, now extended by work on artificial selection of RNA aptamers and ribozymes, is generating some pessimism concerning the 'naked gene' theories of the origin of life. It is suggested here that the low probability of finding RNA aptamers and ribozymes within pools of random sequences is not as disquieting as the poor gain in efficiency obtained with increases in information content. As acknowledged by Orgel and many other authors, primitive RNA replication and catalysis must have occurred within already complex and dynamic environments. I, thus, propose to pay attention to a number of possibilities that bridge the gap between 'naked gene' theories, on one side, and metabolic theories in which complex systems self-propagate by growth and fragmentation, on the other side. For instance, one can de-emphasize nucleotide-by-nucleotide replication leading to long informational polymers, and view instead long random polymers as storage devices, from which shorter oligomers are excised. Catalytic tasks would be mainly performed by complexes associating two or more oligomers belonging to the same or to different chemical families. It is proposed that the problems of stability, binding affinity, reactivity, and specificity could be easier to handle by heterogeneous complexes of short oligomers than by long, single-stranded polymers. Finally, I point out that replication errors in a primitive replication context should include incorporations of alternative nucleotides with interesting, chemically reactive groups. In this way, an RNA sequence could be at the same time an inert sequence when copied without error, and a ribozyme, when a chemically reactive nucleotide is inadvertently introduced during replication.

  1. The Quick and the Dead: A Guide to Fast Phasing of Small Ribozyme and Riboswitch Crystal Structures.

    Science.gov (United States)

    Jenkins, Jermaine L; Wedekind, Joseph E

    2016-01-01

    Ribozymes and riboswitches are examples of non-protein-coding (nc)RNA molecules that achieve biological activity by adopting complex three-dimensional folds. Visualization of such molecules at near-atomic resolution can enhance our understanding of how chemical groups are organized spatially, thereby providing novel insight into function. This approach has its challenges, which mainly entail sample crystallization followed by the application of empirical, structure-determination methods that often include experimental "phasing" of X-ray diffraction data. A paucity of high-quality crystals or a low symmetry space group are factors that demand rapid assessment of phasing potential during an ongoing experiment in order to assure a successful outcome. Here we describe the process of evaluating the anomalous signal-to-noise as a prelude to single wavelength or multiwavelength anomalous diffraction (SAD or MAD) phasing. Test cases include an autolytic 62-mer RNA enzyme known as the hairpin ribozyme, and a 33-mer riboswitch that binds the modified guanine metabolite preQ1. The crystals were derivatized with iridium (III) hexammine and osmium (III) pentaammine triflate, respectively. Each data set was then subjected to the XPREP and SHELX programs to assess the anomalous signal-to-noise and to locate the heavy-atom substructure. Subsequent noise filtering was conducted in SHELXE or RESOLVE. The methods described are applicable to the rapid phasing of RNA X-ray diffraction data, and contrast the efficacy of in-house X-rays with those attainable from synchrotron-radiation sources in terms of the potential to plan for and execute an experimental structure determination.

  2. Multiple Translational Products from a Five-Nucleotide Ribozyme

    Science.gov (United States)

    Turk, R. M.; Yarus, M.

    2010-04-01

    This work shows that a tiny RNA enzyme can catalyze one of the key reactions in modern protein synthesis. The small size and minimal chemical requirements for this ribozyme support the idea of facile assembly and function of RNA enzymes during the origin of life on Earth.

  3. Ionic interactions and the global conformations of the hammerhead ribozyme

    DEFF Research Database (Denmark)

    Bassi, G S; Møllegaard, N E; Murchie, A I;

    1995-01-01

    Here we investigate the global conformation of the hammerhead ribozyme. Electrophoretic studies demonstrate that the structure is folded in response to the concentration and type of ions present. Folding based on colinear alignment of arms II and III is suggested, with a variable angle subtended...

  4. Evolution in vitro: analysis of a lineage of ribozymes

    Science.gov (United States)

    Lehman, N.; Joyce, G. F.

    1993-01-01

    Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

  5. Multicatalyst system in asymmetric catalysis

    CERN Document Server

    Zhou, Jian

    2014-01-01

    This book introduces multi-catalyst systems by describing their mechanism and advantages in asymmetric catalysis.  Helps organic chemists perform more efficient catalysis with step-by-step methods  Overviews new concepts and progress for greener and economic catalytic reactions  Covers topics of interest in asymmetric catalysis including bifunctional catalysis, cooperative catalysis, multimetallic catalysis, and novel tandem reactions   Has applications for pharmaceuticals, agrochemicals, materials, and flavour and fragrance

  6. Kinetics and Catalysis Demonstrations.

    Science.gov (United States)

    Falconer, John L.; Britten, Jerald A.

    1984-01-01

    Eleven videotaped kinetics and catalysis demonstrations are described. Demonstrations include the clock reaction, oscillating reaction, hydrogen oxidation in air, hydrogen-oxygen explosion, acid-base properties of solids, high- and low-temperature zeolite reactivity, copper catalysis of ammonia oxidation and sodium peroxide decomposition, ammonia…

  7. Catalysis seen in action

    NARCIS (Netherlands)

    Tromp, M.

    2015-01-01

    Synchrotron radiation techniques are widely applied in materials research and heterogeneous catalysis. In homogeneous catalysis, its use so far is rather limited despite its high potential. Here, insights in the strengths and limitations of X-ray spectroscopy technique in the field of homogeneous ca

  8. Concepts in Heterogeneous Catalysis

    Science.gov (United States)

    1974-06-01

    The group Vill metals have vacant atomic d-orbilals (holes in the d-band) which were ex- peeled to promote celuemiorplion and catalysisA by...Houston, Texas, February 24.26 1971. Mango , F. D., Advances in Catalysis, 19 (1969). Mango , F. D. and i. H. Schachtschnelder, J. Am. Chem. Soc., 89

  9. Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

    Directory of Open Access Journals (Sweden)

    David G Nickens

    Full Text Available BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxyribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2>A(1>A(3>A(4>A(0>A(6>A(8>A(10 and T(2>T(3>T(4>T(6 approximately T(1>T(8>T(10. Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90% joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI did not support ligation when

  10. Tomographic PIV measurements of a regenerating hairpin vortex

    Science.gov (United States)

    Sabatino, D. R.; Rossmann, T.

    2016-01-01

    The three-dimensional formation and regeneration of a hairpin vortex in a laminar boundary layer is studied in a free-surface water channel. The vortex is generated by fluid injection through a narrow slot into a laminar boundary layer (Re_{δ ^*} = 485) and recorded with tomographic particle image velocimetry. The swirling strength based on the λ _2 criterion shows that the hairpin initially forms at the upstream edge of the elongated ring vortex produced by the injection. The elongated ring vortex decays while the hairpin vortex strengthens. Because the hairpin vortex is of sufficient strength, it forms a kink in the legs as a result of inviscid induction. A bridging structure forms between the legs initially upstream of the kink. As this structure dissipates, another bridging structure forms downstream of the kink and closes the vortex loop between the legs. This pinches off the original hairpin head such that two distinct vortices result. The formation of the secondary hairpin head does not appear to be preceded by a reduction in the spanwise gap between the legs or significant change in height above the wall as has been seen when exposed to a mean turbulent profile. Instead, the formation is preceded by the stretching of the hairpin legs downstream of the kink, exposes the ejected fluid between the legs to boundary layer flow producing conditions similar to those that formed the initial hairpin vortex.

  11. Molecular water oxidation catalysis

    CERN Document Server

    Llobet, Antoni

    2014-01-01

    Photocatalytic water splitting is a promising strategy for capturing energy from the sun by coupling light harvesting and the oxidation of water, in order to create clean hydrogen fuel. Thus a deep knowledge of the water oxidation catalysis field is essential to be able to come up with useful energy conversion devices based on sunlight and water splitting. Molecular Water Oxidation Catalysis: A Key Topic for New Sustainable Energy Conversion Schemes presents a comprehensive and state-of-the-art overview of water oxidation catalysis in homogeneous phase, describing in detail the most importan

  12. Surface and nanomolecular catalysis

    CERN Document Server

    Richards, Ryan

    2006-01-01

    Using new instrumentation and experimental techniques that allow scientists to observe chemical reactions and molecular properties at the nanoscale, the authors of Surface and Nanomolecular Catalysis reveal new insights into the surface chemistry of catalysts and the reaction mechanisms that actually occur at a molecular level during catalysis. While each chapter contains the necessary background and explanations to stand alone, the diverse collection of chapters shows how developments from various fields each contributed to our current understanding of nanomolecular catalysis as a whole. The

  13. Catalysis seen in action.

    Science.gov (United States)

    Tromp, Moniek

    2015-03-06

    Synchrotron radiation techniques are widely applied in materials research and heterogeneous catalysis. In homogeneous catalysis, its use so far is rather limited despite its high potential. Here, insights in the strengths and limitations of X-ray spectroscopy technique in the field of homogeneous catalysis are given, including new technique developments. A relevant homogeneous catalyst, used in the industrially important selective oligomerization of ethene, is taken as a worked-out example. Emphasis is placed on time-resolved operando X-ray absorption spectroscopy with outlooks to novel high energy resolution and emission techniques. All experiments described have been or can be done at the Diamond Light Source Ltd (Didcot, UK).

  14. Eukaryotic penelope-like retroelements encode hammerhead ribozyme motifs.

    Science.gov (United States)

    Cervera, Amelia; De la Peña, Marcos

    2014-11-01

    Small self-cleaving RNAs, such as the paradigmatic Hammerhead ribozyme (HHR), have been recently found widespread in DNA genomes across all kingdoms of life. In this work, we found that new HHR variants are preserved in the ancient family of Penelope-like elements (PLEs), a group of eukaryotic retrotransposons regarded as exceptional for encoding telomerase-like retrotranscriptases and spliceosomal introns. Our bioinformatic analysis revealed not only the presence of minimalist HHRs in the two flanking repeats of PLEs but also their massive and widespread occurrence in metazoan genomes. The architecture of these ribozymes indicates that they may work as dimers, although their low self-cleavage activity in vitro suggests the requirement of other factors in vivo. In plants, however, PLEs show canonical HHRs, whereas fungi and protist PLEs encode ribozyme variants with a stable active conformation as monomers. Overall, our data confirm the connection of self-cleaving RNAs with eukaryotic retroelements and unveil these motifs as a significant fraction of the encoded information in eukaryotic genomes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Reconciling Ligase Ribozyme Activity with Fatty Acid Vesicle Stability

    Directory of Open Access Journals (Sweden)

    Fabrizio Anella

    2014-12-01

    Full Text Available The “RNA world” and the “Lipid world” theories for the origin of cellular life are often considered incompatible due to the differences in the environmental conditions at which they can emerge. One obstacle resides in the conflicting requirements for divalent metal ions, in particular Mg2+, with respect to optimal ribozyme activity, fatty acid vesicle stability and protection against RNA strand cleavage. Here, we report on the activity of a short L1 ligase ribozyme in the presence of myristoleic acid (MA vesicles at varying concentrations of Mg2+. The ligation rate is significantly lower at low-Mg2+ conditions. However, the loss of activity is overcompensated by the increased stability of RNA leading to a larger amount of intact ligated substrate after long reaction periods. Combining RNA ligation assays with fatty acid vesicles we found that MA vesicles made of 5 mM amphiphile are stable and do not impair ligase ribozyme activity in the presence of approximately 2 mM Mg2+. These results provide a scenario in which catalytic RNA and primordial membrane assembly can coexist in the same environment.

  16. Investigating a new generation of ribozymes in order to target HCV.

    Directory of Open Access Journals (Sweden)

    Michel V Lévesque

    Full Text Available For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with which to target HCV (+ RNA strands. These ribozymes resulted from the coupling of a specific on/off adaptor (SOFA to the ribozyme domain derived from the Hepatitis Delta Virus (HDV. The former switches cleavage activity "on" solely in the presence of the desired RNA substrate, while the latter was the first catalytic RNA reported to function naturally in human cells, specifically in hepatocytes. In order to maximize the chances for success, a step-by-step approach was used for both the design and the selection of the ribozymes. This approach included the use of both bioinformatics and biochemical methods for the identification of the sites possessing the greatest potential for targeting, and the subsequent in vitro testing of the cleavage activities of the corresponding SOFA-HDV ribozymes. These efforts led to a significant improvement in the ribozymes' designs. The ability of the resulting SOFA-HDV ribozymes to inhibit HCV replication was further examined using a luciferase-based replicon. Although some of the ribozymes exhibited high levels of cleavage activity in vitro, none appears to be a potential long term inhibitor in cellulo. Analysis of recent discoveries in the cellular biology of HCV might explain this failure, as well as provide some ideas on the potential limits of using nucleic acid-based drugs to control the propagation of HCV. Finally, the above conclusions received support from experiments performed using a collection of SOFA

  17. Mg(2+) Binding Promotes SLV as a Scaffold in Varkud Satellite Ribozyme SLI-SLV Kissing Loop Junction.

    Science.gov (United States)

    Bergonzo, Christina; Cheatham, Thomas E

    2017-07-25

    Though the structure of the substrate stem loop I (SLI)-stem loop V (SLV) kissing loop junction of the Varkud Satellite ribozyme has been experimentally characterized, the dynamics of this Mg(2+)-dependent loop-loop interaction have been elusive. Specifically, each hairpin loop contains a U-turn motif, but only SLV shows a conformational shift triggered by Mg(2+) ion association. Here, we use molecular dynamics simulations to analyze the binding and dynamics of this kissing loop junction. We show that SLV acts as a scaffold, providing stability to the junction. Mg(2+) ions associate with SLV when it is part of the junction in a manner similar to when it is unbound, but there is no specificity in Mg(2+) binding for the SLI loop. This suggests that the entropic penalty of ordering the larger SLI is too high, allowing SLV to act as a scaffold for multiple substrate loop sequences. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Research on Catalysis.

    Science.gov (United States)

    Bartholomew, Calvin H.; Hecker, William C.

    1984-01-01

    The objectives and philosophy of the Catalysis Laboratory at Brigham Young University are discussed. Also discusses recent and current research activities at the laboratory as well as educational opportunities, research facilities, and sources of research support. (JN)

  19. Flanking sequences with an essential role in hydrolysis of a self-cleaving group I-like ribozyme

    DEFF Research Database (Denmark)

    Einvik, C; Nielsen, Henrik; Nour, R

    2000-01-01

    DiGIR1 is a group I-like ribozyme derived from the mobile twin ribozyme group I intron DiSSU1 in the nuclear ribosomal DNA of the myxomycete Didymium iridis. This ribozyme is responsible for intron RNA processing in vitro and in vivo at two internal sites close to the 5'-end of the intron endo...

  20. A natural fast-cleaving branching ribozyme from the amoeboflagellate Naegleria pringsheimi

    DEFF Research Database (Denmark)

    Tang, Yunjia; Nielsen, Henrik; Birgisdottir, Asa Birna

    2011-01-01

    The GIR1 branching ribozyme constitutes a separate class of naturally occurring ribozymes. Most studies have been performed with the single GIR1 known from the myxomycete Didymium iridis whereas the large number of GIR1s found in the amoeboflagellate Naegleria has remained largely uncharacterized...

  1. Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene

    Institute of Scientific and Technical Information of China (English)

    Zhi-Ming Hap; Jin-Yan Luo; Jin Cheng; Quan-Yin Wang; Guang-Xiao Yang

    2003-01-01

    AIM: To design a hammerhead ribozyme targeting humantelomerase reverse transcriptase (hTERT) and clone it's genefor future use in the study of tumor gene therapy.METHODS: Using the software RNAstructure, the secondarystructure of hTERT mRNA was predicted and the cleavagesite of ribozyme was selected. A hammerhead ribozymetargeting this site was designed and bimolecular fold betweenthe ribozyme and hTERT was predicted. The DNA encodingthe ribozyme was synthesized and cloned into pGEMEX-1and the sequence of the ribozyme gene was confirmed byDNA sequencing.RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosenas the cleavage site of the ribozyme. The designed ribozymewas comprised of 22nt catalytic core and 17nt flankingsequence. Computer-aided prediction suggested that theribozyme and hTERT mRNA could cofold into a properconformation. Endonuclease restriction and DNA sequencingconfirmed the correct insertion of the ribozyme gene intothe vector pGEMEX-1.CONCLUSION: This fundamental work of successfuldesigning and cloning of an anti-hTERT hammerheadribozyme has paved the way for further study of inhibitingtumor cell growth by cleaving hTERT mRNA with ribozyme.

  2. How the discovery of ribozymes cast RNA in the roles of both chicken and egg in origin-of-life theories.

    Science.gov (United States)

    Sankaran, Neeraja

    2012-12-01

    Scientific theories about the origin-of-life theories have historically been characterized by the chicken-and-egg problem of which essential aspect of life was the first to appear, replication or self-sustenance. By the 1950s the question was cast in molecular terms and DNA and proteins had come to represent the carriers of the two functions. Meanwhile, RNA, the other nucleic acid, had played a capricious role in origin theories. Because it contained building blocks very similar to DNA, biologists recognized early that RNA could store information in its linear sequences. With the discovery in the 1980s that RNA molecules were capable of biological catalysis, a function hitherto ascribed to proteins alone, RNA took on the role of the single entity that could act as both chicken and egg. Within a few years of the discovery of these catalytic RNAs (ribozymes) scientists had formulated an RNA World hypothesis that posited an early phase in the evolution of life where all key functions were performed by RNA molecules. This paper traces the history the role of RNA in origin-of-life theories with a focus on how the discovery of ribozymes influenced the discourse.

  3. Structural insights into substrate recognition by the Neurospora Varkud satellite ribozyme: importance of U-turns at the kissing-loop junction.

    Science.gov (United States)

    Bouchard, Patricia; Legault, Pascale

    2014-01-14

    Substrate recognition by the Neurospora Varkud satellite ribozyme depends on the formation of a magnesium-dependent kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain. From mutagenesis studies, it has been established that this I/V kissing-loop interaction involves three Watson-Crick base pairs and is associated with a structural rearrangement of the SLI substrate that facilitates catalysis. Here, we report the NMR structural characterization of this I/V kissing-loop using isolated stem-loops. NMR studies were performed on different SLI/SLV complexes containing a common SLV and shiftable, preshifted, or double-stranded SLI variants. These studies confirm the presence of three Watson-Crick base pairs at the kissing-loop junction and provide evidence for the structural rearrangement of shiftable SLI variants upon SLV binding. NMR structure determination of an SLI/SLV complex demonstrates that both the SLI and SLV loops adopt U-turn structures, which facilitates intermolecular Watson-Crick base pairing. Several other interactions at the I/V interface, including base triples and base stacking, help create a continuously stacked structure. These NMR studies provide a structural basis to understand the stability of the I/V kissing-loop interaction and lead us to propose a kinetic model for substrate activation in the VS ribozyme.

  4. Experimental cancer gene therapy by multiple anti-survivin hammerhead ribozymes

    Institute of Scientific and Technical Information of China (English)

    Qi Fei; Yuwen Ke; Xuebiao Yao; Jingde Zhu; Hongyu Zhang; Lili Fu; Xinlan Dai; Baomei Gao; Min Ni; Chao Ge; Jinjun Li; Xia Ding

    2008-01-01

    To improve the efficacy of gene therapy for cancer, we designed four hammerhead ribozyme adenoviruses (R1 to R4) targeting the exposed regions of survivin mRNA. In addition to the in vitro characterization, which included a determination of the sequence specificity of cleavage by primer extension, assays for cell proliferation and for in vivo tumor growth were used to score for ribozyme efficiency.The resulting suppression of survivin expression induced mitotic catastrophe and cell death via the caspase-3-dependent pathway. Importantly, administration of the ribozyme adenoviruses inhibited tumor growth in a hepato-cellular carcinoma xenograft mouse model. Co-expression of R1, R3 and R4 ribozymes synergistically suppressed survivin and, as this combination targets all major forms of the survivin transcripts, produced the most potent anti-cancer effects. The adenoviruses carrying the multiple hammerhead ribozymes described in this report offered a robust gene therapy strategy against cancer.

  5. Generation and Development of RNA Ligase Ribozymes with Modular Architecture Through “Design and Selection”

    Directory of Open Access Journals (Sweden)

    Yuki Fujita

    2010-08-01

    Full Text Available In vitro selection with long random RNA libraries has been used as a powerful method to generate novel functional RNAs, although it often requires laborious structural analysis of isolated RNA molecules. Rational RNA design is an attractive alternative to avoid this laborious step, but rational design of catalytic modules is still a challenging task. A hybrid strategy of in vitro selection and rational design has been proposed. With this strategy termed “design and selection,” new ribozymes can be generated through installation of catalytic modules onto RNA scaffolds with defined 3D structures. This approach, the concept of which was inspired by the modular architecture of naturally occurring ribozymes, allows prediction of the overall architectures of the resulting ribozymes, and the structural modularity of the resulting ribozymes allows modification of their structures and functions. In this review, we summarize the design, generation, properties, and engineering of four classes of ligase ribozyme generated by design and selection.

  6. Destabilization of the TAR hairpin leads to extension of the polyA hairpin and inhibition of HIV-1 polyadenylation.

    Science.gov (United States)

    Vrolijk, Martine M; Harwig, Alex; Berkhout, Ben; Das, Atze T

    2009-02-11

    Two hairpin structures that are present at both the 5' and 3' end of the HIV-1 RNA genome have important functions in the viral life cycle. The TAR hairpin binds the viral Tat protein and is essential for Tat-mediated activation of transcription. The adjacent polyA hairpin encompasses the polyadenylation signal AAUAAA and is important for the regulation of polyadenylation. Specifically, this RNA structure represses polyadenylation at the 5' side, and enhancer elements on the 3' side overcome this suppression. We recently described that the replication of an HIV-1 variant that does not need TAR for transcription was severely impaired by destabilization of the TAR hairpin, even though a complete TAR deletion was acceptable. In this study, we show that the TAR-destabilizing mutations result in reduced 3' polyadenylation of the viral transcripts due to an extension of the adjacent polyA hairpin. Thus, although the TAR hairpin is not directly involved in polyadenylation, mutations in TAR can affect this process. The stability of the HIV-1 TAR hairpin structure is important for the proper folding of the viral RNA transcripts. This study illustrates how mutations that are designed to study the function of a specific RNA structure can change the structural presentation of other RNA domains and thus affect viral replication in an indirect way.

  7. Investigating the role of chain and linker length on the catalytic activity of an H 2 production catalyst containing a β-hairpin peptide

    Energy Technology Data Exchange (ETDEWEB)

    Reback, Matthew L.; Ginovska, Bojana; Buchko, Garry W.; Dutta, Arnab; Priyadarshani, Nilusha; Kier, Brandon L.; Helm, Monte L.; Raugei, Simone; Shaw, Wendy J.

    2016-06-02

    Building on our recent report of an active H2 production catalyst [Ni(PPh2NProp-peptide)2]2+ (Prop=para-phenylpropionic acid, peptide (R10)=WIpPRWTGPR-NH2, p=D-proline, and P2N=1-aza-3,6-diphosphacycloheptane) that contains structured -hairpin peptides, here we investigate how H2 production is effected by: (1) the length of the hairpin (eight or ten residues) and (2) limiting the flexibility between the peptide and the core complex by altering the length of the linker: para-phenylpropionic acid (three carbons) or para-benzoic acid (one carbon). Reduction of the peptide chain length from ten to eight residues increases or maintains the catalytic current for H2 production for all complexes, suggesting a non-productive steric interaction at longer peptide lengths. While the structure of the hairpin appears largely intact for the complexes, NMR data are consistent with differences in dynamic behavior which may contribute to the observed differences in catalytic activity. Molecular dynamics simulations demonstrate that complexes with a one-carbon linker have the desired effect of restricting the motion of the hairpin relative to the complex; however, the catalytic currents are significantly reduced compared to complexes containing a three-carbon linker as a result of the electron withdrawing nature of the -COOH group. These results demonstrate the complexity and interrelated nature of the outer coordination sphere on catalysis.

  8. Phenotypic inheritance induced by hairpin RNA in Drosophila

    Institute of Scientific and Technical Information of China (English)

    Huaguang Li; Yi Lu

    2009-01-01

    Phenotypic inheritance induced by RNA has been docu-mented in mouse and Caenorhabditis elegans. Here we report a similar inheritance in Drosophila. Mutant phe-notypes of eye defects and antenna duplication gener-ated from the crossing of one RNA interference (RNAi)transgenic line harboring one hairpin RNA transgene with a GAL4 driver line were inherited independently of the GAL4 driver. Hairpin RNA injection exper-iments demonstrated that the hairpin RNA could induce heritable mutant-like phenotypes on the eye and antenna. The penetrance of mutant phenotypes was reduced when the mutants were crossed to agol and piwi mutants. Our data suggest that hairpin RNA can induce phenotypic inheritance in Drosophila.

  9. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    Science.gov (United States)

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  10. Catalysis for alternative energy generation

    CERN Document Server

    2012-01-01

    Summarizes recent problems in using catalysts in alternative energy generation and proposes novel solutions  Reconsiders the role of catalysis in alternative energy generation  Contributors include catalysis and alternative energy experts from across the globe

  11. Asymmetric catalysis with helical polymers

    NARCIS (Netherlands)

    Megens, Rik P.; Roelfes, Gerard

    Inspired by nature, the use of helical biopolymer catalysts has emerged over the last years as a new approach to asymmetric catalysis. In this Concept article the various approaches and designs and their application in asymmetric catalysis will be discussed.

  12. Asymmetric catalysis with helical polymers

    NARCIS (Netherlands)

    Megens, Rik P.; Roelfes, Gerard

    2011-01-01

    Inspired by nature, the use of helical biopolymer catalysts has emerged over the last years as a new approach to asymmetric catalysis. In this Concept article the various approaches and designs and their application in asymmetric catalysis will be discussed.

  13. Preface: Catalysis Today

    DEFF Research Database (Denmark)

    Li, Yongdan

    2016-01-01

    This special issue of Catalysis Today with the theme “Sustain-able Energy” results from a great success of the session “Catalytic Technologies Accelerating the Establishment of Sustainable and Clean Energy”, one of the two sessions of the 1st International Symposium on Catalytic Science and Techn......This special issue of Catalysis Today with the theme “Sustain-able Energy” results from a great success of the session “Catalytic Technologies Accelerating the Establishment of Sustainable and Clean Energy”, one of the two sessions of the 1st International Symposium on Catalytic Science...... and Technology in Sustainable Energy and Environment, held in Tianjin, China during October8–10, 2014. This biennial symposium offers an international forum for discussing and sharing the cutting-edge researches and the most recent breakthroughs in energy and environmental technologies based on catalysis...

  14. Design principles for ligand-sensing, conformation-switching ribozymes.

    Directory of Open Access Journals (Sweden)

    Xi Chen

    2009-12-01

    Full Text Available Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer with a catalytic signal generator (ribozyme. Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications.

  15. Analysis of solvent nucleophile isotope effects: evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis.

    Science.gov (United States)

    Cassano, Adam G; Anderson, Vernon E; Harris, Michael E

    2004-08-17

    ribozyme catalyzed reaction provides support for nucleophilic activation by metal ion catalysis.

  16. Isotopes in heterogeneous catalysis

    CERN Document Server

    Hargreaves, Justin SJ

    2006-01-01

    The purpose of this book is to review the current, state-of-the-art application of isotopic methods to the field of heterogeneous catalysis. Isotopic studies are arguably the ultimate technique in in situ methods for heterogeneous catalysis. In this review volume, chapters have been contributed by experts in the field and the coverage includes both the application of specific isotopes - Deuterium, Tritium, Carbon-14, Sulfur-35 and Oxygen-18 - as well as isotopic techniques - determination of surface mobility, steady state transient isotope kinetic analysis, and positron emission profiling.

  17. Nanomaterials in catalysis

    CERN Document Server

    Serp, Philippe; Somorjai, Gabor A; Chaudret, Bruno

    2012-01-01

    Nanocatalysis has emerged as a field at the interface between homogeneous and heterogeneous catalysis and offers unique solutions to the demanding requirements for catalyst improvement. Heterogeneous catalysis represents one of the oldest commercial applications of nanoscience and nanoparticles of metals, semiconductors, oxides, and other compounds have been widely used for important chemical reactions. The main focus of this fi eld is the development of well-defined catalysts, which may include both metal nanoparticles and a nanomaterial as the support. These nanocatalysts should display the

  18. Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

    Directory of Open Access Journals (Sweden)

    Ulrich F. Müller

    2017-01-01

    Full Text Available Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

  19. Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Qing Xie; Xia-Qiu Zhou; Shan Jiang; You-Xin Jin

    2004-01-01

    AIM: To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activityin vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases.METHODS: Anti-caspase-7 ribozymes targeting sites 333and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained byin vitro transcription were cloned into pBSKneo U6' and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experimentsin vitro.RESULTS: Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed byin vitro transcription.In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394.CONCLUSION: Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.

  20. Pollution Control by Catalysis

    DEFF Research Database (Denmark)

    Eriksen, Kim Michael; Fehrmann, Rasmus

    1998-01-01

    The report summarises the results of two years of collaboration supported by INTAS between Department of Chemistry,DTU,DK , IUSTI,Universite de Provence,FR, ICE/HT University 6of Patras,GR, and Boreskov Institute of Catalysis,RU.The project has been concerned with mechanistic studies of deNOx and...

  1. Pollution Control by Catalysis

    DEFF Research Database (Denmark)

    Eriksen, Kim Michael; Fehrmann, Rasmus

    1998-01-01

    The report summarises the results of two years of collaboration supported by INTAS between Department of Chemistry,DTU,DK , IUSTI,Universite de Provence,FR, ICE/HT University 6of Patras,GR, and Boreskov Institute of Catalysis,RU.The project has been concerned with mechanistic studies of deNOx and...

  2. Anion-π catalysis.

    Science.gov (United States)

    Zhao, Yingjie; Beuchat, César; Domoto, Yuya; Gajewy, Jadwiga; Wilson, Adam; Mareda, Jiri; Sakai, Naomi; Matile, Stefan

    2014-02-05

    The introduction of new noncovalent interactions to build functional systems is of fundamental importance. We here report experimental and theoretical evidence that anion-π interactions can contribute to catalysis. The Kemp elimination is used as a classical tool to discover conceptually innovative catalysts for reactions with anionic transition states. For anion-π catalysis, a carboxylate base and a solubilizer are covalently attached to the π-acidic surface of naphthalenediimides. On these π-acidic surfaces, transition-state stabilizations up to ΔΔGTS = 31.8 ± 0.4 kJ mol(-1) are found. This value corresponds to a transition-state recognition of KTS = 2.7 ± 0.5 μM and a catalytic proficiency of 3.8 × 10(5) M(-1). Significantly increasing transition-state stabilization with increasing π-acidity of the catalyst, observed for two separate series, demonstrates the existence of "anion-π catalysis." In sharp contrast, increasing π-acidity of the best naphthalenediimide catalysts does not influence the more than 12 000-times weaker substrate recognition (KM = 34.5 ± 1.6 μM). Together with the disappearance of Michaelis-Menten kinetics on the expanded π-surfaces of perylenediimides, this finding supports that contributions from π-π interactions are not very important for anion-π catalysis. The linker between the π-acidic surface and the carboxylate base strongly influences activity. Insufficient length and flexibility cause incompatibility with saturation kinetics. Moreover, preorganizing linkers do not improve catalysis much, suggesting that the ideal positioning of the carboxylate base on the π-acidic surface is achieved by intramolecular anion-π interactions rather than by an optimized structure of the linker. Computational simulations are in excellent agreement with experimental results. They confirm, inter alia, that the stabilization of the anionic transition states (but not the neutral ground states) increases with the π-acidity of the

  3. Group I Intron Internal Guide Sequence Binding Strength as a Component of Ribozyme Network Formation

    Directory of Open Access Journals (Sweden)

    Laura Elizabeth Satterwhite

    2016-09-01

    Full Text Available Origins-of-life research requires searching for a plausible transition from simple chemicals to larger macromolecules that can both hold information and catalyze their own production. We have previously shown that some group I intron ribozymes possess the ability to help synthesize other ribozyme genotypes by recombination reactions in small networks in an autocatalytic fashion. By simplifying these recombination reactions, using fluorescent anisotropy, we quantified the thermodynamic binding strength between two nucleotides of two group I intron RNA fragments for all 16 possible genotype combinations. We provide evidence that the binding strength (KD between the 3-nucleotide internal guide sequence (IGS of one ribozyme and its complement in another is correlated to the catalytic ability of the ribozyme. This work demonstrates that one can begin to deconstruct the thermodynamic basis of information in prebiotic RNA systems.

  4. A BRIEF HISTORY OF INDUSTRIAL CATALYSIS

    Energy Technology Data Exchange (ETDEWEB)

    Heinemann, Heinz

    1979-06-01

    This history covers: catalytic cracking and other acid catalysed reactions; zeolite catalysis; dual functional catalysis; hydrogenation catalysis and hydrogen production; catalytic hydrocarbon dehydrogenation; catalytic alkylation and dealkylation; catalytic coal liquefaction and gasification; heterogeneous oxidation, arnmoxidation, chlorination, and oxychlorination catalysis; olefin disproportionation catalysis; industrial homogeneous catalysis; catalytic polymerization; catalysis for motor vehicle emission control; fuel cell catalysis; and the profession of the catalytic chemist or engineer. The discussion is mostly limited to the rapid growth of industrial catalysis between the second World War and 1978.

  5. Hybridization-based biosensor containing hairpin probes and use thereof

    Science.gov (United States)

    Miller, Benjamin L.; Strohsahl, Christopher M.

    2010-10-12

    A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

  6. Emergence of hairpins in the conformations of a confined polymer

    CERN Document Server

    Werner, E; Muralidhar, A; Frykholm, K; Smithe, T St Clere; Fritzsche, J; Westerlund, F; Dorfman, K D; Mehlig, B

    2016-01-01

    If a semiflexible polymer confined to a narrow channel bends around by 180 degrees, the polymer is said to exhibit a hairpin. The equilibrium extension statistics in either the limit where hairpins are rare or in the limit where they are common have been characterized in detail. In this article we consider the intermediate situation, where hairpins are rare but common enough to influence the extension statistics. We study the equilibrium distribution of the extension, as well as the approach to equilibrium, by a combination of theoretical analysis, Monte Carlo simulations, and experiments on DNA coated by the protein RecA, which enhances the stiffness of DNA by approximately one order of magnitude. We find good agreement between the model and simulations. The model also provides excellent agreement with experimental data provided that we assume a persistence length of 2 $\\mu$m for the RecA-DNA filament.

  7. Magnetic Catalysis in Graphene

    CERN Document Server

    Winterowd, Christopher; Zafeiropoulos, Savvas

    2015-01-01

    One of the most important developments in condensed matter physics in recent years has been the discovery and characterization of graphene. A two-dimensional layer of Carbon arranged in a hexagonal lattice, graphene exhibits many interesting electronic properties, most notably that the low energy excitations behave as massless Dirac fermions. These excitations interact strongly via the Coulomb interaction and thus non-perturbative methods are necessary. Using methods borrowed from lattice QCD, we study the graphene effective theory in the presence of an external magnetic field. Graphene, along with other $(2+1)$-dimensional field theories, has been predicted to undergo spontaneous breaking of flavor symmetry including the formation of a gap as a result of the external magnetic field. This phenomenon is known as magnetic catalysis. Our study investigates magnetic catalysis using a fully non-perturbative approach.

  8. Solid Base Catalysis

    CERN Document Server

    Ono, Yoshio

    2011-01-01

    The importance of solid base catalysts has come to be recognized for their environmentally benign qualities, and much significant progress has been made over the past two decades in catalytic materials and solid base-catalyzed reactions. The book is focused on the solid base. Because of the advantages over liquid bases, the use of solid base catalysts in organic synthesis is expanding. Solid bases are easier to dispose than liquid bases, separation and recovery of products, catalysts and solvents are less difficult, and they are non-corrosive. Furthermore, base-catalyzed reactions can be performed without using solvents and even in the gas phase, opening up more possibilities for discovering novel reaction systems. Using numerous examples, the present volume describes the remarkable role solid base catalysis can play, given the ever increasing worldwide importance of "green" chemistry. The reader will obtain an overall view of solid base catalysis and gain insight into the versatility of the reactions to whic...

  9. Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures.

    Science.gov (United States)

    Abi, Alireza; Ferapontova, Elena E

    2013-04-01

    Genetic analysis of infectious and genetic diseases and cancer diagnostics require the development of efficient tools for fast and reliable analysis of single-nucleotide polymorphism (SNP) in targeted DNA and RNA sequences often responsible for signalling disease onset. Here, we highlight the main trends in the development of electrochemical genosensors for sensitive and selective detection of SNP that are based on hairpin DNA architectures exhibiting better SNP recognition properties compared with linear DNA probes. SNP detection by electrochemical hairpin DNA beacons is discussed, and comparative analysis of the existing SNP sensing strategies based on enzymatic and nanoparticle signal amplification schemes is presented.

  10. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins.

    Science.gov (United States)

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-06-24

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  11. Robust suppression of HIV replication by intracellularly expressed reverse transcriptase aptamers is independent of ribozyme processing.

    Science.gov (United States)

    Lange, Margaret J; Sharma, Tarun K; Whatley, Angela S; Landon, Linda A; Tempesta, Michael A; Johnson, Marc C; Burke, Donald H

    2012-12-01

    RNA aptamers that bind human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) also inhibit viral replication, making them attractive as therapeutic candidates and potential tools for dissecting viral pathogenesis. However, it is not well understood how aptamer-expression context and cellular RNA pathways govern aptamer accumulation and net antiviral bioactivity. Using a previously-described expression cassette in which aptamers were flanked by two "minimal core" hammerhead ribozymes, we observed only weak suppression of pseudotyped HIV. To evaluate the importance of the minimal ribozymes, we replaced them with extended, tertiary-stabilized hammerhead ribozymes with enhanced self-cleavage activity, in addition to noncleaving ribozymes with active site mutations. Both the active and inactive versions of the extended hammerhead ribozymes increased inhibition of pseudotyped virus, indicating that processing is not necessary for bioactivity. Clonal stable cell lines expressing aptamers from these modified constructs strongly suppressed infectious virus, and were more effective than minimal ribozymes at high viral multiplicity of infection (MOI). Tertiary stabilization greatly increased aptamer accumulation in viral and subcellular compartments, again regardless of self-cleavage capability. We therefore propose that the increased accumulation is responsible for increased suppression, that the bioactive form of the aptamer is one of the uncleaved or partially cleaved transcripts, and that tertiary stabilization increases transcript stability by reducing exonuclease degradation.

  12. Catalysis and biocatalysis program

    Science.gov (United States)

    Ingham, J. D.

    1993-01-01

    This final report presents a summary of research activities and accomplishments for the Catalysis and Biocatalysis Program, which was renamed the Biological and Chemical Technologies Research (BCTR) Program, currently of the Advanced Industrial Concepts Division (AICD), Office of Industrial Technologies of the Department of Energy (DOE). The Program was formerly under the Division of Energy Conversion and Utilization Technologies (ECUT) until the DOE reorganization in April, 1990. The goals of the BCTR Program are consistent with the initial ECUT goals, but represent an increased effort toward advances in chemical and biological technology transfer. In addition, the transition reflects a need for the BCTR Program to assume a greater R&D role in chemical catalysis as well as a need to position itself for a more encompassing involvement in a broader range of biological and chemical technology research. The mission of the AICD is to create a balanced Program of high risk, long-term, directed interdisciplinary research and development that will improve energy efficiency and enhance fuel flexibility in the industrial sector. Under AICD, the DOE Catalysis and Biocatalysis Program sponsors research and development in furthering industrial biotechnology applications and promotes the integrated participation of universities, industrial companies, and government research laboratories.

  13. Dendritic structure DNA for specific metal ion biosensor based on catalytic hairpin assembly and a sensitive synergistic amplification strategy.

    Science.gov (United States)

    Zhao, Jianmin; Jing, Pei; Xue, Shuyan; Xu, Wenju

    2017-01-15

    In this work, a sensitive electrochemical biosensing to Pb(2+) was proposed based on the high specificity of DNAzymes to Pb(2+). The response signal was efficiently amplified by the catalytic hairpin assembly induced by strand replacement reaction and the formation of dendritic structure DNA (DSDNA) by layer-by-layer assembly. Firstly, in the presence of Pb(2+), the substrate strand (S1) of the Pb(2+)-specific DNAzymes was specifically cleaved by Pb(2+). Secondly, one of the two fragments (rS1) introduced into the electrode surface was hybridized with a hairpin DNA (H1) and further replaced by another hairpin DNA (H2) by the hybridization reaction of H1 with H2. The released rS1 then induced the next hybridization with H1. After repeated cycles, the catalytic recycling assembly of H2 with H1 was completed. Thirdly, two bioconjugates of Pt@Pd nanocages (Pt@PdNCs) labeled with DNA S3/S4 and electroactive toluidine blue (Tb) (Tb-S3-Pt@PdNCs and Tb-S4-Pt@PdNCs) were captured onto the resultant electrode surface through the hybridization of S3 and H2, S3 and S4, resulting in the formation of DSDNA triggered by layer-by-layer assembly. This formed DSDNA greatly facilitated the immobilization of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrin (MnTMPyP) as mimicking enzyme. Under the synergistic catalysis of Pt@PdNCs and MnTMPyP to H2O2 reduction, the effective signal amplification of the developed Pb(2+) biosensor was achieved. As a result, the sensitive detection of the proposed electrochemical strategy for Pb(2+) was greatly improved in the range of 0.1pM-200nM with a detection limit of 0.033pM.

  14. Cleavage of an amide bond by a ribozyme

    Science.gov (United States)

    Dai, X.; De Mesmaeker, A.; Joyce, G. F.; Miller, S. L. (Principal Investigator)

    1995-01-01

    A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

  15. A hammerhead ribozyme inhibits ADE1 gene expression in yeast.

    Science.gov (United States)

    Ferbeyre, G; Bratty, J; Chen, H; Cedergren, R

    1995-03-21

    To study factors that affect in vivo ribozyme (Rz) activity, a model system has been devised in Saccharomyces cerevisiae based on the inhibition of ADE1 gene expression. This gene was chosen because Rz action can be evaluated visually by the Red phenotype produced when the activity of the gene product is inhibited. Different plasmid constructs allowed the expression of the Rz either in cis or in trans with respect to ADE1. Rz-related inhibition of ADE1 expression was correlated with a Red phenotype and a diminution of ADE1 mRNA levels only when the Rz gene was linked 5' to ADE1. The presence of the expected 3' cleavage fragment was demonstrated using a technique combining RNA ligation and PCR. This yeast system and detection technique are suited to the investigation of general factors affecting Rz-catalyzed inhibition of gene expression under in vivo conditions.

  16. Whirlwinds and hairpins in the atmospheric surface layer

    NARCIS (Netherlands)

    Oncley, Steven P.; Hartogensis, O.K.; Tong, Chenning

    2016-01-01

    Vortices in the atmospheric surface layer are characterized using observations at unprecedented resolution from a fixed array of 31 turbulence sensors. During the day, these vortices likely are dust devils, though no visual observations are available for confirmation. At night, hairpin vortices

  17. Light-triggered β-hairpin folding and unfolding

    NARCIS (Netherlands)

    Schrader, Tobias E.; Schreier, Wolfgang J.; Cordes, Thorben; Koller, Florian O.; Babitzki, Galina; Denschlag, Robert; Renner, Christian; Löweneck, Markus; Dong, Shou-Liang; Moroder, Luis; Tavan, Paul; Zinth, Wolfgang

    2007-01-01

    A light-switchable peptide is transformed with ultrashort pulses from a β-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular

  18. Thermodynamics of a Simple Three-Dimensional DNA Hairpin Model

    CERN Document Server

    Kremer, Kellan; Boggess, Erin; Mask, Walker; Saucedo, Tony; Hansen, JJ; Appelgate, Ian; Jurgensen, Taylor; Santos, Aaron

    2016-01-01

    We characterize the equation of state for a simple three-dimensional DNA hairpin model using a Metropolis Monte Carlo algorithm. This algorithm was run at constant temperature and fixed separation between the terminal ends of the strand. From the equation of state, we compute the compressibility, thermal expansion coefficient, and specific heat along with adiabatic path.

  19. Whirlwinds and hairpins in the atmospheric surface layer

    NARCIS (Netherlands)

    Oncley, Steven P.; Hartogensis, O.K.; Tong, Chenning

    2016-01-01

    Vortices in the atmospheric surface layer are characterized using observations at unprecedented resolution from a fixed array of 31 turbulence sensors. During the day, these vortices likely are dust devils, though no visual observations are available for confirmation. At night, hairpin vortices appe

  20. Raising the Speed Limit for β-Hairpin Formation

    Science.gov (United States)

    Davis, Caitlin M.; Xiao, Shifeng; Raleigh, Daniel P.; Dyer, R. Brian

    2012-01-01

    Understanding the folding of the β-hairpin is a crucial step in studying how β-rich proteins fold. We have studied CLN025, an optimized ten residue synthetic peptide, which adopts a compact, well-structured β-hairpin conformation. Formation of the component β-sheet and β-turn structures of CLN025 was probed independently using a combination of equilibrium Fourier transform infrared spectroscopy and laser-induced temperature jump coupled with time-resolved infrared and fluorescence spectroscopies. We find that CLN025 is an ultrafast folder due to its small free energy barrier to folding and that it exceeds the predicted speed limit for β-hairpin formation by an order of magnitude. We also find that the folding mechanism cannot be described by a simple two-state model, but rather is a heterogeneous process involving two independent parallel processes. Formation of stabilizing cross-strand hydrophobic interactions and turn alignment occur competitively, with relaxation lifetimes of 82 ± 10 and 124 ± 10 ns, respectively, at the highest probed temperature. The ultrafast and heterogeneous folding kinetics observed for CLN025 provide evidence for folding on a nearly barrierless free energy landscape, and recalibrate the speed limit for the formation of a β-hairpin. PMID:22873643

  1. Heuristic RNA pseudoknot prediction including intramolecular kissing hairpins

    Science.gov (United States)

    Sperschneider, Jana; Datta, Amitava; Wise, Michael J.

    2011-01-01

    Pseudoknots are an essential feature of RNA tertiary structures. Simple H-type pseudoknots have been studied extensively in terms of biological functions, computational prediction, and energy models. Intramolecular kissing hairpins are a more complex and biologically important type of pseudoknot in which two hairpin loops form base pairs. They are hard to predict using free energy minimization due to high computational requirements. Heuristic methods that allow arbitrary pseudoknots strongly depend on the quality of energy parameters, which are not yet available for complex pseudoknots. We present an extension of the heuristic pseudoknot prediction algorithm DotKnot, which covers H-type pseudoknots and intramolecular kissing hairpins. Our framework allows for easy integration of advanced H-type pseudoknot energy models. For a test set of RNA sequences containing kissing hairpins and other types of pseudoknot structures, DotKnot outperforms competing methods from the literature. DotKnot is available as a web server under http://dotknot.csse.uwa.edu.au. PMID:21098139

  2. Inorganic Reaction Mechanisms Part II: Homogeneous Catalysis

    Science.gov (United States)

    Cooke, D. O.

    1976-01-01

    Suggests several mechanisms for catalysis by metal ion complexes. Discusses the principal factors of importance in these catalysis reactions and suggests reactions suitable for laboratory study. (MLH)

  3. β-hairpin-mediated nucleation of polyglutamine amyloid formation

    Science.gov (United States)

    Kar, Karunakar; Hoop, Cody L.; Drombosky, Kenneth W.; Baker, Matthew A.; Kodali, Ravindra; Arduini, Irene; van der Wel, Patrick C. A.; Horne, W. Seth; Wetzel, Ronald

    2013-01-01

    The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington’s disease (HD). Here we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (trpzip, disulfide, D-Pro-Gly, Coulombic attraction, L-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well-correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n* = 4 for a simple, unbroken Q23 sequence to approximate unitary n* values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of SSNMR 13C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn* values remain quite low (~ 10−10) and there is no evidence for significant embellishment of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases. PMID:23353826

  4. Mechanochemistry, catalysis, and catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Butyagin, P.Yu.

    1987-07-01

    The physical basis of mechanochemistry and the reasons for the initiation and acceleration of chemical reactions upon the mechanical treatment of solids have been considered. The phenomenon of mechanical catalysis has been described in the example case of the oxidation of CO on oxide surfaces, and the nature of the active sites and the laws governing the mechanically activated chemisorption of gases on cleavage and friction surfaces of solids have been examined. The possibilities of the use of the methods of mechanochemistry in processes used to prepare catalysts have been analyzed in examples of decomposition reactions of inorganic compounds and solid-phase synthesis.

  5. Electron Transfer Chain Catalysis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@ Electron-transfer chain (ETC) catalysis belongs to the family of chain reactions where the electron is the catalyst. The ETC mechanism could be initiated by chemical activation, electrochemistry, or photolysis. If this pathway is applied to the preparation of organometallic complexes, it utilizes the greatly enhanced reactivity of organometallic 17e and 19e radicals. The chemical propagation is followed by the cross electron-transfer while the electron-transfer step is also followed by the chemical propagation, creating a loop in which reactants are facilely transformed into products. Interestingly the overall reaction is without any net redox change.

  6. Electron Transfer Chain Catalysis

    Institute of Scientific and Technical Information of China (English)

    LIU; LingKang

    2001-01-01

    Electron-transfer chain (ETC) catalysis belongs to the family of chain reactions where the electron is the catalyst. The ETC mechanism could be initiated by chemical activation, electrochemistry, or photolysis. If this pathway is applied to the preparation of organometallic complexes, it utilizes the greatly enhanced reactivity of organometallic 17e and 19e radicals. The chemical propagation is followed by the cross electron-transfer while the electron-transfer step is also followed by the chemical propagation, creating a loop in which reactants are facilely transformed into products. Interestingly the overall reaction is without any net redox change.  ……

  7. Catalysis and prebiotic RNA synthesis

    Science.gov (United States)

    Ferris, James P.

    1993-01-01

    The essential role of catalysis for the origins of life is discussed. The status of the prebiotic synthesis of 2',5'- and 3'5'-linked oligomers of RNA is reviewed. Examples of the role of metal ion and mineral catalysis in RNA oligomer formation are discussed.

  8. Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy.

    Science.gov (United States)

    Aitha, Mahesh; Moritz, Lindsay; Sahu, Indra D; Sanyurah, Omar; Roche, Zahilyn; McCarrick, Robert; Lorigan, Gary A; Bennett, Brian; Crowder, Michael W

    2015-04-01

    Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron-electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20-35 Å away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.

  9. Folding Dynamics of an α Helix and a β Hairpin

    Science.gov (United States)

    Hofrichter, James

    1998-03-01

    What processes limit the rate at which proteins fold? In an effort to address this question we have begun to study the dynamics of the formation of loops, α helices and the minimal β structural element, a β hairpin, which must occur on the pathway from random coils to folded proteins. Because these processes occur on time scales of 10-5-10-9 seconds and experimental access to these time scales has been limited, the kinetics of these processes have not been extensively studied. The expectation is that a more complete understanding of the dynamics of these microprocesses will provide constraints on possible mechanisms for the overall folding of more complex structures. We have explored the kinetics of the helix-coil transition of a synthetic, 21-residue peptide: Ac-WAAAH^+(AAARA)_3A-NH2 and of the folding of a 16 residue β hairpin from protein G B1 using the nanosecond temperature jump technique. Both processes were studied by monitoring tryptophan fluorescence. In the helical peptide, the quantum yield of tryptophan decreases as a result of the interaction between tryptophan in position 1 with the protonated histidine in position 5. In the native conformation of the hairpin, it increases because it forms part of a hydrophobic cluster which stabilizes the native conformation (in a peptide in which a dansylated lysine is incorporated at the C-terminus the fluorescence is quenched). At 300 K, the relaxation time for the helix-coil transition is ~ 250 ns and that for the hairpin-coil transition is ~ 2.2 μs, about 10 times slower. The apparent activation energies are 6.8 kcal/mol for the helix and 10 kcal/mol for the hairpin. We have developed simple kinetic models for these processes which incorporate the sequence- and position-dependent properties known from equilibrium studies and the single-sequence approximation. These models provide a remarkably consistent picture of the dynamics, permitting us to extract information on both the microscopic rates for the c

  10. Circular RNAs with hammerhead ribozymes encoded in eukaryotic genomes: The enemy at home.

    Science.gov (United States)

    de la Peña, Marcos; Cervera, Amelia

    2017-04-27

    A new family of non-autonomous retrotransposons with self-cleaving hammerhead ribozymes, the so called retrozymes, has recently been found encoded in diverse plant genomes. These retroelements can be actively transcribed, and their RNAs accumulate in the cells as abundant non-coding circular RNAs (circRNAs) of small size (600-1000 nt). Related circRNAs with self-cleaving ribozymes had already been described in plants, and belong to a group of infectious RNA agents with an uncertain origin: the viroids and viroid-like satellites of plant RNA viruses. These pathogenic circRNAs show many structural similarities with retrozyme circRNAs, and both have been found to occur in flowering plants as heterogeneous RNA molecules of positive and negative polarities. Taking all these data together, we hypothesize that circRNAs encoded by genomic retrozymes could have given origin to infectious circRNAs with self-cleaving ribozymes. Moreover, we propose that retrozymes in time could have evolved from the ancient family of Penelope-like retroelements, which also harbour hammerhead ribozymes. Putative retrozyme sequences with hammerhead ribozymes have been detected as well in metazoan genomes, opening the door to a common occurrence of circRNAs with self-cleaving motifs among eukaryotes.

  11. Efficient inhibition of hepatitis B virus replication by hepatitis delta virus ribozymes delivered by targeting retrovirus

    Directory of Open Access Journals (Sweden)

    Chen Long-Hua

    2010-03-01

    Full Text Available Abstract Background Hepatitis delta virus (HDV ribozyme is an attractive molecular tool that can specifically recognize and catalyze the self-cleavage of the viral RNA phosphodiester backbone. However, a major obstacle in the medical application of the HDV ribozyme is the lack of specificity in the delivery of the ribozyme to defined target cells. Results The objective of this study was to determine whether retroviral vectors can deliver the HDV ribozyme into the target cells and to elucidate whether HDV ribozyme plays a role in hepatitis B virus (HBV replication. In our study, the transduction of helper-free pseudotyped retrovirus, which showed a broad host range, in human hepatoma cells was performed under 2 conditions, that is, in the presence of polymerized human serum albumin (pHSA and in the absence of pHSA. The transduction ability in the presence of pHSA was higher than in the absence of pHSA. Moreover, HBsAg and HBeAg levels after transductions with pHSA were significantly lower than those in the absence of pHSA, thus indicating that the recombinant retrovirus had HBV-specific cleavage activity and targeted HepG2215 cells. Conclusions These data suggest that this system provides a new approach for targeting hepatocytes and has a great potential in gene therapy for HBV infection.

  12. Cell cycle arrest promotes trans-hammerhead ribozyme action in yeast.

    Science.gov (United States)

    Ferbeyre, G; Bratty, J; Chen, H; Cedergren, R

    1996-08-09

    A hammerhead ribozyme designed to cleave the yeast ADE1 mRNA has been expressed in yeast under the control of a galactose-inducible promoter. RNA prepared from the galactose-induced yeast cultures possesses an activity that cleaves ADE1 mRNA in vitro. However, in spite of high expression levels of the ribozyme, no cleavage activity could be demonstrated in vivo. On the other hand, when the yeast cells expressing hammerhead RNA were treated with the alpha-factor mating pheromone, the level of ADE1 mRNA was reduced by 50%. Similar reductions were observed when this strain was cultured in the presence of lithium acetate or in nitrogen-free medium. Moreover, control experiments in which disabled hammerhead genes were expressed showed no such reductions. Extension of the length of the flanking recognition arms of the ribozyme from a total of 10 to 16 or 24 nucleotides diminished the inhibitory effect of the ribozyme. These data suggest that ribozymes are able to cleave a trans-RNA target in yeast.

  13. [Ribozyme riboswitch based gene expression regulation systems for gene therapy applications: progress and challenges].

    Science.gov (United States)

    Feng, Jing-Xian; Wang, Jia-wen; Lin, Jun-sheng; Diao, Yong

    2014-11-01

    Robust and efficient control of therapeutic gene expression is needed for timing and dosing of gene therapy drugs in clinical applications. Ribozyme riboswitch provides a promising building block for ligand-controlled gene-regulatory system, based on its property that exhibits tunable gene regulation, design modularity, and target specificity. Ribozyme riboswitch can be used in various gene delivery vectors. In recent years, there have been breakthroughs in extending ribozyme riboswitch's application from gene-expression control to cellular function and fate control. High throughput screening platforms were established, that allow not only rapid optimization of ribozyme riboswitch in a microbial host, but also straightforward transfer of selected devices exhibiting desired activities to mammalian cell lines in a predictable manner. Mathematical models were employed successfully to explore the performance of ribozyme riboswitch quantitively and its rational design predictably. However, to progress toward gene therapy relevant applications, both precision rational design of regulatory circuits and the biocompatibility of regulatory ligand are still of crucial importance.

  14. The Formation of Packets of Hairpins in Shear Flows

    Science.gov (United States)

    Cohen, Jacob; Karp, Michael; Shukhman, Ilia

    2009-11-01

    In the present work we utilize a recently developed new method in an attempt to understand the generation of packets of hairpin vortices from a pair of counter rotating streamwise vortices embedded in uniform shear flow. This analytical-based solution method is capable of following (numerically) the evolution of finite-amplitude localized vortical disturbances embedded in shear flows. Due to their localization in space, the surrounding base flow is assumed to have homogeneous shear to leading order. The method can solve in a novel way the interaction between a general family of unbounded planar homogeneous shear flows and any localized disturbance. The solution is carried out using Lagrangian variables in Fourier space which is convenient and enables fast computations. The revealed mechanism for generation of packets of hairpins seems to be universal and has been observed in the past both in fully developed wall-bounded shear flows as well as in wall-bounded transitional shear flows.

  15. Retrozymes are a unique family of non-autonomous retrotransposons with hammerhead ribozymes that propagate in plants through circular RNAs.

    Science.gov (United States)

    Cervera, Amelia; Urbina, Denisse; de la Peña, Marcos

    2016-06-23

    Catalytic RNAs, or ribozymes, are regarded as fossils of a prebiotic RNA world that have remained in the genomes of modern organisms. The simplest ribozymes are the small self-cleaving RNAs, like the hammerhead ribozyme, which have been historically considered biological oddities restricted to some RNA pathogens. Recent data, however, indicate that small self-cleaving ribozymes are widespread in genomes, although their functions are still unknown. We reveal that hammerhead ribozyme sequences in plant genomes form part of a new family of small non-autonomous retrotransposons with hammerhead ribozymes, referred to as retrozymes. These elements contain two long terminal repeats of approximately 350 bp, each harbouring a hammerhead ribozyme that delimitates a variable region of 600-1000 bp with no coding capacity. Retrozymes are actively transcribed, which gives rise to heterogeneous linear and circular RNAs that accumulate differentially depending on the tissue or developmental stage of the plant. Genomic and transcriptomic retrozyme sequences are highly heterogeneous and share almost no sequence homology among species except the hammerhead ribozyme motif and two small conserved domains typical of Ty3-gypsy long terminal repeat retrotransposons. Moreover, we detected the presence of RNAs of both retrozyme polarities, which suggests events of independent RNA-RNA rolling-circle replication and evolution, similarly to that of infectious circular RNAs like viroids and viral satellite RNAs. Our work reveals that circular RNAs with hammerhead ribozymes are frequently occurring molecules in plant and, most likely, metazoan transcriptomes, which explains the ubiquity of these genomic ribozymes and suggests a feasible source for the emergence of circular RNA plant pathogens.

  16. β-hairpin-mediated nucleation of polyglutamine amyloid formation

    OpenAIRE

    Kar, Karunakar; Hoop, Cody L.; Drombosky, Kenneth W.; Baker, Matthew A.; Kodali, Ravindra; Arduini, Irene; van der Wel, Patrick C.A.; Horne, W. Seth; Wetzel, Ronald

    2013-01-01

    The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington’s disease (HD). Here we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (trpzip, disulfide, D-Pro-Gly, Coulombic attraction, L-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well...

  17. Spin-modified catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Choudhary, R. [Department of Physics and Astronomy and NCMN, University of Nebraska, Lincoln, Nebraska 68588 (United States); School of Basic Sciences, Indian Institute of Technology Mandi, Mandi 175001, Himachal Pradesh (India); Manchanda, P.; Enders, A.; Balamurugan, B.; Sellmyer, D. J.; Skomski, R., E-mail: rskomski@unl.edu [Department of Physics and Astronomy and NCMN, University of Nebraska, Lincoln, Nebraska 68588 (United States); Kashyap, A. [School of Basic Sciences, Indian Institute of Technology Mandi, Mandi 175001, Himachal Pradesh (India); Sykes, E. C. H. [Department of Chemistry, Pearson Chemistry Laboratory, Tufts University, Medford, Massachusetts 02155 (United States)

    2015-05-07

    First-principle calculations are used to explore the use of magnetic degrees of freedom in catalysis. We use the Vienna Ab-Initio Simulation Package to investigate both L1{sub 0}-ordered FePt and CoPt bulk materials and perform supercell calculations for FePt nanoclusters containing 43 atoms. As the catalytic activity of transition-metal elements and alloys involves individual d levels, magnetic alloying strongly affects the catalytic performance, because it leads to shifts in the local densities of states and to additional peaks due to magnetic-moment formation. The peak shift persists in nanoparticles but is surface-site specific and therefore depends on cluster size. Our research indicates that small modifications in stoichiometry and cluster size are a useful tool in the search for new catalysts.

  18. Simulations of chemical catalysis

    Science.gov (United States)

    Smith, Gregory K.

    This dissertation contains simulations of chemical catalysis in both biological and heterogeneous contexts. A mixture of classical, quantum, and hybrid techniques are applied to explore the energy profiles and compare possible chemical mechanisms both within the context of human and bacterial enzymes, as well as exploring surface reactions on a metal catalyst. A brief summary of each project follows. Project 1 - Bacterial Enzyme SpvC The newly discovered SpvC effector protein from Salmonella typhimurium interferes with the host immune response by dephosphorylating mitogen-activated protein kinases (MAPKs) with a beta-elimination mechanism. The dynamics of the enzyme substrate complex of the SpvC effector is investigated with a 3.2 ns molecular dynamics simulation, which reveals that the phosphorylated peptide substrate is tightly held in the active site by a hydrogen bond network and the lysine general base is positioned for the abstraction of the alpha hydrogen. The catalysis is further modeled with density functional theory (DFT) in a truncated active-site model at the B3LYP/6-31 G(d,p) level of theory. The truncated model suggested the reaction proceeds via a single transition state. After including the enzyme environment in ab initio QM/MM studies, it was found to proceed via an E1cB-like pathway, in which the carbanion intermediate is stabilized by an enzyme oxyanion hole provided by Lys104 and Tyr158 of SpvC. Project 2 - Human Enzyme CDK2 Phosphorylation reactions catalyzed by kinases and phosphatases play an indispensable role in cellular signaling, and their malfunctioning is implicated in many diseases. Ab initio quantum mechanical/molecular mechanical studies are reported for the phosphoryl transfer reaction catalyzed by a cyclin-dependent kinase, CDK2. Our results suggest that an active-site Asp residue, rather than ATP as previously proposed, serves as the general base to activate the Ser nucleophile. The corresponding transition state features a

  19. Asymmetric trienamine catalysis: new opportunities in amine catalysis.

    Science.gov (United States)

    Kumar, Indresh; Ramaraju, Panduga; Mir, Nisar A

    2013-02-07

    Amine catalysis, through HOMO-activating enamine and LUMO-activating iminium-ion formation, is receiving increasing attention among other organocatalytic strategies, for the activation of unmodified carbonyl compounds. Particularly, the HOMO-raising activation concept has been applied to the greatest number of asymmetric transformations through enamine, dienamine, and SOMO-activation strategies. Recently, trienamine catalysis, an extension of amine catalysis, has emerged as a powerful tool for synthetic chemists with a novel activation strategy for polyenals/polyenones. In this review article, we discuss the initial developments of trienamine catalysis for highly asymmetric Diels-Alder reactions with different dienophiles and emerging opportunities for other types of cycloadditions and cascade reactions.

  20. Traveling hairpin-shaped fluid vortices in plane Couette flow.

    Science.gov (United States)

    Deguchi, K; Nagata, M

    2010-11-01

    Traveling-wave solutions are discovered in plane Couette flow. They are obtained when the so-called steady hairpin vortex state found recently by Gibson [J. Fluid Mech. 638, 243 (2009)] and Itano and Generalis [Phys. Rev. Lett. 102, 114501 (2009)] is continued to sliding Couette flow geometry between two concentric cylinders by using the radius ratio as a homotopy parameter. It turns out that in the plane Couette flow geometry two traveling waves having the phase velocities with opposite signs are associated with their appearance from the steady hairpin vortex state, where the amplitude of the phase velocities increases gradually from zero as the Reynolds number is increased. The solutions obviously inherit the streaky structure of the hairpin vortex state, but shape preserving flow patterns propagate in the streamwise direction. Other striking features of the solution are asymmetric mean flow profiles and strong quasistreamwise vortices which occupy the vicinity of only the top or bottom moving boundary, depending on the sign of the phase velocity. Furthermore, we find that the pitchfork bifurcation associated with the appearance of the solution becomes imperfect when the flow is perturbed by a Poiseuille flow component.

  1. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing.

    Science.gov (United States)

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-09-01

    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

  2. Inhibiting Apoptosis of CTLL-2 Cells to Enhance Their GVL Effects via Anti-Fas Ribozyme

    Institute of Scientific and Technical Information of China (English)

    Min ZHANG; Fang LIU; Lin-Bo LIU; Yong YOU; Zhi-Chao CHEN; Ping ZOU

    2004-01-01

    To investigate the inhibition role of anti-Fas hammerhead ribozyme on fas expression and Fas-mediated apoptosis of CTL cell line CTLL-2 cells,the cDNA of an anti-Fas hammerhead ribozyme was synthesized,its expression plasmid was constructed and transfected into CTLL-2 cells by electroporation.fas expression of CTLL-2 cells was detected by RT-PCR and Western blot.CTLL-2 cell viability was measured using MTT assay when co-cultured with mouse T cell leukemia cell line EL4 cells that highly expressed Fas ligand(FasL).Meanwhile,caspase-3 proteolytic activity was detected,and cell apoptosis was measured by flow cytometry and Hochest-PI double staining.Killing activity of CTLL-2 cells was detected by lactate dehydrogenase(LDH)releasing assay in vitro.Results showed that the expression of both Fas mRNA and protein in CTLL-2 cells were decreased after transfection of anti-Fas ribozyme.Compared with mocktransfected group and mutant ribozyme-transfected group,viability of CTLL-2 cells co-cultured with EL4 cells was increased significantly and cells killing activity was enhanced after transfected with anti-Fas ribozyme,while the caspase-3 activity and apoptosis rate was significantly decreased.The results demonstrated anti-Fas ribozyme could efficiently cleave Fas and inhibit Fas-mediated apoptosis of CTLL-2 cells to improve their viability.Our study made a basis for enhancing CTLL-2 cells anti-leukemia effect in DLI.

  3. DNA-based hybrid catalysis.

    Science.gov (United States)

    Rioz-Martínez, Ana; Roelfes, Gerard

    2015-04-01

    In the past decade, DNA-based hybrid catalysis has merged as a promising novel approach to homogeneous (asymmetric) catalysis. A DNA hybrid catalysts comprises a transition metal complex that is covalently or supramolecularly bound to DNA. The chiral microenvironment and the second coordination sphere interactions provided by the DNA are key to achieve high enantioselectivities and, often, additional rate accelerations in catalysis. Nowadays, current efforts are focused on improved designs, understanding the origin of the enantioselectivity and DNA-induced rate accelerations, expanding the catalytic scope of the concept and further increasing the practicality of the method for applications in synthesis. Herein, the recent developments will be reviewed and the perspectives for the emerging field of DNA-based hybrid catalysis will be discussed.

  4. A Survey Course in Catalysis.

    Science.gov (United States)

    Skaates, J. M.

    1982-01-01

    Describes a 10-week survey course in catalysis for chemical engineering and chemistry students designed to show how modern chemistry and chemical engineering interact in the ongoing development of industrial catalysts. Includes course outline and instructional strategies. (Author/JN)

  5. Asymmetric cation-binding catalysis

    DEFF Research Database (Denmark)

    Oliveira, Maria Teresa; Lee, Jiwoong

    2017-01-01

    and KCN, are selectively bound to the catalyst, providing exceptionally high enantioselectivities for kinetic resolutions, elimination reactions (fluoride base), and Strecker synthesis (cyanide nucleophile). Asymmetric cation-binding catalysis was recently expanded to silicon-based reagents, enabling...... solvents, thus increasing their applicability in synthesis. The expansion of this concept to chiral polyethers led to the emergence of asymmetric cation-binding catalysis, where chiral counter anions are generated from metal salts, particularly using BINOL-based polyethers. Alkali metal salts, namely KF...

  6. Editorial: Nanoscience makes catalysis greener

    KAUST Repository

    Polshettiwar, Vivek

    2012-01-09

    Green chemistry by nanocatalysis: Catalysis is a strategic field of science because it involves new ways of meeting energy and sustainability challenges. The concept of green chemistry, which makes the science of catalysis even more creative, has become an integral part of sustainability. This special issue is at the interface of green chemistry and nanocatalysis, and features excellent background articles as well as the latest research results. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Entropy and Enzyme Catalysis.

    Science.gov (United States)

    Åqvist, Johan; Kazemi, Masoud; Isaksen, Geir Villy; Brandsdal, Bjørn Olav

    2017-02-21

    The role played by entropy for the enormous rate enhancement achieved by enzymes has been debated for many decades. There are, for example, several confirmed cases where the activation free energy is reduced by around 10 kcal/mol due to entropic effects, corresponding to a rate enhancement of ∼10(7) compared to the uncatalyzed reaction. However, despite substantial efforts from both the experimental and theoretical side, no real consensus has been reached regarding the origin of such large entropic contributions to enzyme catalysis. Another remarkable instance of entropic effects is found in enzymes that are adapted by evolution to work at low temperatures, near the freezing point of water. These cold-adapted enzymes invariably show a more negative entropy and a lower enthalpy of activation than their mesophilic orthologs, which counteracts the exponential damping of reaction rates at lower temperature. The structural origin of this universal phenomenon has, however, remained elusive. The basic problem with connecting macroscopic thermodynamic quantities, such as activation entropy and enthalpy derived from Arrhenius plots, to the 3D protein structure is that the underlying detailed (microscopic) energetics is essentially inaccessible to experiment. Moreover, attempts to calculate entropy contributions by computer simulations have mostly focused only on substrate entropies, which do not provide the full picture. We have recently devised a new approach for accessing thermodynamic activation parameters of both enzyme and solution reactions from computer simulations, which turns out to be very successful. This method is analogous to the experimental Arrhenius plots and directly evaluates the temperature dependence of calculated reaction free energy profiles. Hence, by extensive molecular dynamics simulations and calculations of up to thousands of independent free energy profiles, we are able to extract activation parameters with sufficient precision for making

  8. Enhanced Micellar Catalysis LDRD.

    Energy Technology Data Exchange (ETDEWEB)

    Betty, Rita G.; Tucker, Mark D; Taggart, Gretchen; Kinnan, Mark K.; Glen, Crystal Chanea; Rivera, Danielle; Sanchez, Andres; Alam, Todd Michael

    2012-12-01

    The primary goals of the Enhanced Micellar Catalysis project were to gain an understanding of the micellar environment of DF-200, or similar liquid CBW surfactant-based decontaminants, as well as characterize the aerosolized DF-200 droplet distribution and droplet chemistry under baseline ITW rotary atomization conditions. Micellar characterization of limited surfactant solutions was performed externally through the collection and measurement of Small Angle X-Ray Scattering (SAXS) images and Cryo-Transmission Electron Microscopy (cryo-TEM) images. Micellar characterization was performed externally at the University of Minnesotas Characterization Facility Center, and at the Argonne National Laboratory Advanced Photon Source facility. A micellar diffusion study was conducted internally at Sandia to measure diffusion constants of surfactants over a concentration range, to estimate the effective micelle diameter, to determine the impact of individual components to the micellar environment in solution, and the impact of combined components to surfactant phase behavior. Aerosolized DF-200 sprays were characterized for particle size and distribution and limited chemical composition. Evaporation rates of aerosolized DF-200 sprays were estimated under a set of baseline ITW nozzle test system parameters.

  9. Intracellular Immunization of Human Fetal Cord Blood Stem/Progenitor Cells with a Ribozyme Against Human Immunodeficiency Virus Type 1

    Science.gov (United States)

    Yu, Mang; Leavitt, Mark C.; Maruyama, Midori; Yamada, Osamu; Young, Dennis; Ho, Anthony D.; Wong-Staal, Flossie

    1995-01-01

    Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34^+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.

  10. The glmS ribozyme cofactor is a general acid-base catalyst.

    Science.gov (United States)

    Viladoms, Júlia; Fedor, Martha J

    2012-11-21

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.

  11. An efficient ligation reaction promoted by a Varkud Satellite ribozyme with extended 5'- and 3'-termini.

    Science.gov (United States)

    Jones, F D; Ryder, S P; Strobel, S A

    2001-12-15

    The Neurospora Varkud Satellite (VS) RNA is capable of promoting a reversible self-cleavage reaction important for its replication pathway. In vivo the VS RNA performs a cis-cleavage reaction to generate monomeric length transcripts that are subsequently ligated to produce circular VS RNA. The predominant form of VS RNA observed in vivo is the closed circular form, though minimal VS ribozyme self-cleavage constructs lack detectable ligation activity. MFOLD analysis of the entire VS RNA sequence revealed an extended region 5' and 3' of the minimal self-cleaving region that could anneal to form a complementary helix, which we have termed helix 7. In full-length VS RNA, this helix appears to span over 40 bp of sequence and brings the 5'- and 3'-ends of the RNA into proximity for the ligation reaction. Here we report a variant of the VS ribozyme with an extended 5'- and 3'-terminus capable of forming a truncated helix 7 that promotes the ligation reaction in vitro. Through mutation and selection of this RNA we have identified a ribozyme containing two point mutations in the truncated helix 7 that ligates with >70% efficiency. These results show that an additional helical element absent in current VS ribozyme constructs is likely to be important for the ligation activity of VS RNA.

  12. Reversal of HCC Drug Resistance by Using Hammerhead Ribozymes against Multidrug Resistance 1 Gene

    Institute of Scientific and Technical Information of China (English)

    QIAO Sen; WANG Hai; CHEN Xiaoping

    2005-01-01

    To reverse multidrug resistance(MDR) of HepG2 by anti-MDR1 hammerhead ribozyme,an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase Ⅲ promoter.The expression of mdr1/Pgp and Rz was detected in HepG2, HepG2 multidrug-resistant cell line and HepG2 Rz-transfected cells by semi quantitative RT-PCR and Western blot methods. Moreover, MTT assay was employed to detect the sensitivity of these ribozyme-transfected cells, and Rhodamine123 (Rh123) was used to test the function of Pgp. The Rz- transfected HepG2 cells became doxorubicin-sensitive, which was concomitant with the decreased MDR1 expression. The study showed that the retrovirus vector encoding the anti-MDR1 ribozyme may be applicable to the treatment of MDR cells.

  13. Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

    Science.gov (United States)

    Chen, H; Ferbeyre, G; Cedergren, R

    1997-05-01

    We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

  14. NMR-spectroscopic characterization of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme.

    Science.gov (United States)

    Fürtig, Boris; Richter, Christian; Schell, Peter; Wenter, Philipp; Pitsch, Stefan; Schwalbe, Harald

    2008-01-01

    In order to relate the conformational dynamics of the hammerhead ribozyme to its biological function the cleavage reaction catalyzed by the hammerhead ribozyme was monitored by time-resolved nuclear magnetic resonance (NMR) spectroscopy. For this purpose, the two nucleosides around the scissile phosphodiester bond were selectively (13)C labelled in multi-step organic syntheses starting from uniformly (13)C-labelled glucose. The phosphoamidites were incorporated using phosphoamidite chemistry in the hammerhead substrate strand. In addition, the 2'-OH group on the 5'-side of the hammerhead substrate strand was labelled with a photolabile protecting group. This labelling strategy enabled a detailed characterisation of the nucleotides around the scissile phosphodiester bond in the ground state conformation of the hammerhead ribozyme in the absence and presence of Mg(2+) ions as well as of the product state. Photochemical induction of the reaction in situ was further characterized by time-resolved NMR spectroscopy. The detailed structural and dynamic investigations revealed that the conformation of the hammerhead ribozyme is significantly affected by addition of Mg(2+) leading to an ensemble of conformations where dynamic transitions between energetically similar conformations occur on the ms-timescale in the presence of Mg(2+). The dynamic transitions are localized around the catalytic core. Cleavage from this ensemble cannot be described by mono-exponential kinetics but follows bi-exponential kinetics. A model is described to take into account these experimental data.

  15. Kinetic Monte Carlo method applied to nucleic acid hairpin folding.

    Science.gov (United States)

    Sauerwine, Ben; Widom, Michael

    2011-12-01

    Kinetic Monte Carlo on coarse-grained systems, such as nucleic acid secondary structure, is advantageous for being able to access behavior at long time scales, even minutes or hours. Transition rates between coarse-grained states depend upon intermediate barriers, which are not directly simulated. We propose an Arrhenius rate model and an intermediate energy model that incorporates the effects of the barrier between simulated states without enlarging the state space itself. Applying our Arrhenius rate model to DNA hairpin folding, we demonstrate improved agreement with experiment compared to the usual kinetic Monte Carlo model. Further improvement results from including rigidity of single-stranded stacking.

  16. Identify Beta-Hairpin Motifs with Quadratic Discriminant Algorithm Based on the Chemical Shifts.

    Science.gov (United States)

    YongE, Feng; GaoShan, Kou

    2015-01-01

    Successful prediction of the beta-hairpin motif will be helpful for understanding the of the fold recognition. Some algorithms have been proposed for the prediction of beta-hairpin motifs. However, the parameters used by these methods were primarily based on the amino acid sequences. Here, we proposed a novel model for predicting beta-hairpin structure based on the chemical shift. Firstly, we analyzed the statistical distribution of chemical shifts of six nuclei in not beta-hairpin and beta-hairpin motifs. Secondly, we used these chemical shifts as features combined with three algorithms to predict beta-hairpin structure. Finally, we achieved the best prediction, namely sensitivity of 92%, the specificity of 94% with 0.85 of Mathew's correlation coefficient using quadratic discriminant analysis algorithm, which is clearly superior to the same method for the prediction of beta-hairpin structure from 20 amino acid compositions in the three-fold cross-validation. Our finding showed that the chemical shift is an effective parameter for beta-hairpin prediction, suggesting the quadratic discriminant analysis is a powerful algorithm for the prediction of beta-hairpin.

  17. Catalysis of Protein Disulfide Bond Isomerization in a Homogeneous Substrate†

    Science.gov (United States)

    Kersteen, Elizabeth A.; Barrows, Seth R.; Raines, Ronald T.

    2008-01-01

    Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a _-hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N-and C-terminus contain a fluorescence donor (tryptophan) and acceptor (N_-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E°_ = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys—Gly—His—Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/KM = 1.7 _ 105 M–1M s–1, which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that

  18. Catalysis of protein disulfide bond isomerization in a homogeneous substrate.

    Science.gov (United States)

    Kersteen, Elizabeth A; Barrows, Seth R; Raines, Ronald T

    2005-09-13

    Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a beta hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N and C terminus contain a fluorescence donor (tryptophan) and acceptor (N(epsilon)-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E(o') = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys-Gly-His-Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/K(M) = 1.7 x 10(5) M(-1) s(-1), which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude

  19. The discodermolide hairpin structure flows from conformationally stable modular motifs.

    Science.gov (United States)

    Jogalekar, Ashutosh S; Kriel, Frederik H; Shi, Qi; Cornett, Ben; Cicero, Daniel; Snyder, James P

    2010-01-14

    (+)-Discodermolide (DDM), a polyketide macrolide from marine sponge, is a potent microtubule assembly promoter. Reported solid-state, solution, and protein-bound DDM conformations reveal the unusual result that a common hairpin conformational motif exists in all three microenvironments. No other flexible microtubule binding agent exhibits such constancy of conformation. In the present study, we combine force-field conformational searches with NMR deconvolution in different solvents to compare DDM conformers with those observed in other environments. While several conformational families are perceived, the hairpin form dominates. The stability of this motif is dictated primarily by steric factors arising from repeated modular segments in DDM composed of the C(Me)-CHX-C(Me) fragment. Furthermore, docking protocols were utilized to probe the DDM binding mode in beta-tubulin. A previously suggested pose is substantiated (Pose-1), while an alternative (Pose-2) has been identified. SAR analysis for DDM analogues differentiates the two poses and suggests that Pose-2 is better able to accommodate the biodata.

  20. Nonlinear effects in asymmetric catalysis.

    Science.gov (United States)

    Satyanarayana, Tummanapalli; Abraham, Susan; Kagan, Henri B

    2009-01-01

    There is a need for the preparation of enantiomerically pure compounds for various applications. An efficient approach to achieve this goal is asymmetric catalysis. The chiral catalyst is usually prepared from a chiral auxiliary, which itself is derived from a natural product or by resolution of a racemic precursor. The use of non-enantiopure chiral auxiliaries in asymmetric catalysis seems unattractive to preparative chemists, since the anticipated enantiomeric excess (ee) of the reaction product should be proportional to the ee value of the chiral auxiliary (linearity). In fact, some deviation from linearity may arise. Such nonlinear effects can be rich in mechanistic information and can be synthetically useful (asymmetric amplification). This Review documents the advances made during the last decade in the use of nonlinear effects in the area of organometallic and organic catalysis.

  1. Operando research in heterogeneous catalysis

    CERN Document Server

    Groot, Irene

    2017-01-01

    This book is devoted to the emerging field of techniques for visualizing atomic-scale properties of active catalysts under actual working conditions, i.e. high gas pressures and high temperatures. It explains how to understand these observations in terms of the surface structures and dynamics and their detailed interplay with the gas phase. This provides an important new link between fundamental surface physics and chemistry, and applied catalysis. The book explains the motivation and the necessity of operando studies, and positions these with respect to the more traditional low-pressure investigations on the one hand and the reality of industrial catalysis on the other. The last decade has witnessed a rapid development of new experimental and theoretical tools for operando studies of heterogeneous catalysis. The book has a strong emphasis on the new techniques and illustrates how the challenges introduced by the harsh, operando conditions are faced for each of these new tools. Therefore, one can also read th...

  2. The Role of XPG in Processing (CAGn/(CTGn DNA Hairpins

    Directory of Open Access Journals (Sweden)

    Hou Caixia

    2011-03-01

    Full Text Available Abstract Background During DNA replication or repair, disease-associated (CAGn/(CTGn expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Recent studies identified a nick-directed (CAGn/(CTGn hairpin repair (HPR system that removes (CAGn/(CTGn hairpins from human cells via endonucleolytic incisions. Because the process is highly similar to the mechanism by which XPG and XPF endonucleases remove bulky DNA lesions during nucleotide excision repair, we assessed the potential role of XPG in conducting (CAGn/(CTGn HPR. Results To determine if the XPG endonuclease is involved in (CAGn/(CTGn hairpin removal, two XPG-deficient cell lines (GM16024 and AG08802 were examined for their ability to process (CAGn/(CTGn hairpins in vitro. We demonstrated that the GM16024 cell line processes all hairpin substrates as efficiently as HeLa cells, and that the AG08802 cell line is partially defective in HPR. Analysis of repair intermediates revealed that nuclear extracts from both XPG-deficient lines remove CAG/CTG hairpins via incisions, but the incision products are distinct from those generated in HeLa extracts. We also show that purified recombinant XPG protein greatly stimulates HPR in XPG-deficient extracts by promoting an incision 5' to the hairpin. Conclusions Our results strongly suggest that 1 human cells possess multiple pathways to remove (CAGn/(CTGn hairpins located in newly synthesized (or nicked DNA strand; and 2 XPG, although not essential for (CAGn/(CTGn hairpin removal, stimulates HPR by facilitating a 5' incision to the hairpin. This study reveals a novel role for XPG in genome-maintenance and implicates XPG in diseases caused by trinucleotide repeat expansion.

  3. Photoredox Catalysis in Organic Chemistry

    Science.gov (United States)

    2016-01-01

    In recent years, photoredox catalysis has come to the forefront in organic chemistry as a powerful strategy for the activation of small molecules. In a general sense, these approaches rely on the ability of metal complexes and organic dyes to convert visible light into chemical energy by engaging in single-electron transfer with organic substrates, thereby generating reactive intermediates. In this Perspective, we highlight the unique ability of photoredox catalysis to expedite the development of completely new reaction mechanisms, with particular emphasis placed on multicatalytic strategies that enable the construction of challenging carbon–carbon and carbon–heteroatom bonds. PMID:27477076

  4. Photoredox Catalysis in Organic Chemistry.

    Science.gov (United States)

    Shaw, Megan H; Twilton, Jack; MacMillan, David W C

    2016-08-19

    In recent years, photoredox catalysis has come to the forefront in organic chemistry as a powerful strategy for the activation of small molecules. In a general sense, these approaches rely on the ability of metal complexes and organic dyes to convert visible light into chemical energy by engaging in single-electron transfer with organic substrates, thereby generating reactive intermediates. In this Perspective, we highlight the unique ability of photoredox catalysis to expedite the development of completely new reaction mechanisms, with particular emphasis placed on multicatalytic strategies that enable the construction of challenging carbon-carbon and carbon-heteroatom bonds.

  5. Catalysis and sustainable (green) chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Centi, Gabriele; Perathoner, Siglinda [Dipartimento di Chimica Industriale ed Ingegneria dei Materiali, University of Messina, Salita Sperone 31, 98166 Messina (Italy)

    2003-01-15

    Catalysis is a key technology to achieve the objectives of sustainable (green) chemistry. After introducing the concepts of sustainable (green) chemistry and a brief assessment of new sustainable chemical technologies, the relationship between catalysis and sustainable (green) chemistry is discussed and illustrated via an analysis of some selected and relevant examples. Emphasis is also given to the concept of catalytic technologies for scaling-down chemical processes, in order to develop sustainable production processes which reduce the impact on the environment to an acceptable level that allows self-depuration processes of the living environment.

  6. EMSL and Institute for Integrated Catalysis (IIC) Catalysis Workshop

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Charles T.; Datye, Abhaya K.; Henkelman, Graeme A.; Lobo, Raul F.; Schneider, William F.; Spicer, Leonard D.; Tysoe, Wilfred T.; Vohs, John M.; Baer, Donald R.; Hoyt, David W.; Thevuthasan, Suntharampillai; Mueller, Karl T.; Wang, Chong M.; Washton, Nancy M.; Lyubinetsky, Igor; Teller, Raymond G.; Andersen, Amity; Govind, Niranjan; Kowalski, Karol; Kabius, Bernd C.; Wang, Hongfei; Campbell, Allison A.; Shelton, William A.; Bylaska, Eric J.; Peden, Charles HF; Wang, Yong; King, David L.; Henderson, Michael A.; Rousseau, Roger J.; Szanyi, Janos; Dohnalek, Zdenek; Mei, Donghai; Garrett, Bruce C.; Ray, Douglas; Futrell, Jean H.; Laskin, Julia; DuBois, Daniel L.; Kuprat, Laura R.; Plata, Charity

    2011-05-24

    Within the context of significantly accelerating scientific progress in research areas that address important societal problems, a workshop was held in November 2010 at EMSL to identify specific and topically important areas of research and capability needs in catalysis-related science.

  7. EMSL and Institute for Integrated Catalysis (IIC) Catalysis Workshop

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Charles T.; Datye, Abhaya K.; Henkelman, Graeme A.; Lobo, Raul F.; Schneider, William F.; Spicer, Leonard D.; Tysoe, Wilfred T.; Vohs, John M.; Baer, Donald R.; Hoyt, David W.; Thevuthasan, Suntharampillai; Mueller, Karl T.; Wang, Chong M.; Washton, Nancy M.; Lyubinetsky, Igor; Teller, Raymond G.; Andersen, Amity; Govind, Niranjan; Kowalski, Karol; Kabius, Bernd C.; Wang, Hongfei; Campbell, Allison A.; Shelton, William A.; Bylaska, Eric J.; Peden, Charles HF; Wang, Yong; King, David L.; Henderson, Michael A.; Rousseau, Roger J.; Szanyi, Janos; Dohnalek, Zdenek; Mei, Donghai; Garrett, Bruce C.; Ray, Douglas; Futrell, Jean H.; Laskin, Julia; DuBois, Daniel L.; Kuprat, Laura R.; Plata, Charity

    2011-05-24

    Within the context of significantly accelerating scientific progress in research areas that address important societal problems, a workshop was held in November 2010 at EMSL to identify specific and topically important areas of research and capability needs in catalysis-related science.

  8. The 5'-proximal hairpin of turnip yellow mosaic virus RNA: its role in translation and encapsidation.

    Science.gov (United States)

    Bink, Hugo H J; Schirawski, Jan; Haenni, Anne-Lise; Pleij, Cornelis W A

    2003-07-01

    The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5' untranslated region, it was observed that stabilization of the 5'-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5'-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5' cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis.

  9. Additive Effects on Asymmetric Catalysis.

    Science.gov (United States)

    Hong, Liang; Sun, Wangsheng; Yang, Dongxu; Li, Guofeng; Wang, Rui

    2016-03-23

    This review highlights a number of additives that can be used to make asymmetric reactions perfect. Without changing other reaction conditions, simply adding additives can lead to improved asymmetric catalysis, such as reduced reaction time, improved yield, or/and increased selectivity.

  10. Binding Energy and Enzymatic Catalysis.

    Science.gov (United States)

    Hansen, David E.; Raines, Ronald T.

    1990-01-01

    Discussed is the fundamental role that the favorable free energy of binding of the rate-determining transition state plays in catalysis. The principle that all of the catalytic factors discussed are realized by the use of this binding energy is reviewed. (CW)

  11. Construct Hairpin RNA to Fight Against Rice Dwarf Virus

    Institute of Scientific and Technical Information of China (English)

    MAZhong-Liang; YANGHuai-Yi; WANGRong; TIENPo

    2004-01-01

    Hairpin RNA (hpRNA) can induce post-transcriptional gene silencing, and was constructed with128-754 bp of segment 8 of rice dwarf virus then placed under the control of the CaMV35S promoter andused to transform rice cultivar "Zhonghua 11" via Agrobacterium tumefaciens. A total of 12 independentlines containing hpRNA were obtained as demonstrated by Southern blotting analysis. Challenge inocula-tion with rice dwarf virus (RDV) viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plantscontaining the hpRNA transgene displayed high resistance or delayed and attenuated viral symptoms. Incontrast, transgenic lines expressing sense RNA showed severe symptoms similar to control plants trans-formed with the vector alone. These results suggest that hpRNA confers high resistance to RDV intransgenic rice.

  12. Short hairpin-loop-structured oligodeoxynucleotides reduce HSV-1 replication

    Directory of Open Access Journals (Sweden)

    Heinrich Jochen

    2009-04-01

    Full Text Available Abstract The Herpes simplex virus (HSV is known as an infectious agent and widespread in the human population. The symptoms of HSV infections can range from mild to life threatening, especially in immune-compromised individuals. HSV infections are commonly treated with the guanosine analogue Aciclovir, but reports of resistance are increasing. Efforts are made to establish single-stranded antisense oligodeoxynucleotides (as and small interfering ribonucleic acids (siRNAs for antiviral treatment. Recently, another class of short interfering nucleic acids, partially double-stranded hairpin loop-structured 54 mer oligodeoxynucleotides (ODNs, was shown to allow hydrolysis of HIV RNA by binding to the viral RNA. This leads to a substrate for the viral RNase H. To assess the potential of such ODNs for inhibition of HSV-1 replication, five partially double-stranded ODNs were designed based on the sequences of known siRNAs against HSV-1 with antiviral activity. Three of them are directed against early and two against leaky late genes. Primary human lung fibroblasts, MRC-5, and African green monkey kidney cells, Vero, were transfected with ODNs and subsequently infected. The effect on HSV-1 replication was determined by analyzing the virus titer in cell culture supernatants by quantitative PCR and plaque assays. An inhibitory effect was observed with all five selected ODNs, with two cases showing statistical significance in both cell types. The observed effect was sequence-specific and dose dependent. In one case the ODN was more efficient than a previously described siRNA directed against the same target site in the mRNA of UL5, a component of the helicase/primase complex. HSV-1 virions and ODNs can be applied simultaneously without transfection reagent, but at a 50-fold higher concentration to Vero cells with similar efficiencies. The results underline the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.

  13. Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro

    Institute of Scientific and Technical Information of China (English)

    Shan Jiang; Qing Xie; Wei Zhang; Xia-Qiu Zhou; You-Xin Jin

    2005-01-01

    AIM: To prepare and identify specific anti-mouse caspase12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis.METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software,and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37 ℃ in reaction buffer (40 mmol/L Tris-HCL,pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea).RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37 ℃,pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%,for Rz218 the value was 36.66%.CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis.

  14. Signal amplification of glucosamine-6-phosphate based on ribozyme glmS.

    Science.gov (United States)

    Zhao, Yongyun; Chen, Haodong; Du, Feng; Yasmeen, Afshan; Dong, Juan; Cui, Xin; Tang, Zhuo

    2014-12-15

    Ribozyme glmS based isothermal amplification assay is developed for the colorimetric detection of glucosamine-6-phosphate (GlcN6P). Upon binding to the metabolite target GlcN6P, self-cleavage of glmS ribozyme is initiated to release RNA fragment that can trigger the cascade signal amplification to release large amount of G-quadruplex DNAzymes as reporter for colorimetric detection. Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. This assay not only offers a convenient detection of GlcN6P with high specificity and sensitivity, but also provides a platform for high-throughput screening of antibiotics based on glmS riboswitches.

  15. Molecular catalysis science: Perspective on unifying the fields of catalysis.

    Science.gov (United States)

    Ye, Rong; Hurlburt, Tyler J; Sabyrov, Kairat; Alayoglu, Selim; Somorjai, Gabor A

    2016-05-10

    Colloidal chemistry is used to control the size, shape, morphology, and composition of metal nanoparticles. Model catalysts as such are applied to catalytic transformations in the three types of catalysts: heterogeneous, homogeneous, and enzymatic. Real-time dynamics of oxidation state, coordination, and bonding of nanoparticle catalysts are put under the microscope using surface techniques such as sum-frequency generation vibrational spectroscopy and ambient pressure X-ray photoelectron spectroscopy under catalytically relevant conditions. It was demonstrated that catalytic behavior and trends are strongly tied to oxidation state, the coordination number and crystallographic orientation of metal sites, and bonding and orientation of surface adsorbates. It was also found that catalytic performance can be tuned by carefully designing and fabricating catalysts from the bottom up. Homogeneous and heterogeneous catalysts, and likely enzymes, behave similarly at the molecular level. Unifying the fields of catalysis is the key to achieving the goal of 100% selectivity in catalysis.

  16. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

    OpenAIRE

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-01-01

    Engineered site-specific RNA cleavage is widely used for gene regulation, RNA mapping, and synthetic RNA production. Here the authors extend the range of engineered recognition selectivity to include cleavage of sequence motifs containing naturally occurring base modifications. They describe and implement a designer hammerhead ribozyme that cleaves a target sequence 1 nt from a site of adenosine to inosine (A-to-I) or cytosine to uracil (C-to-U) editing in synthetic or physiological mRNA cont...

  17. Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme.

    Science.gov (United States)

    Krasovska, Maryna V; Sefcikova, Jana; Réblová, Kamila; Schneider, Bohdan; Walter, Nils G; Sponer, Jirí

    2006-07-15

    The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.

  18. Magnesium-Dependent Active-Site Conformational Selection in the Diels-Alderase Ribozyme

    Energy Technology Data Exchange (ETDEWEB)

    Berezniak, Tomasz [University of Heidelberg; Zahran, Mai [ORNL; Imhof, Petra [University of Heidelberg; Jaeschke, Andres [Free University of Berlin; Smith, Jeremy C [ORNL

    2010-10-01

    The Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme, accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition, a reaction with broad application in organic chemistry. Here, the Diels-Alderase ribozyme is examined via molecular dynamics (MD) simulations in both crystalline and aqueous solution environments. The simulations indicate that the catalytic pocket is highly dynamic. At low Mg(2+) ion concentrations, inactive states with the catalytic pocket closed dominate. Stabilization of the enzymatically active, open state of the catalytic pocket requires a high concentration of Mg(2+) ions (e.g., 54 mM), with cations binding to specific phosphate sites on the backbone of the residues bridging the opposite strands of the pocket. The free energy profile for pocket opening at high Mg(2+) cation concentration exhibits a double minimum, with a barrier to opening of approximately 5.5 kJ/mol and the closed state approximately 3 kJ/mol lower than the open state. Selection of the open state on substrate binding leads to the catalytic activity of the ribozyme. The simulation results explain structurally the experimental observation that full catalytic activity depends on the Mg(2+) ion concentration

  19. Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

    Institute of Scientific and Technical Information of China (English)

    LIU; Bailin(刘柏林); QU; Yi(屈艺); LIU; Shuqiu(刘菽秋); OUYANG; Xuesong(欧阳雪松)

    2002-01-01

    Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme(telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase(hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings(PDS). After 19-20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ effectively inhibited the telomerase activity in transplanted tumor, promoted apoptosis of the transplanted tumor cells, and decreased the tumor size significantly. These results indicate that teloRZ can effectively inhibit telomerase activity and growth of tumor cells, and suggest the potential use of this ribozyme in anti-cancer therapy.

  20. Spiegelzymes: sequence specific hydrolysis of L-RNA with mirror image hammerhead ribozymes and DNAzymes.

    Directory of Open Access Journals (Sweden)

    Eliza Wyszko

    Full Text Available In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes, which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers, which we had previously developed and for which it has been shown that they are non-toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.

  1. Structural Investigation of the GlmS Ribozyme Bound to Its Catalytic Cofactor

    Energy Technology Data Exchange (ETDEWEB)

    Cochrane,J.; Lipchock, S.; Strobel, S.

    2007-01-01

    The GlmS riboswitch is located in the 5'-untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase. The GlmS riboswitch is a ribozyme with activity triggered by binding of the metabolite GlcN6P. Presented here is the structure of the GlmS ribozyme (2.5 {angstrom} resolution) with GlcN6P bound in the active site. The GlmS ribozyme adopts a compact double pseudoknot tertiary structure, with two closely packed helical stacks. Recognition of GlcN6P is achieved through coordination of the phosphate moiety by two hydrated magnesium ions as well as specific nucleobase contacts to the GlcN6P sugar ring. Comparison of this activator bound and the previously published apoenzyme complex supports a model in which GlcN6P does not induce a conformational change in the RNA, as is typical of other riboswitches, but instead functions as a catalytic cofactor for the reaction. This demonstrates that RNA, like protein enzymes, can employ the chemical diversity of small molecules to promote catalytic activity.

  2. Characterization of the trans Watson-Crick GU base pair located in the catalytic core of the antigenomic HDV ribozyme.

    Directory of Open Access Journals (Sweden)

    Dominique Lévesque

    Full Text Available The HDV ribozyme's folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U(23 and G(28 nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U(23 and G(28 can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction.

  3. Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    XU; Renhuan; (

    2001-01-01

    [1]Andrew, F., Gerard, E., A license to kill, Cell, 1996, 85: 781-784.[2]Thornberry, N. A., Lazebnik, Y., Caspases: Enemies within, Science, 1998, 281: 1312-1316.[3]Kijima, H., Ishida, H., Ohkawa, T. et al., Therapeutic application of ribozymes, Pharmacol. Ther., 1995, 68: 247-264.[4]Phylactou, L. A., Kilpatrick, M. W., Wood, M. J., Ribozymes as therapeutic tools for genetic disease, Hum. Mol. Genet., 1998, 7(10): 1649-1653.[5]Bettrand, E., Pictet, R ., Grange, T., Can heamerhead ribozymes be efficient tools inactivate gene function? Nucleic Acids Res., 1994, 22: 293-300.[6]Lieber, A., Strauss, M., Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library, Mol. Cell Biol., 1995, 15: 540-551.[7]Xu, R. H., Zhou, X. Q., Xie, Q. et al., Preparation and identification of hammerhead ribozyme in vitro against rat caspase-3 mRNA fragment, Chin. J. Hepatol., 2000,8: 361-363.[8]Liu, J., Jin, Y. X., Wang, D. B., A novel vector for abundant expression of antisense RNA, triplex-forming RNA and ribozyme in vivo, High Technology Letters, 2000, 6: 84-88.[9]Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989.[10]Porter, A. G., J?nicke, R. U., Emerging roles of caspase-3 in apoptosis, Cell Death Differ, 1999, 6: 99-104.[11]Cryns, V., Yuan, J., Proteases to die for, Genes Dev., 1998, 12: 1551-1570.[12]Narendra, K. V., Anikumar, R. K., Fritz, E., Recent developments in the hammerhead ribozyme field, Nucleic Acids Research, 1998, 26: 5237-5242.

  4. Identification and tracking of hairpin vortex auto-generation in turbulent wall-bounded flow

    Science.gov (United States)

    Huang, Yangzi; Green, Melissa

    2016-11-01

    Hairpin vortices have been widely accepted as component structures of turbulent boundary layers. Their properties (size, vorticity, energy) and dynamic phenomena (origin, growth, breakdown) have been shown to correlate to the complex, multi-scaled turbulent motions observed in both experiments and simulations. As established in the literature, the passage of a hairpin vortex creates a wall-normal ejection of fluid, which encounters the high-speed freestream resulting in near-wall shear and increased drag. A previously generated simulation of an isolated hairpin vortex is used to study the auto-generation of a secondary vortex structure. Eulerian methods such as the Q criterion and Γ2 function, as well as Lagrangian methods are used to visualize the three-dimensional hairpin vortices and the auto-generation process. The circulation development and wall-normal location of both primary and secondary hairpin heads are studied to determine if there is a correlation between the strength and height of the primary hairpin vortex with the secondary hairpin vortex auto-generation.

  5. Insights into enzymatic thiamin catalysis

    OpenAIRE

    Wikner, Christer

    1997-01-01

    Thiamin diphosphate, the biologically active form of vitamin B,, functions as a cofactor in various enzymes in the cell. The protein enhances the reactivity of the cofactor by binding it in a very specific manner. In this work, based upon information from the crystal structure, the mechanism of the thiamin dependent enzyme transketolase from yeast has been investigated by various methods. In enzymatic thiamin catalysis, the protein has three major tasks in the formation of a...

  6. Stable RNA hairpins in 88 coding regions of human mRNA

    Institute of Scientific and Technical Information of China (English)

    PAN Min; WANG Chuanming; LIU Ciquan

    2004-01-01

    RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R=G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

  7. Reaction Selectivity in Heterogeneous Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Somorjai, Gabor A.; Kliewer, Christopher J.

    2009-02-02

    The understanding of selectivity in heterogeneous catalysis is of paramount importance to our society today. In this review we outline the current state of the art in research on selectivity in heterogeneous catalysis. Current in-situ surface science techniques have revealed several important features of catalytic selectivity. Sum frequency generation vibrational spectroscopy has shown us the importance of understanding the reaction intermediates and mechanism of a heterogeneous reaction, and can readily yield information as to the effect of temperature, pressure, catalyst geometry, surface promoters, and catalyst composition on the reaction mechanism. DFT calculations are quickly approaching the ability to assist in the interpretation of observed surface spectra, thereby making surface spectroscopy an even more powerful tool. HP-STM has revealed three vitally important parameters in heterogeneous selectivity: adsorbate mobility, catalyst mobility, and selective site-blocking. The development of size controlled nanoparticles from 0.8 to 10 nm, of controlled shape, and of controlled bimetallic composition has revealed several important variables for catalytic selectivity. Lastly, DFT calculations may be paving the way to guiding the composition choice for multi-metallic heterogeneous catalysis for the intelligent design of catalysts incorporating the many factors of selectivity we have learned.

  8. The group I-like ribozyme DiGIR1 mediates alternative processing of pre-rRNA transcripts in Didymium iridis

    DEFF Research Database (Denmark)

    Vader, Anna; Johansen, Steinar; Nielsen, Henrik

    2002-01-01

    During starvation induced encystment, cells of the myxomycete Didymium iridis accumulate a 7.5-kb RNA that is the result of alternative processing of pre-rRNA. The 5' end corresponds to an internal processing site cleaved by the group I-like ribozyme DiGIR1, located within the twin-ribozyme intron...

  9. Structure, dynamics, and function of the hammerhead ribozyme in bulk water and at a clay mineral surface from replica exchange molecular dynamics.

    Science.gov (United States)

    Swadling, Jacob B; Wright, David W; Suter, James L; Coveney, Peter V

    2015-03-01

    Compared with proteins, the relationship between structure, dynamics, and function of RNA enzymes (known as ribozymes) is far less well understood, despite the fact that ribozymes are found in many organisms and are often conceived as "molecular fossils" of the first self-replicating molecules to have arisen on Earth. To investigate how ribozymal function is governed by structure and dynamics, we study the full hammerhead ribozyme in bulk water and in an aqueous clay mineral environment by computer simulation using replica-exchange molecular dynamics. Through extensive sampling of the major conformational states of the hammerhead ribozyme, we are able to show that the hammerhead manifests a free-energy landscape reminiscent of that which is well known in proteins, exhibiting a "funnel" topology that guides the ribozyme into its globally most stable conformation. The active-site geometry is found to be closely correlated to the tertiary structure of the ribozyme, thereby reconciling conflicts between previously proposed mechanisms for the self-scission of the hammerhead. The conformational analysis also accounts for the differences reported experimentally in the catalytic activity of the hammerhead ribozyme, which is reduced when interacting with clay minerals as compared with bulk water.

  10. DiGIR1 and NaGIR1: naturally occurring group I-like ribozymes with unique core organization and evolved biological role

    DEFF Research Database (Denmark)

    Johansen, Steinar; Einvik, Christer; Nielsen, Henrik

    2002-01-01

    introns (twin-ribozyme introns) in distantly related organisms. The Didymium GIR1 (DiGIR1) and Naegleria GIR1 (NaGIR1) share fundamental features in structural organization and reactivity, and display significant differences when compared to the related group I splicing ribozymes. GIR1 lacks...

  11. Efficient hammerhead ribozyme-mediated cleavage of the structured hepatitis B virus encapsidation signal in vitro and in cell extracts, but not in intact cells.

    Science.gov (United States)

    Beck, J; Nassal, M

    1995-01-01

    Hepatitis B virus (HBV), the causative agent of B-type hepatitis in man, is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. An essential highly conserved cis-element present twice on this RNA is the encapsidation signal epsilon, a stem-loop structure that is critical for pregenome packaging and reverse transcription. Epsilon is hence an attractive target for antiviral therapy. Its structure, however, is a potential obstacle to antivirals whose action depends on hybridization, e.g. ribozymes. Here we demonstrate effective in vitro cleavage inside epsilon by hammerhead ribozymes containing flanking sequences complementary to an adjacent less structured region. Upon co-transfection with a HBV expression construct corresponding ribozymes embedded in a U6 snRNA context led to a significant, though modest, reduction in the steady-state level of HBV pregenomes. Inactive ribozyme mutants revealed that antisense effects contributed substantially to this reduction, however, efficient epsilon cleavage by the intracellularly expressed ribozymes was observed in Mg(2+)-supplemented cell lysates. Artificial HBV pregenomes carrying the ribozymes in cis and model RNAs lacking all HBV sequences except epsilon exhibited essentially the same behaviour. Hence, neither the absence of co-localization of ribozyme and target nor a viral component, but rather a cellular factor(s), is responsible for the strikingly different ribozyme activities inside cells and in cellular extracts. Images PMID:8559651

  12. Nanometallic chemistry: deciphering nanoparticle catalysis from the perspective of organometallic chemistry and homogeneous catalysis.

    Science.gov (United States)

    Yan, Ning; Yuan, Yuan; Dyson, Paul J

    2013-10-07

    Nanoparticle (NP) catalysis is traditionally viewed as a sub-section of heterogeneous catalysis. However, certain properties of NP catalysts, especially NPs dispersed in solvents, indicate that there could be benefits from viewing them from the perspective of homogeneous catalysis. By applying the fundamental approaches and concepts routinely used in homogeneous catalysis to NP catalysts it should be possible to rationally design new nanocatalysts with superior properties to those currently in use.

  13. Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome.

    Science.gov (United States)

    Nawtaisong, Pruksa; Keith, James; Fraser, Tresa; Balaraman, Velmurugan; Kolokoltsov, Andrey; Davey, Robert A; Higgs, Stephen; Mohammed, Ahmed; Rongsriyam, Yupha; Komalamisra, Narumon; Fraser, Malcolm J

    2009-06-04

    Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNA(val) promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

  14. Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

    Directory of Open Access Journals (Sweden)

    Mohammed Ahmed

    2009-06-01

    Full Text Available Abstract Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

  15. Cosmic strings and baryon decay catalysis

    Science.gov (United States)

    Gregory, Ruth; Perkins, W. B.; Davis, A.-C.; Brandenberger, R. H.

    1989-01-01

    Cosmic strings, like monopoles, can catalyze proton decay. For integer charged fermions, the cross section for catalysis is not amplified, unlike in the case of monopoles. The catalysis processes are reviewed both in the free quark and skyrmion pictures and the implications for baryogenesis are discussed. A computation of the cross section for monopole catalyzed skyrmion decay is presented using classical physics. Also discussed are some effects which can screen catalysis processes.

  16. Cosmic strings and baryon decay catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Gregory, R.; Perkins, W.B.; Davis, A.C.; Brandenberger, R.H. (Fermi National Accelerator Lab., Batavia, IL (USA); Cambridge Univ. (UK); Brown Univ., Providence, RI (USA). Dept. of Physics)

    1989-09-01

    Cosmic strings, like monopoles, can catalyze proton decay. For integer charged fermions, the cross section for catalysis is not amplified, unlike in the case of monopoles. We review the catalysis processes both in the free quark and skyrmion pictures and discuss the implications for baryogenesis. We present a computation of the cross section for monopole catalyzed skyrmion decay using classical physics. We also discuss some effects which can screen catalysis processes. 32 refs., 1 fig.

  17. Cosmic strings and baryon decay catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Gregory, R.; Perkins, W.B.; Davis, A.C.; Brandenberger, R.H. (Fermi National Accelerator Lab., Batavia, IL (USA); Cambridge Univ. (UK); Brown Univ., Providence, RI (USA). Dept. of Physics)

    1989-09-01

    Cosmic strings, like monopoles, can catalyze proton decay. For integer charged fermions, the cross section for catalysis is not amplified, unlike in the case of monopoles. We review the catalysis processes both in the free quark and skyrmion pictures and discuss the implications for baryogenesis. We present a computation of the cross section for monopole catalyzed skyrmion decay using classical physics. We also discuss some effects which can screen catalysis processes. 32 refs., 1 fig.

  18. Cooperative catalysis designing efficient catalysts for synthesis

    CERN Document Server

    Peters, René

    2015-01-01

    Written by experts in the field, this is a much-needed overview of the rapidly emerging field of cooperative catalysis. The authors focus on the design and development of novel high-performance catalysts for applications in organic synthesis (particularly asymmetric synthesis), covering a broad range of topics, from the latest progress in Lewis acid / Br?nsted base catalysis to e.g. metal-assisted organocatalysis, cooperative metal/enzyme catalysis, and cooperative catalysis in polymerization reactions and on solid surfaces. The chapters are classified according to the type of cooperating acti

  19. Solid acid catalysis from fundamentals to applications

    CERN Document Server

    Hattori, Hideshi

    2014-01-01

    IntroductionTypes of solid acid catalystsAdvantages of solid acid catalysts Historical overviews of solid acid catalystsFuture outlookSolid Acids CatalysisDefinition of acid and base -Brnsted acid and Lewis acid-Acid sites on surfacesAcid strengthRole of acid sites in catalysisBifunctional catalysisPore size effect on catalysis -shape selectivity-Characterization of Solid Acid Catalysts Indicator methodTemperature programmed desorption (TPD) of ammoniaCalorimetry of adsorption of basic moleculesInfrare

  20. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations

    Science.gov (United States)

    Jia, Yanwei; Sanchez, J. Aquiles; Wangh, Lawrence J.

    2014-01-01

    Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals. PMID:25082368

  1. Maintenance of the flip sequence orientation of the ears in the parvoviral left-end hairpin is a nonessential consequence of the critical asymmetry in the hairpin stem.

    Science.gov (United States)

    Li, Lei; Cotmore, Susan F; Tattersall, Peter

    2012-11-01

    Parvoviral terminal hairpins are essential for viral DNA amplification but are also implicated in multiple additional steps in the viral life cycle. The palindromes at the two ends of the minute virus of mice (MVM) genome are dissimilar and are processed by different resolution mechanisms that selectively direct encapsidation of predominantly negative-sense progeny genomes and conserve a single Flip sequence orientation at the 3' (left) end of such progeny. The sequence and predicted structure of these 3' hairpins are highly conserved within the genus Parvovirus, exemplified by the 121-nucleotide left-end sequence of MVM, which folds into a Y-shaped hairpin containing small internal palindromes that form the "ears" of the Y. To explore the potential role(s) of this hairpin in the viral life cycle, we constructed infectious clones with the ear sequences either inverted, to give the antiparallel Flop orientation, or with multiple transversions, conserving their base composition but changing their sequence. These were compared with a "bubble" mutant, designed to activate the normally silent origin in the inboard arm of the hairpin, thus potentially rendering symmetric the otherwise asymmetric junction resolution mechanism that drives maintenance of Flip. This mutant exhibited a major defect in viral duplex and single-strand DNA replication, characterized by the accumulation of covalently closed turnaround forms of the left end, and was rapidly supplanted by revertants that restored asymmetry. In contrast, both sequence and orientation changes in the hairpin ears were tolerated, suggesting that maintaining the Flip orientation of these structures is a consequence of, but not the reason for, asymmetric left-end processing.

  2. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    Science.gov (United States)

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

  3. Uridylation of RNA Hairpins by Tailor Confines the Emergence of MicroRNAs in Drosophila

    OpenAIRE

    Reimão-Pinto, Madalena M.; Ignatova, Valentina; Burkard, Thomas R.; Hung, Jui-Hung; Manzenreither, Raphael A.; Sowemimo, Ivica; Herzog, Veronika A.; Reichholf, Brian; Fariña-Lopez, Sara; Ameres, Stefan L.

    2015-01-01

    Summary Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. Here, we report Tailor, an uridylyltransferase that is required for the majority of 3′ end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3′ overhang of microRNA hairpins, which...

  4. Recognition of hairpin DNA from coil DNA by electrospray mass spectrometry with annealing strategy

    Institute of Scientific and Technical Information of China (English)

    Bo Zheng; Yi Quan Liu; Gu Yuan

    2012-01-01

    This research presented an annealing strategy to identify hairpin DNA from coil DNA with the same base composition but different arrangements using electrospray mass spectrometry (ESI-MS).A series of single-stranded DNA were annealed with their complementary sequences,respectively.All the five pairs of hairpin DNA and coil DNA were unambiguously distinguished by ESIMS with annealing strategy.This research offers a potential method to probe the DNA structure by comparing with mass spectral characteristics.

  5. Simulations of beta-hairpin folding confined to spherical pores using distributed computing.

    Science.gov (United States)

    Klimov, D K; Newfield, D; Thirumalai, D

    2002-06-11

    We report the thermodynamics and kinetics of an off-lattice Go model beta-hairpin from Ig-binding protein confined to an inert spherical pore. Confinement enhances the stability of the hairpin due to the decrease in the entropy of the unfolded state. Compared with their values in the bulk, the rates of hairpin formation increase in the spherical pore. Surprisingly, the dependence of the rates on the pore radius, R(s), is nonmonotonic. The rates reach a maximum at R(s)/R(g,N)(b) approximately equal to 1.5, where R(g,N)(b) is the radius of gyration of the folded beta-hairpin in the bulk. The denatured state ensemble of the encapsulated beta-hairpin is highly structured even at substantially elevated temperatures. Remarkably, a profound effect of confinement is evident even when the beta-hairpin occupies less than a 10th of the sphere volume. Our calculations show that the emergence of substantial structure in the denatured state of proteins in inert pores is a consequence of confinement. In contrast, the structure of the bulk denatured state ensemble depends dramatically on the extent of denaturation.

  6. Design of monomeric water-soluble β-hairpin and β-sheet peptides.

    Science.gov (United States)

    Jiménez, M Angeles

    2014-01-01

    Since the first report in 1993 (JACS 115, 5887-5888) of a peptide able to form a monomeric β-hairpin structure in aqueous solution, the design of peptides forming either β-hairpins (two-stranded antiparallel β-sheets) or three-stranded antiparallel β-sheets has become a field of growing interest and activity. These studies have yielded great insights into the principles governing the stability and folding of β-hairpins and antiparallel β-sheets. This chapter provides an overview of the reported β-hairpin/β-sheet peptides focussed on the applied design criteria, reviews briefly the factors contributing to β-hairpin/β-sheet stability, and describes a protocol for the de novo design of β-sheet-forming peptides based on them. Guidelines to select appropriate turn and strand residues and to avoid self-association are provided. The methods employed to check the success of new designed peptides are also summarized. Since NMR is the best technique to that end, NOEs and chemical shifts characteristic of β-hairpins and three-stranded antiparallel β-sheets are given.

  7. The membrane integration of a naturally occurring alpha-helical hairpin.

    Science.gov (United States)

    Nagy, Akos; Turner, R James

    2007-05-04

    Helical hairpins, two closely spaced helical membrane spanning segments separated by a short surface turn, are thought to be common in integral membrane proteins. Here, we study the membrane integration of a naturally occurring helical hairpin from the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1. This sequence is only slightly longer and significantly less hydrophobic than a previously identified minimal poly-leucine model hairpin structure. Using site directed mutagenesis we document the importance of the turn propensity of the amino acids in the intervening surface turn but, somewhat surprisingly, our results indicate that the formation of this natural hairpin apparently does not depend on specific helix-helix interactions. Our results suggest that helical hairpins may be formed quite readily from even minimally hydrophobic sequences separated by a short, sufficiently strong, turn signal, and that current methods for predicting integral membrane protein topology may miss many similar short helical hairpin sequences. Thus the occurrence of these structures may be much more common than presently thought.

  8. Short-term sequence evolution and vertical inheritance of the Naegleria twin-ribozyme group I intron

    Directory of Open Access Journals (Sweden)

    De Jonckheere Johan F

    2006-05-01

    Full Text Available Abstract Background Ribosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516 has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2 and a homing endonuclease gene (NaHEG. NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA. Results To elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, contains hallmarks of a degenerated NaHEG. Phylogenetic analyses performed on separate data sets representing NaGIR1, NaGIR2, NaHEG, and ITS1-5.8S-ITS2 ribosomal DNA are consistent with an overall vertical inheritance pattern of the intron within the Naegleria genus. Conclusion The Nae.S516 twin-ribozyme intron was gained early in the Naegleria evolution with subsequent vertical inheritance. The intron was lost in the majority of isolates (70%, leaving a widespread but scattered distribution pattern. Why the apparent asexual Naegleria amoebae harbors active intron homing endonucleases, dependent on sexual reproduction for its function, remains a puzzle.

  9. Fundamental concepts in heterogeneous catalysis

    CERN Document Server

    Norskov, Jens K; Abild-Pedersen, Frank; Bligaard, Thomas

    2014-01-01

    This book is based on a graduate course and suitable as a primer for any newcomer to the field, this book is a detailed introduction to the experimental and computational methods that are used to study how solid surfaces act as catalysts.   Features include:First comprehensive description of modern theory of heterogeneous catalysisBasis for understanding and designing experiments in the field   Allows reader to understand catalyst design principlesIntroduction to important elements of energy transformation technologyTest driven at Stanford University over several semesters

  10. Indenylmetal Catalysis in Organic Synthesis.

    Science.gov (United States)

    Trost, Barry M; Ryan, Michael C

    2017-03-06

    Synthetic organic chemists have a long-standing appreciation for transition metal cyclopentadienyl complexes, of which many have been used as catalysts for organic transformations. Much less well known are the contributions of the benzo-fused relative of the cyclopentadienyl ligand, the indenyl ligand, whose unique properties have in many cases imparted differential reactivity in catalytic processes toward the synthesis of small molecules. In this Review, we present examples of indenylmetal complexes in catalysis and compare their reactivity to their cyclopentadienyl analogues, wherever possible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Cobalt particle size effects in catalysis

    NARCIS (Netherlands)

    den Breejen, J.P.

    2010-01-01

    Aim of the work described in this thesis was first to investigate cobalt particle size effects in heterogeneous catalysis. The main focus was to provide a deeper understanding of the origin of the cobalt particle size effects in Fischer-Tropsch (FT) catalysis in which synthesis gas (H2/CO) is conver

  12. DOE Laboratory Catalysis Research Symposium - Abstracts

    Energy Technology Data Exchange (ETDEWEB)

    Dunham, T.

    1999-02-01

    The conference consisted of two sessions with the following subtopics: (1) Heterogeneous Session: Novel Catalytic Materials; Photocatalysis; Novel Processing Conditions; Metals and Sulfides; Nuclear Magnetic Resonance; Metal Oxides and Partial Oxidation; Electrocatalysis; and Automotive Catalysis. (2) Homogeneous Catalysis: H-Transfer and Alkane Functionalization; Biocatalysis; Oxidation and Photocatalysis; and Novel Medical, Methods, and Catalyzed Reactions.

  13. Cobalt particle size effects in catalysis

    NARCIS (Netherlands)

    den Breejen, J.P.

    2010-01-01

    Aim of the work described in this thesis was first to investigate cobalt particle size effects in heterogeneous catalysis. The main focus was to provide a deeper understanding of the origin of the cobalt particle size effects in Fischer-Tropsch (FT) catalysis in which synthesis gas (H2/CO) is

  14. Asymmetric catalysis : ligand design and microwave acceleration

    OpenAIRE

    Bremberg, Ulf

    2000-01-01

    This thesis deals partly with the design and synthesis ofligands for use in asymmetric catalysis, and partly with theapplication of microwave heating on metal-based asymmetriccatalytic reactions. Enantiomerically pure pyridyl alcohols and bipyridylalcohols were synthesized from the chiral pool for future usein asymmetric catalysis. Lithiated pyridines were reacted withseveral chiral electrophiles, yielding diastereomeric mixturesthat could be separated without the use of resolutiontechniques....

  15. Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures

    Science.gov (United States)

    El-Murr, Nizar; Maurel, Marie-Christine; Rihova, Martina; Vergne, Jacques; Hervé, Guy; Kato, Mikio; Kawamura, Kunio

    2012-09-01

    The "RNA world" hypothesis proposes that—early in the evolution of life—RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg2+, which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg2+ on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na+ or K+), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

  16. RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

    Science.gov (United States)

    Miao, Zhichao; Adamiak, Ryszard W; Antczak, Maciej; Batey, Robert T; Becka, Alexander J; Biesiada, Marcin; Boniecki, Michał J; Bujnicki, Janusz; Chen, Shi-Jie; Cheng, Clarence Yu; Chou, Fang-Chieh; Ferré-D'Amaré, Adrian R; Das, Rhiju; Dawson, Wayne K; Feng, Ding; Dokholyan, Nikolay V; Dunin-Horkawicz, Stanisław; Geniesse, Caleb; Kappel, Kalli; Kladwang, Wipapat; Krokhotin, Andrey; Łach, Grzegorz E; Major, François; Mann, Thomas H; Magnus, Marcin; Pachulska-Wieczorek, Katarzyna; Patel, Dinshaw J; Piccirilli, Joseph A; Popenda, Mariusz; Purzycka, Katarzyna J; Ren, Aiming; Rice, Greggory M; Santalucia, John; Sarzynska, Joanna; Szachniuk, Marta; Tandon, Arpit; Trausch, Jeremiah J; Tian, Siqi; Wang, Jian; Weeks, Kevin M; Williams, Benfeard; Xiao, Yi; Xu, Xiaojun; Zhang, Dong; Zok, Tomasz; Westhof, Eric

    2017-01-30

    RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF) and one set describes large conformational changes between ligand-free and ligand-bound states; the Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All the puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal notable need for algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

  17. Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

    Institute of Scientific and Technical Information of China (English)

    Run-Ping Gao; David R Brigstock

    2009-01-01

    AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-b1 to the culture medium. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen Ⅰ, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen Ⅰ, and an increase in produced and secreted CCN2 or extracellular collagen Ⅰ protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen Ⅰ protein. Furthermore, the TGF-b1-induced increase in mRNA or protein for CCN2 or collagen Ⅰ was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-b1-induced collagen Ⅰ production in human HSCs and regulates entry of the cells into Sphase.

  18. Ribozyme Activity of RNA Nonenzymatically Polymerized from 3′,5′-Cyclic GMP

    Directory of Open Access Journals (Sweden)

    Samanta Pino

    2013-12-01

    Full Text Available 3′,5′-Cyclic GMP spontaneously nonenzymatically polymerizes in a base-catalyzed reaction affording G oligonucleotides. When reacted with fully or partially sequence-complementary RNA (oligo C, the abiotically generated oligo G RNA displays a typical ribozyme activity consisting of terminal ligation accompanied by cleavage of an internal phosphate site of the donor oligonucleotide stem upon attack of the acceptor 3′ terminal OH. This reaction is dubbed Ligation following Intermolecular Cleavage (LIC. In a prebiotic perspective, the ability of oligo G polynucleotides to react with other sequences outlines a simple and possible evolutionary scenario based on the autocatalytic properties of RNA.

  19. "Nanocrystal bilayer for tandem catalysis"

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Yusuke; Tsung, Chia Kuang; Huang, Wenyu; Huo, Ziyang; E.Habas, Susan E; Soejima, Tetsuro; Aliaga, Cesar E; Samorjai, Gabor A; Yang, Peidong

    2011-01-24

    Supported catalysts are widely used in industry and can be optimized by tuning the composition and interface of the metal nanoparticles and oxide supports. Rational design of metal-metal oxide interfaces in nanostructured catalysts is critical to achieve better reaction activities and selectivities. We introduce here a new class of nanocrystal tandem catalysts that have multiple metal-metal oxide interfaces for the catalysis of sequential reactions. We utilized a nanocrystal bilayer structure formed by assembling platinum and cerium oxide nanocube monolayers of less than 10 nm on a silica substrate. The two distinct metal-metal oxide interfaces, CeO2-Pt and Pt-SiO2, can be used to catalyse two distinct sequential reactions. The CeO2-Pt interface catalysed methanol decomposition to produce CO and H2, which were subsequently used for ethylene hydroformylation catalysed by the nearby Pt-SiO2 interface. Consequently, propanal was produced selectively from methanol and ethylene on the nanocrystal bilayer tandem catalyst. This new concept of nanocrystal tandem catalysis represents a powerful approach towards designing high-performance, multifunctional nanostructured catalysts

  20. Nanocrystal bilayer for tandem catalysis.

    Science.gov (United States)

    Yamada, Yusuke; Tsung, Chia-Kuang; Huang, Wenyu; Huo, Ziyang; Habas, Susan E; Soejima, Tetsuro; Aliaga, Cesar E; Somorjai, Gabor A; Yang, Peidong

    2011-05-01

    Supported catalysts are widely used in industry and can be optimized by tuning the composition and interface of the metal nanoparticles and oxide supports. Rational design of metal-metal oxide interfaces in nanostructured catalysts is critical to achieve better reaction activities and selectivities. We introduce here a new class of nanocrystal tandem catalysts that have multiple metal-metal oxide interfaces for the catalysis of sequential reactions. We utilized a nanocrystal bilayer structure formed by assembling platinum and cerium oxide nanocube monolayers of less than 10 nm on a silica substrate. The two distinct metal-metal oxide interfaces, CeO(2)-Pt and Pt-SiO(2), can be used to catalyse two distinct sequential reactions. The CeO(2)-Pt interface catalysed methanol decomposition to produce CO and H(2), which were subsequently used for ethylene hydroformylation catalysed by the nearby Pt-SiO(2) interface. Consequently, propanal was produced selectively from methanol and ethylene on the nanocrystal bilayer tandem catalyst. This new concept of nanocrystal tandem catalysis represents a powerful approach towards designing high-performance, multifunctional nanostructured catalysts.

  1. Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

    DEFF Research Database (Denmark)

    Birgisdottir, Asa B; Nielsen, Henrik; Beckert, Bertrand;

    2011-01-01

    processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor...

  2. Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination.

    OpenAIRE

    Washio, T.; Sasayama, J; Tomita, M.

    1998-01-01

    Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes. Instead of trying to detect each individual hairpin, we averaged the free energy values around the stop codons over the entire genome to predict how extensively the organism relies on hairpin formation in the process of transcription termination. The free energy values of Escherichia coli K-12 sho...

  3. Uridylation of RNA Hairpins by Tailor Confines the Emergence of MicroRNAs in Drosophila

    Science.gov (United States)

    Reimão-Pinto, Madalena M.; Ignatova, Valentina; Burkard, Thomas R.; Hung, Jui-Hung; Manzenreither, Raphael A.; Sowemimo, Ivica; Herzog, Veronika A.; Reichholf, Brian; Fariña-Lopez, Sara; Ameres, Stefan L.

    2015-01-01

    Summary Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. Here, we report Tailor, an uridylyltransferase that is required for the majority of 3′ end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3′ overhang of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3′ guanosine. In contrast to mirtrons, conserved Drosophila precursor microRNAs are significantly depleted in 3′ guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3′ ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila. PMID:26145176

  4. Formation mechanism of hairpin vortices in the wake of a truncated square cylinder in a duct

    CERN Document Server

    Dousset, Vincent

    2010-01-01

    We investigate the laminar shedding of hairpin vortices in the wake of a truncated square cylinder placed in a duct, for Reynolds numbers around the critical threshold of the onset of vortex shedding. We single out the formation mechanism of the hairpin vortices by means of a detailed analysis of the flow patterns in the steady regime. We show that unlike in previous studies of similar structures, the dynamics of the hairpin vortices is entwined with that of the counter-rotating pair of streamwise vortices, which we found to be generated in the bottom part of the near wake (these are usually referred to as base vortices). In particular, once the hairpin structure is released, the base vortices attach to it, forming its legs, so these are streamwise, and not spanwise as previously observed in unconfined wakes or behind cylinders of lower aspect ratios. We also single out a trail of Omega-shaped vortices, generated between successive hairpin vortices through a mechanism that is analogous to that active in near-...

  5. The role of loop stacking in the dynamics of DNA hairpin formation

    CERN Document Server

    Mosayebi, Majid; Ouldridge, Thomas E; Louis, Ard A; Doye, Jonathan P K

    2014-01-01

    We study the dynamics of DNA hairpin formation using oxDNA, a nucleotide-level coarse-grained model of DNA. In particular, we explore the effects of the loop stacking interactions and non-native base pairing on the hairpin closing times. We find a non-monotonic variation of the hairpin closing time with temperature, in agreement with the experimental work of Wallace et al. [Proc. Nat. Acad. Sci. USA 2001, 98, 5584-5589]. The hairpin closing process involves the formation of an initial nucleus of one or two bonds between the stems followed by a rapid zippering of the stem. At high temperatures, typically above the hairpin melting temperature, an effective negative activation enthalpy is observed because the nucleus has a lower enthalpy than the open state. By contrast, at low temperatures, the activation enthalpy becomes positive mainly due to the increasing energetic cost of bending a loop that becomes increasingly highly stacked as the temperature decreases. We show that stacking must be very strong to induc...

  6. Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

    Directory of Open Access Journals (Sweden)

    Kai Hoehlig

    Full Text Available 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.

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

    Science.gov (United States)

    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

  8. Effects of blocking androgen receptor expression with specific hammerhead ribozyme on in vitro growth of prostate cancer cell line

    Institute of Scientific and Technical Information of China (English)

    童强松; 赵军; 陈朝晖; 曾甫清; 鲁功成

    2003-01-01

    Objective To study the possibility of gene therapy for prostate cancer by blocking androgen receptor (AR) gene expression using a specific hammerhead ribozyme (RZ).Methods The hammerhead ribozyme expression vector pcDNA-hAR-RZ, specific to AR mRNA, was constructed and transfected into the prostate cancer cell line LNCaP by using lipofectamine. Androgen receptor expression was measured by RT-PCR and immunohistochemical methods. Cellular proliferation activities were assayed using the tetrazolium bromide colorimetry method; cell cycle changes were observed by flow cytometry; and cell apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling method. Results One to seven days after transfection with the ribozyme expression vector, AR mRNA expression at molecular and protein levels in LNCaP cells decreased by 32.6%-40.7% (P<0.05) and 21.0%-87.64% (P<0.05) respectively, and cell proliferation was inhibited by 18.28%-35.34% (P<0.05). Meanwhile, the cell cycle was arrested at the G2/M stage, and apoptotic morphological changes occurred with an apoptosis rate of 25.17% (P<0.01).Conclusion Ribozyme specific against AR mRNA is capable of inhibiting the expression AR and inducing the apoptosis in prostate cancer cells.

  9. Palladium catalysis for energy applications

    Energy Technology Data Exchange (ETDEWEB)

    Pfefferle, L. D.; Datye, Abhaya

    2001-03-01

    Palladium (Pd) is an attractive catalyst for a range of new combustion applications comprising primary new technologies for future industrial energy needs, including gas turbine catalytic combustion, auto exhaust catalysts, heating and fuel cells. Pd poses particular challenges because it changes both chemical state and morphology as a function of temperature and reactant environment and those changes result in positive and negative changes in activity. Interactions with the support, additives, water, and contaminants as well as carbon formation have also been observed to affect Pd catalyst performance. This report describes the results of a 3.5 year project that resolves some of the conflicting reports in the literature about the performance of Pd-based catalysis.

  10. Nanocrystal assembly for tandem catalysis

    Science.gov (United States)

    Yang, Peidong; Somorjai, Gabor; Yamada, Yusuke; Tsung, Chia-Kuang; Huang, Wenyu

    2014-10-14

    The present invention provides a nanocrystal tandem catalyst comprising at least two metal-metal oxide interfaces for the catalysis of sequential reactions. One embodiment utilizes a nanocrystal bilayer structure formed by assembling sub-10 nm platinum and cerium oxide nanocube monolayers on a silica substrate. The two distinct metal-metal oxide interfaces, CeO.sub.2--Pt and Pt--SiO.sub.2, can be used to catalyze two distinct sequential reactions. The CeO.sub.2--Pt interface catalyzed methanol decomposition to produce CO and H.sub.2, which were then subsequently used for ethylene hydroformylation catalyzed by the nearby Pt--SiO.sub.2 interface. Consequently, propanal was selectively produced on this nanocrystal bilayer tandem catalyst.

  11. Inverse Magnetic/Shear Catalysis

    CERN Document Server

    McInnes, Brett

    2015-01-01

    It is well known that very large magnetic fields are generated when the Quark-Gluon Plasma is formed during peripheral heavy-ion collisions. Lattice, holographic, and other studies strongly suggest that these fields may, for observationally relevant field values, induce ``inverse magnetic catalysis'', signalled by a lowering of the critical temperature for the chiral/deconfinement transition. The theoretical basis of this effect has recently attracted much attention; yet so far these investigations have not included another, equally dramatic consequence of the peripheral collision geometry: the QGP acquires a large angular momentum vector, parallel to the magnetic field. Here we use holographic techniques to argue that the angular momentum can also, independently, have an effect on transition temperatures, and we obtain a rough estimate of the relative effects of the presence of both a magnetic field and an angular momentum density. We find that the shearing angular momentum reinforces the effect of the magne...

  12. Asymmetric catalysis with short-chain peptides.

    Science.gov (United States)

    Lewandowski, Bartosz; Wennemers, Helma

    2014-10-01

    Within this review article we describe recent developments in asymmetric catalysis with peptides. Numerous peptides have been established in the past two decades that catalyze a wide variety of transformations with high stereoselectivities and yields, as well as broad substrate scope. We highlight here catalytically active peptides, which have addressed challenges that had thus far remained elusive in asymmetric catalysis: enantioselective synthesis of atropoisomers and quaternary stereogenic centers, regioselective transformations of polyfunctional substrates, chemoselective transformations, catalysis in-flow and reactions in aqueous environments.

  13. Inhibition of Hepatitis B Virus Replication and Expression in Vitro and in Vivo by the Hammerhead Ribozymes Targeted Different Sites

    Institute of Scientific and Technical Information of China (English)

    Wei; Dai; Rong; Zhou; Hong; Yu; Xiao-juan; Li

    2012-01-01

    Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the

  14. Phototriggered DNA hairpin formation in a stilbenediether-linked bis(oligonucleotide) conjugate

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, F.D.; Liu, X.

    1999-12-22

    Photochemical switches have found numerous biological applications, but have received only limited application to the control of DNA base pairing processes. The authors report here that a hairpin-forming bis(oligonucleotide) conjugate possessing a stilbenediether linker undergoes reversible trans {leftrightarrow} cis photoisomerization when both the cis and trans conjugates are in the random coil conformation, but undergoes one-way cis {r{underscore}arrow} trans isomerization when both are in hairpin conformations. Thus base-pairing inhibits trans {r{underscore}arrow} cis but not cis {r{underscore}arrow} trans isomerization. Moreover, photoisomerization of the random coil cis conjugate under conditions where the trans conjugate forms a stable hairpin results in phototriggered base pairing. The use of base pairing to effect one-way photoisomerization and of photoisomerization to effect base pairing are without precedent.

  15. Predicting 3D Structure, Flexibility, and Stability of RNA Hairpins in Monovalent and Divalent Ion Solutions

    Science.gov (United States)

    Shi, Ya-Zhou; Jin, Lei; Wang, Feng-Hua; Zhu, Xiao-Long; Tan, Zhi-Jie

    2015-01-01

    A full understanding of RNA-mediated biology would require the knowledge of three-dimensional (3D) structures, structural flexibility, and stability of RNAs. To predict RNA 3D structures and stability, we have previously proposed a three-bead coarse-grained predictive model with implicit salt/solvent potentials. In this study, we further develop the model by improving the implicit-salt electrostatic potential and including a sequence-dependent coaxial stacking potential to enable the model to simulate RNA 3D structure folding in divalent/monovalent ion solutions. The model presented here can predict 3D structures of RNA hairpins with bulges/internal loops (RNA hairpins with bulge loops of different lengths at several divalent/monovalent ion conditions. In addition, the model successfully predicts the stability of RNA hairpins with various loops/stems in divalent/monovalent ion solutions. PMID:26682822

  16. DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

    Science.gov (United States)

    Franch, Oskar; Iacovelli, Federico; Falconi, Mattia; Juul, Sissel; Ottaviani, Alessio; Benvenuti, Claudia; Biocca, Silvia; Ho, Yi-Ping; Knudsen, Birgitta R.; Desideri, Alessandro

    2016-07-01

    In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous

  17. Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood (Utah); (UMM)

    2012-09-05

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

  18. Micelle Catalysis of an Aromatic Substitution Reaction

    Science.gov (United States)

    Corsaro, Gerald; Smith J. K.

    1976-01-01

    Describes an experiment in which the iodonation of aniline reaction is shown to undergo catalysis in solution of sodium lauryl sulfate which forms micelles with negatively charged pseudo surfaces. (MLH)

  19. A Course in Kinetics and Catalysis.

    Science.gov (United States)

    Bartholomew, C. H.

    1981-01-01

    Describes a one-semester, three-credit hour course integrating the fundamentals of kinetics and the scientific/engineering principles of heterogeneous catalysis. Includes course outline, list of texts, background readings, and topical journal articles. (SK)

  20. Current trends of surface science and catalysis

    CERN Document Server

    Park, Jeong Young

    2014-01-01

    Including detail on applying surface science in renewable energy conversion, this book covers the latest results on model catalysts including single crystals, bridging "materials and pressure gaps", and hot electron flows in heterogeneous catalysis.

  1. Bioorthogonal catalysis: Rise of the nanobots

    Science.gov (United States)

    Unciti-Broceta, Asier

    2015-07-01

    Bioorthogonal catalysis provides new ways of mediating artificial transformations in living environs. Now, researchers have developed a nanodevice whose catalytic activity can be regulated by host-guest chemistry.

  2. Special section on Nano-Catalysis

    CSIR Research Space (South Africa)

    Makgwane, PR

    2013-01-01

    Full Text Available This special issue on nano-catalysis was devoted to the development and application of nanosized structured catalysts materials in various fields such as chemical transformation, environmental cleaning and energy generation supply as a concept tool...

  3. DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

    DEFF Research Database (Denmark)

    Franch, Oskar; Iacovelli, Federico; Falconi, Mattia

    2016-01-01

    and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724–9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin...... forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening–closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation...

  4. Loop residues and catalysis in OMP synthase

    DEFF Research Database (Denmark)

    Wang, Gary P.; Hansen, Michael Riis; Grubmeyer, Charles

    2012-01-01

    (preceding paper in this issue, DOI 10.1021/bi300083p)]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure...... of the catalytic loop, which when closed, produces rapid and reversible catalysis....

  5. Phosphine catalysis of allenes with electrophiles.

    Science.gov (United States)

    Wang, Zhiming; Xu, Xingzhu; Kwon, Ohyun

    2014-05-07

    Nucleophilic phosphine catalysis of allenes with electrophiles is one of the most powerful and straightforward synthetic strategies for the generation of highly functionalized carbocycle or heterocycle structural motifs, which are present in a wide range of bioactive natural products and medicinally important substances. The reaction topologies can be controlled through a judicious choice of the phosphine catalyst and the structural variations of starting materials. This Tutorial Review presents selected examples of nucleophilic phosphine catalysis using allenes and electrophiles.

  6. Advancing Sustainable Catalysis with Magnetite Surface ...

    Science.gov (United States)

    This article surveys the recent developments in the synthesis, surface modification, and synthetic applications of magnetitenanoparticles. The emergence of iron(II,III) oxide (triiron tetraoxide or magnetite; Fe3O4, or FeO•Fe2O3) nanoparticles as a sustainable support in heterogeneous catalysis is highlighted. Use of an oxide of earth-abundant iron for various applications in catalysis and environmental remediation.

  7. Recent advances in homogeneous nickel catalysis.

    Science.gov (United States)

    Tasker, Sarah Z; Standley, Eric A; Jamison, Timothy F

    2014-05-15

    Tremendous advances have been made in nickel catalysis over the past decade. Several key properties of nickel, such as facile oxidative addition and ready access to multiple oxidation states, have allowed the development of a broad range of innovative reactions. In recent years, these properties have been increasingly understood and used to perform transformations long considered exceptionally challenging. Here we discuss some of the most recent and significant developments in homogeneous nickel catalysis, with an emphasis on both synthetic outcome and mechanism.

  8. New developments in oxidation catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Rosowski, F. [BASF SE, Ludwigshafen (Germany)

    2011-07-01

    The impact of heterogeneous catalysis on the economy can be depicted by the global revenue of the chemical industry in 2006, which accounted for 2200 billion Euros with a share of all chemical products produced applying heterogeneous catalysis of about two thirds. [1] The range of products is enormous and they contribute greatly to the quality of our lifes. The advancement in the development of basic and intermediate chemical products is crucially dependent on either the further development of existing catalyst systems or the development of new catalysts and key to success for the chemical industry. Within the context of oxidation catalysis, the following driving forces are guiding research activities: There is a continuous desire to increase the selectivity of a given process in response to both economic as well as ecological needs and taking advantage of higher efficiencies in terms of cost savings and a better utilization of raw materials. A second motivation focuses on raw material change to all abundant and competitive feedstocks requiring both new developments in catalyst design as well as process technology. A more recent motivation refers to the use of metal oxide redox systems which are key to success for the development of novel technologies allowing for the separation of carbon dioxide and the use of carbon dioxide as a feedstock molecule as well as storing renewable energy in a chemical. To date, general ab initio approaches are known for the design of novel catalytic materials only for a few chemical reactions, whereas most industrial catalytic processes have been developed by empirical methods. [2] The development of catalytic materials are either based on the targeted synthesis of catalytic lead structures as well as high throughput methods that allow for the screening of a large range of parameters. [3 - 5] The successful development of catalysts together with reactor technology has led to both significant savings in raw materials and emissions. The

  9. Residues in the central β-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding

    NARCIS (Netherlands)

    Satapathy, Ajit K.; Kochaniak, Anna B.; Mukherjee, Sourav; Crampton, Donald J.; Oijen, Antoine van; Richardson, Charles C.

    2010-01-01

    The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic β-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the β-hairpin loop to determine their role during dTTPase-driven

  10. The 5′-Proximal Hairpin of Turnip Yellow Mosaic Virus RNA: Its Role in Translation and Encapsidation

    Science.gov (United States)

    Bink, Hugo H. J.; Schirawski, Jan; Haenni, Anne-Lise; Pleij, Cornelis W. A.

    2003-01-01

    The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5′ untranslated region, it was observed that stabilization of the 5′-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5′-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5′ cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis. PMID:12805444

  11. Specific acid catalysis and Lewis acid catalysis of Diels–Alder reactions in aqueous media

    NARCIS (Netherlands)

    Mubofu, Egid B.; Engberts, Jan B.F.N.

    2004-01-01

    A comparative study of specific acid catalysis and Lewis acid catalysis of Diels–Alder reactions between dienophiles (1, 4 and 6) and cyclopentadiene (2) in water and mixed aqueous media is reported. The reactions were performed in water with copper(II) nitrate as the Lewis acid catalyst whereas

  12. Specific acid catalysis and Lewis acid catalysis of Diels-Alder reactions in aqueous media

    NARCIS (Netherlands)

    Mubofu, E.B.; Engberts, J.B.F.N.

    A comparative study of specific acid catalysis and Lewis acid catalysis of Diells-Alder reactions between dienophiles (1, 4 and 6) and cyclopentadiene (2) in water and mixed aqueous media is reported. The reactions were performed in water with copper(II) nitrate as the Lewis acid catalyst whereas

  13. Acceptorless Dehydrogenation of N-Heterocycles by Merging Visible-Light Photoredox Catalysis and Cobalt Catalysis.

    Science.gov (United States)

    He, Ke-Han; Tan, Fang-Fang; Zhou, Chao-Zheng; Zhou, Gui-Jiang; Yang, Xiao-Long; Li, Yang

    2017-03-06

    Herein, the first acceptorless dehydrogenation of tetrahydroquinolines (THQs), indolines, and other related N-heterocycles, by merging visible-light photoredox catalysis and cobalt catalysis at ambient temperature, is described. The potential applications to organic transformations and hydrogen-storage materials are demonstrated. Primary mechanistic investigations indicate that the catalytic cycle occurs predominantly by an oxidative quenching pathway.

  14. Specific acid catalysis and Lewis acid catalysis of Diels-Alder reactions in aqueous media

    NARCIS (Netherlands)

    Mubofu, E.B.; Engberts, J.B.F.N.

    2004-01-01

    A comparative study of specific acid catalysis and Lewis acid catalysis of Diells-Alder reactions between dienophiles (1, 4 and 6) and cyclopentadiene (2) in water and mixed aqueous media is reported. The reactions were performed in water with copper(II) nitrate as the Lewis acid catalyst whereas hy

  15. Specific acid catalysis and Lewis acid catalysis of Diels–Alder reactions in aqueous media

    NARCIS (Netherlands)

    Mubofu, Egid B.; Engberts, Jan B.F.N.

    2004-01-01

    A comparative study of specific acid catalysis and Lewis acid catalysis of Diels–Alder reactions between dienophiles (1, 4 and 6) and cyclopentadiene (2) in water and mixed aqueous media is reported. The reactions were performed in water with copper(II) nitrate as the Lewis acid catalyst whereas hyd

  16. ISOTOPE METHODS IN HOMOGENEOUS CATALYSIS.

    Energy Technology Data Exchange (ETDEWEB)

    BULLOCK,R.M.; BENDER,B.R.

    2000-12-01

    The use of isotope labels has had a fundamentally important role in the determination of mechanisms of homogeneously catalyzed reactions. Mechanistic data is valuable since it can assist in the design and rational improvement of homogeneous catalysts. There are several ways to use isotopes in mechanistic chemistry. Isotopes can be introduced into controlled experiments and followed where they go or don't go; in this way, Libby, Calvin, Taube and others used isotopes to elucidate mechanistic pathways for very different, yet important chemistries. Another important isotope method is the study of kinetic isotope effects (KIEs) and equilibrium isotope effect (EIEs). Here the mere observation of where a label winds up is no longer enough - what matters is how much slower (or faster) a labeled molecule reacts than the unlabeled material. The most careti studies essentially involve the measurement of isotope fractionation between a reference ground state and the transition state. Thus kinetic isotope effects provide unique data unavailable from other methods, since information about the transition state of a reaction is obtained. Because getting an experimental glimpse of transition states is really tantamount to understanding catalysis, kinetic isotope effects are very powerful.

  17. Phylogenetic study on structural elements of HIV-1 poly(A region. 1. PolyA and DSE hairpins

    Directory of Open Access Journals (Sweden)

    Zarudnaya M. I.

    2013-11-01

    Full Text Available Genome of human immunodeficiency virus type 1 (HIV-1 is highly heterogeneous. The aim of this work was a phylogenetic study on structural elements of the HIV-1 poly(A region, in particular polyA and DSE hairpins which compose a core poly(A site. Methods. The secondary structure of the HIV-1 core poly(A site has been predicted by the UNAFold program. Results. The structure of the polyA and DSE hairpins has been analysed in 1679 HIV-1 genomes of group M and 18 genomes of simian immunodeficiency virus SIVcpzPtt. We found 244 and 171 different sequences for the HIV-1 polyA and DSE hairpins, respectively. However 70 % of the HIV-1 isolates studied contain one of 7 variants of the polyA hairpin which occur with a frequency 5 % (main variants and 79 % of the isolates contain one of 7 main variants of the DSE hairpin. We also revealed subtype and country specific mutations in these hairpins. We found that the SIV polyA hairpin most closely resembles that found in HIV-1 genomes of B/C subtypes. Conclusions. The results of our large-scale phylogenetic study support some structural models of the HIV-1 5' UTR, in particular the tertiary interaction between the polyA hairpin and the matrix region in HIV-1 gRNA. Possibly, the DSE hairpin appeared in the course of viral evolution of the HIV-1 group M. An exposure of the U/GU-rich element in the apical loop of DSE hairpin could significantly increase the efficiency of pre-mRNA polyadenylation in this HIV-1 group.

  18. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.

    Science.gov (United States)

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-05-26

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.

  19. Inducible Expression and Splicing of Candida Group Ⅰ Ribozyme in E.coli

    Institute of Scientific and Technical Information of China (English)

    SHANG Yuan; WANG Chen; ZHANG Yi

    2005-01-01

    The Ca. LSU intron flanking a 129 bp exon upstream and a 100 bp exon downstream was inserted into the lacZ gene on pRS426 to transform E. coli. Northern blot analysis and RT-PCR showed that splicing of Ca. LSU in E.coli is efficient upon inducible expression of the precursor RNA. In contrast, co-transcriptional self-splicing of the intron in vitro is much less active. Therefore, this E. coli splicing system can be used as a better model to investigate the effect of the ribozyme inhibitors on Ca. LSU splicing in living cell. We examined the effects of neomycin sulfate and pentamidine on Ca. LSU splicing in E. coli, and found that these drugs does-dependently inhibit the intron splicing.However, heomycin is more potent than pentamidine in this action.

  20. Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing.

    Science.gov (United States)

    Auyeung, Vincent C; Ulitsky, Igor; McGeary, Sean E; Bartel, David P

    2013-02-14

    To use microRNAs to downregulate mRNA targets, cells must first process these ~22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.

  1. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

    Directory of Open Access Journals (Sweden)

    Gregory Upert

    2012-01-01

    Full Text Available Human immunodeficiency virus-1 (HIV-1 replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR, to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP cation-based vectors that efficiently deliver nucleotide analogs (PNAs into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

  2. Evolutions of hairpin vortexes over a superhydrophobic surface in turbulent boundary layer flow

    Science.gov (United States)

    Zhang, Jingxian; Tian, Haiping; Yao, Zhaohui; Hao, Pengfei; Jiang, Nan

    2016-09-01

    Turbulent flows over a superhydrophobic surface and a smooth surface have been measured and studied by particle image velocimetry technology at Reθ = 990. The Reynolds shear stress distributions over the two surfaces are significantly different. Specifically, for the superhydrophobic surface, the Reynolds shear stress is suppressed in the near-wall region (y/δ curve. Evolutions of hairpin vortexes are analyzed to interpret differences in the Reynolds shear stress, based on some comparisons in the low-speed streaks and Q2/Q4 (ejection/sweep) events. The results show that, in the near wall region, the turbulent coherent structures (low-speed streaks and hairpin vortex) over the superhydrophobic surface are more stable and flat, due to the suppression in the strength and the lifting effect of the hairpin vortex. In the outer region, the superhydrophobic surface lifts the hairpin vortex away from the wall with a value of 0.14δ in our experiment, which makes the Q4 events occur further from the wall and contribute less to skin friction.

  3. Effect of the reflectional symmetry on the coherent hole transport across DNA hairpins

    Science.gov (United States)

    Zarea, Mehdi; Berlin, Yuri; Ratner, Mark A.

    2017-03-01

    The coherent hole transfer in three types of DNA hairpins containing strands with adenine (A) and guanine (G) nucleobases has been studied. The investigated hairpins involve An+1GGAn, AnGAGAn, or (AG)2nA strands that connect the hole donor and hole acceptor located on opposite ends of hairpins. The positive charge transfer from the photo-excited donor to the acceptor is shown to be slower for An+1GGAn in comparison with AnGAGAn and (AG)2nA sequences. We have revealed that this is due to the reflectional symmetry of the last two sequences with respect to the axis passing through the middle base. As has been demonstrated, the symmetry of the sequence structure manifests itself in the reflectional symmetry of the energy eigenstates. In addition, it has been shown that (AG)2nA is the only symmetric sequence with a zero energy state in the middle of the LUMO tight-binding energy band. Based on our theoretical findings, we predict that the hairpin with this sequence should have the fastest coherent hole transfer rate among the class of base sequences studied.

  4. Evolution of outer membrane beta-barrels from an ancestral beta beta hairpin.

    Science.gov (United States)

    Remmert, M; Biegert, A; Linke, D; Lupas, A N; Söding, J

    2010-06-01

    Outer membrane beta-barrels (OMBBs) are the major class of outer membrane proteins from Gram-negative bacteria, mitochondria, and plastids. Their transmembrane domains consist of 8-24 beta-strands forming a closed, barrel-shaped beta-sheet around a central pore. Despite their obvious structural regularity, evidence for an origin by duplication or for a common ancestry has not been found. We use three complementary approaches to show that all OMBBs from Gram-negative bacteria evolved from a single, ancestral beta beta hairpin. First, we link almost all families of known single-chain bacterial OMBBs with each other through transitive profile searches. Second, we identify a clear repeat signature in the sequences of many OMBBs in which the repeating sequence unit coincides with the structural beta beta hairpin repeat. Third, we show that the observed sequence similarity between OMBB hairpins cannot be explained by structural or membrane constraints on their sequences. The third approach addresses a longstanding problem in protein evolution: how to distinguish between a very remotely homologous relationship and the opposing scenario of "sequence convergence." The origin of a diverse group of proteins from a single hairpin module supports the hypothesis that, around the time of transition from the RNA to the protein world, proteins arose by amplification and recombination of short peptide modules that had previously evolved as cofactors of RNAs.

  5. Binding of DNA hairpins to an assembler-strand as part of a primordial translation device

    Science.gov (United States)

    Baumann, Ulrich

    1987-09-01

    A crucial event in the process leading to the origin of life is the emergence of a simple translation device. To approach experimental realization of this device the binding ability of short DNA hairpins to complementary oligonucleotides fixed on a solid support was investigated. The binding is achieved by base pairing between the loop nucleotides of the hairpins containing different numbers of adenosine residues and oligothymidylates covalently linked to cellulose. The loop has to consist of at least five nucleotides to achieve binding. The exact number of established base pairs was determined in two ways. First, the elution temperatures of hairpins and those of oligoadenylates which had the length of the loop were compared. Secondly, the architecture of the loop was analyzed by means of the single-strand-specific nuclease from mung bean acting as structural probe. Onlyn-2 of n loop nucleotides of a hairpin are able to form base pairs. Therefore, a strong evidence for the formation of a triplet of base pairs between primeval tRNA and mRNA sufficient to stabilize the complex enzyme-free is given.

  6. Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

    Directory of Open Access Journals (Sweden)

    Suzuki Harukazu

    2011-02-01

    Full Text Available Abstract Background DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs and long double-stranded RNAs, generating microRNA (miRNA duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown. Results In this study, we show that human recombinant DICER protein (rDICER processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage. Conclusions The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.

  7. An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

    Science.gov (United States)

    Chartrand, P; Harvey, S C; Ferbeyre, G; Usman, N; Cedergren, R

    1995-10-25

    A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer.

  8. Dodecamer d-AGATCTAGATCT and a homologous hairpin form triplex in the presence of peptide REWER.

    Directory of Open Access Journals (Sweden)

    Amrita Das

    Full Text Available We have designed a dodecamer d-AGATCTAGATCT (RY12 with alternate oligopurines and oligopyrimidines tracts and its homologous 28 bp hairpin oligomer (RY28 that forms a triple helix only in the presence of a pentapeptide REWER. An intermolecular triplex is formed by the single strand invasion of the RY28 duplex by RY12 in the presence of REWER. 5'- oligopurine end of RY12 binds to oligopurine sequence of RY28 in a parallel orientation and its oligopyrimidine stretch then changes strand and adopts an antiparallel orientation with the other strand of the duplex. Evidence for the formation of the triplex come from our studies of the UV melting curves, UV mixing curves, gel retardation assay, and chemical sequencing of 1∶1 mixture of dodecamer and hairpin oligonucleotides in the presence and absence of the peptide REWER. RY12 exists as a duplex that melts at 35°C. The hairpin (RY28 melts at 68°C. 1∶1 mixture of RY12 and RY28 in the absence of REWER gives a biphasic transition curve with thermodynamic properties corresponding to those of the melting of the duplex of RY12 and the hairpin RY28. However, the melting curve of this mixture is triphasic in the presence of the REWER; the thermodynamic parameters associated with the first phase (melting of the duplex of RY12, second phase (melting of the triplex and the third phase (melting of the hairpin show dependence on the molar ratio of peptide to oligonucleotides. Under appropriate conditions, gel retardation assay showed a shifted band that corresponds to a possible triplex. Chemical sequencing of KMnO4 and DEPC treated mixture of RY12, RY28 and REWER revealed the footprint of triplex.

  9. Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

    Directory of Open Access Journals (Sweden)

    Sedlacek Radislav

    2010-10-01

    Full Text Available Abstract Background RNA interference (RNAi is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type. Results Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals. Conclusions We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.

  10. Molecular modeling of heterogeneous catalysis

    Science.gov (United States)

    Gislason, Jason Joseph

    A novel method for modeling heterogeneous catalysis was developed to further facilitate the understanding of catalytic reactor mechanisms. The method employs molecular dynamics simulations, statistical mechanical, and Unity Bond Index - Quadratic Exponential Potential (UBI-QEP) calculations to calculate the rate constants for reactions on metal surfaces. The primary difficulty of molecular dynamics simulations on metal surfaces has been the lack of reliable reactive potential energy surfaces. We have overcome this through the development of the Normalized Bond Index - Reactive Potential Function (NBI-RPF), which can accurately describe the reaction of adsorbates on metal surfaces. The first calculations of rate constants for a reaction on a metal surface using molecular dynamics simulations are presented. This method is applied to the determination of the mechanism for selective hydrogenation of acetylene in an ethylene rich flow. It was determined that the selectivity for acetylene hydrogenation is attributable to the higher reactivity of acetylene versus ethylene with respect to hydrogenation by molecular hydrogen. It was shown that hydrogen transfer from the carbonaceous layer to acetylene or ethylene is insignificant in the hydrogenation process. Molecular dynamics simulations and molecular mechanics calculations were used to determine the diffusion rate constants for dimethylnaphthalene isomers is mordenite. 2,6-dimethylnaphthalene and 2,7-dimethylnaphthalene were found to have similar diffusion rate constants. Grand canonical Monte Carlo calculations were performed on the competitive adsorption of 2,6-dimethylnaphthalene and 2,7-dimethylnaphthalene in type X zeolites exchanged individually with barium, calcium, potassium, and rubidium ions, calcium exchanged MCM-22, and hydrogen form mordenite (MOR), X zeolite, Y zeolite, hypBEB, ZSM- 12, and MCM-22. These calculations showed that barium exchanged X zeolite was the most selective toward 2

  11. Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM.

    Science.gov (United States)

    Radak, Brian K; Lee, Tai-Sung; Harris, Michael E; York, Darrin M

    2015-09-01

    The hepatitis delta virus ribozyme is an efficient catalyst of RNA 2'-O-transphosphorylation and has emerged as a key experimental system for identifying and characterizing fundamental features of RNA catalysis. Recent structural and biochemical data have led to a proposed mechanistic model whereby an active site Mg(2+) ion facilitates deprotonation of the O2' nucleophile, and a protonated cytosine residue (C75) acts as an acid to donate a proton to the O5' leaving group as noted in a previous study. This model assumes that the active site Mg(2+) ion forms an inner-sphere coordination with the O2' nucleophile and a nonbridging oxygen of the scissile phosphate. These contacts, however, are not fully resolved in the crystal structure, and biochemical data are not able to unambiguously exclude other mechanistic models. In order to explore the feasibility of this model, we exhaustively mapped the free energy surfaces with different active site ion occupancies via quantum mechanical/molecular mechanical (QM/MM) simulations. We further incorporate a three-dimensional reference interaction site model for the solvated ion atmosphere that allows these calculations to consider not only the rate associated with the chemical steps, but also the probability of observing the system in the presumed active state with the Mg(2+) ion bound. The QM/MM results predict that a pathway involving metal-assisted nucleophile activation is feasible based on the rate-controlling transition state barrier departing from the presumed metal-bound active state. However, QM/MM results for a similar pathway in the absence of Mg(2+) are not consistent with experimental data, suggesting that a structural model in which the crystallographically determined Mg(2+) is simply replaced with Na(+) is likely incorrect. It should be emphasized, however, that these results hinge upon the assumption of the validity of the presumed Mg(2+)-bound starting state, which has not yet been definitively verified

  12. Asymmetric catalysis based on tropos ligands.

    Science.gov (United States)

    Aikawa, Kohsuke; Mikami, Koichi

    2012-11-21

    All enantiopure atropisomeric (atropos) ligands essentially require enantiomeric resolution or synthetic transformation from a chiral pool. In sharp contrast, the use of tropos (chirally flexible) ligands, which are highly modular, versatile, and easy to synthesize without enantiomeric resolution, has recently been the topic of much interest in asymmetric catalysis. Racemic catalysts bearing tropos ligands can be applied to asymmetric catalysis through enantiomeric discrimination by the addition of a chiral source, which preferentially transforms one catalyst enantiomer into a highly activated catalyst enantiomer. Additionally, racemic catalysts bearing tropos ligands can also be utilized as atropos enantiopure catalysts obtained via the control of chirality by a chiral source followed by the memory of chirality. In this feature article, our results on the asymmetric catalysis via the combination of various central metals and tropos ligands are summarized.

  13. Geometrically induced magnetic catalysis and critical dimensions

    CERN Document Server

    Flachi, Antonino; Vitagliano, Vincenzo

    2015-01-01

    We discuss the combined effect of magnetic fields and geometry in interacting fermionic systems. At leading order in the heat-kernel expansion, the infrared singularity (that in flat space leads to the magnetic catalysis) is regulated by the chiral gap effect and the catalysis is deactivated by effect of the curvature. We discover that an infrared singularity may reappear from higher-order terms in the heat kernel expansion leading to a novel form of geometrically induced magnetic catalysis (absent in flat space). The dynamical mass squared is then modified not only due to the chiral gap effect by an amount proportional to the curvature, but also by a magnetic shift $\\propto (4-D)eB$ where $D$ represents the number of space-time dimensions. We argue that $D=4$ is a critical dimension across which the behaviour of the magnetic shift changes qualitatively.

  14. Progress towards bioorthogonal catalysis with organometallic compounds.

    Science.gov (United States)

    Völker, Timo; Dempwolff, Felix; Graumann, Peter L; Meggers, Eric

    2014-09-22

    The catalysis of bioorthogonal transformations inside living organisms is a formidable challenge--yet bears great potential for future applications in chemical biology and medicinal chemistry. We herein disclose highly active organometallic ruthenium complexes for bioorthogonal catalysis under biologically relevant conditions and inside living cells. The catalysts uncage allyl carbamate protected amines with unprecedented high turnover numbers of up to 270 cycles in the presence of water, air, and millimolar concentrations of thiols. By live-cell imaging of HeLa cells and with the aid of a caged fluorescent probe we could reveal a rapid development of intense fluorescence within the cellular cytoplasm and therefore support the proposed bioorthogonality of the catalysts. In addition, to illustrate the manifold applications of bioorthogonal catalysis, we developed a method for catalytic in-cell activation of a caged anticancer drug, which efficiently induced apoptosis in HeLa cells.

  15. Green chemistry by nano-catalysis

    KAUST Repository

    Polshettiwar, Vivek

    2010-01-01

    Nano-materials are important in many diverse areas, from basic research to various applications in electronics, biochemical sensors, catalysis and energy. They have emerged as sustainable alternatives to conventional materials, as robust high surface area heterogeneous catalysts and catalyst supports. The nano-sized particles increase the exposed surface area of the active component of the catalyst, thereby enhancing the contact between reactants and catalyst dramatically and mimicking the homogeneous catalysts. This review focuses on the use of nano-catalysis for green chemistry development including the strategy of using microwave heating with nano-catalysis in benign aqueous reaction media which offers an extraordinary synergistic effect with greater potential than these three components in isolation. To illustrate the proof-of-concept of this "green and sustainable" approach, representative examples are discussed in this article. © 2010 The Royal Society of Chemistry.

  16. Request for Symposia Support: Advances in Olefin Polymerization Catalysis

    Science.gov (United States)

    2014-11-24

    included, but were not limited to, heterogeneous catalysis , homogeneous catalysis , advances in catalyst activation, methods for polymer topological...SECURITY CLASSIFICATION OF: This Advances in Olefin Polymerization Catalysis symposium was held at the 247th ACS National Meeting and Exposition...March 19, 2014 in Dallas, Texas and consisted of twelve (12) invited/contributed talks. The hosting ACS division was the Division of Catalysis Science

  17. Efficient trans-cleavage by the Schistosoma mansoni SMalpha1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus.

    Science.gov (United States)

    Vazquez-Tello, Alejandro; Castán, Pablo; Moreno, Renata; Smith, James M; Berenguer, José; Cedergren, Robert

    2002-04-01

    The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step. The optimal temperature for trans-cleavage was 70 degrees C. This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo.

  18. Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme.

    Science.gov (United States)

    Klein, Daniel J; Wilkinson, Sara R; Been, Michael D; Ferré-D'Amaré, Adrian R

    2007-10-12

    The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

  19. Experimental and numerical techniques to assess catalysis

    Science.gov (United States)

    Herdrich, G.; Fertig, M.; Petkow, D.; Steinbeck, A.; Fasoulas, S.

    2012-01-01

    Catalytic heating can be a significant portion of the thermal load experienced by a body during re-entry. Under the auspices of the NATO Research and Technology Organisation Applied Vehicle Technologies Panel Task Group AVT-136 an assessment of the current state-of-the-art in the experimental characterization and numerical simulation of catalysis on high-temperature material surfaces has been conducted. This paper gives an extraction of the final report for this effort, showing the facilities and capabilities worldwide to assess catalysis data. A corresponding summary for the modeling activities is referenced in this article.

  20. RNA catalysis and the origins of life

    Science.gov (United States)

    Orgel, Leslie E.

    1986-01-01

    The role of RNA catalysis in the origins of life is considered in connection with the discovery of riboszymes, which are RNA molecules that catalyze sequence-specific hydrolysis and transesterification reactions of RNA substrates. Due to this discovery, theories positing protein-free replication as preceding the appearance of the genetic code are more plausible. The scope of RNA catalysis in biology and chemistry is discussed, and it is noted that the development of methods to select (or predict) RNA sequences with preassigned catalytic functions would be a major contribution to the study of life's origins.

  1. Catalysis by nonmetals rules for catalyst selection

    CERN Document Server

    Krylov, Oleg V

    1970-01-01

    Catalysis by Non-metals: Rules of Catalyst Selection presents the development of scientific principles for the collection of catalysts. It discusses the investigation of the mechanism of chemosorption and catalysis. It addresses a series of properties of solid with catalytic activity. Some of the topics covered in the book are the properties of a solid and catalytic activity in oxidation-reduction reactions; the difference of electronegativities and the effective charges of atoms; the role of d-electrons in the catalytic properties of a solid; the color of solids; and proton-acid and proton-ba

  2. Bioinspired catalysis metal-sulfur complexes

    CERN Document Server

    Weigand, Wolfgang

    2014-01-01

    The growing interest in green chemistry calls for new, efficient and cheap catalysts. Living organisms contain a wide range of remarkably powerful enzymes, which can be imitated by chemists in the search for new catalysts. In bioinspired catalysis, chemists use the basic principles of biological enzymes when creating new catalyst analogues. In this book, an international group of experts cover the topic from theoretical aspects to applications by including a wide variety of examples of different systems. This valuable overview of bioinspired metal-sulfur catalysis is a must-have for all sci

  3. Keynotes in energy-related catalysis

    CERN Document Server

    Kaliaguine, S

    2011-01-01

    Catalysis by solid acids, which includes (modified) zeolites, is of special relevance to energy applications. Acid catalysis is highly important in modern petroleum refining operations - large-scale processes such as fluid catalytic cracking, catalytic reforming, alkylation and olefin oligomerization rely on the transformation of hydrocarbons by acid catalysts. (Modified) zeolites are therefore essential for the improvement of existing processes and for technical innovations in the conversion of crude. There can be little doubt that zeolite-based catalysts will play a major role in the futu

  4. Heterogeneous catalysis at nanoscale for energy applications

    CERN Document Server

    Tao, Franklin (Feng); Kamat, Prashant V

    2015-01-01

    This book presents both the fundamentals concepts and latest achievements of a field that is growing in importance since it represents a possible solution for global energy problems.  It focuses on an atomic-level understanding of heterogeneous catalysis involved in important energy conversion processes. It presents a concise picture for the entire area of heterogeneous catalysis with vision at the atomic- and nano- scales, from synthesis, ex-situ and in-situ characterization, catalytic activity and selectivity, to mechanistic understanding based on experimental exploration and theoretical si

  5. The 5′-Proximal Hairpin of Turnip Yellow Mosaic Virus RNA: Its Role in Translation and Encapsidation

    OpenAIRE

    Bink, Hugo H. J.; Schirawski, Jan; Haenni, Anne-Lise; Pleij, Cornelis W. A.

    2003-01-01

    The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5′ untranslated region, it was observed that stabilization of the 5′-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutat...

  6. Direct sp(3)C-H acroleination of N-aryl-tetrahydroisoquinolines by merging photoredox catalysis with nucleophilic catalysis.

    Science.gov (United States)

    Feng, Zhu-Jia; Xuan, Jun; Xia, Xu-Dong; Ding, Wei; Guo, Wei; Chen, Jia-Rong; Zou, You-Quan; Lu, Liang-Qiu; Xiao, Wen-Jing

    2014-04-07

    Sequence catalysis merging photoredox catalysis (PC) and nucleophilic catalysis (NC) has been realized for the direct sp(3) C-H acroleination of N-aryl-tetrahydroisoquinoline (THIQ). The reaction was performed under very mild conditions and afforded products in 50-91% yields. A catalytic asymmetric variant was proved to be successful with moderate enantioselectivities (up to 83 : 17 er).

  7. Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes

    DEFF Research Database (Denmark)

    Bomholt, Niels; Filichev, Vyacheslav V; Pedersen, Erik Bjerregaard

    2011-01-01

    Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been designed and synthesized. The modifications were incorporated into DNA oligonucleotides via standard DNA phosphoramidite synthesis. The ability of cationic modifications to stabilize palindromic DNA hairpins...... and parallel triplexes were evaluated using gel electrophoresis, circular dichroism and thermal denaturation measurements. The non-nucleosidic modifications were found to increase the thermal stability of palindromic hairpins at pH 8.0 as compared with a nucleosidic tetraloop (TCTC). Incorporation...... of modifications at the 5'-end of a triplex forming oligonucleotide resulted in a significant increase in thermal stability at low pH when the modifications were placed as the 5'-dangling end....

  8. Capping β-hairpin with N-terminal d-amino acid stabilizes peptide scaffold.

    Science.gov (United States)

    Makwana, Kamlesh M; Mahalakshmi, Radhakrishnan

    2016-05-01

    Various strategies exist to stabilize de novo designed synthetic peptide β-hairpins or β-sheets structures, especially at the non-hydrogen bonding position. However, strategies to stabilize strand termini, which are affected by fraying, are highly limited. Here, by substituting N-terminal aliphatic amino acid with its mirror image counterpart, we achieve a significant increase in scaffold stabilization, resulting from the formation of a terminal aliphatic-aromatic hydrophobic CH…pi cluster. Our extensive solution NMR studies support the incorporation of an N-terminal d-aliphatic amino acid in the design of short β-hairpins, while successfully retaining the overall structural scaffold. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 260-266, 2016.

  9. A new tool in peptide engineering: a photoswitchable stilbene-type beta-hairpin mimetic.

    Science.gov (United States)

    Erdélyi, Máté; Karlén, Anders; Gogoll, Adolf

    2005-12-23

    Peptide secondary structure mimetics are important tools in medicinal chemistry, as they provide analogues of endogenous peptides with new physicochemical and pharmacological properties. The development, synthesis, photochemical investigation, and conformational analysis of a stilbene-type beta-hairpin mimetic capable of light-triggered conformational changes have been achieved. In addition to standard spectroscopic techniques (nuclear Overhauser effects, amide temperature coefficients, circular dichroism spectroscopy), the applicability of self-diffusion measurements (longitudinal eddy current delay pulsed-field gradient spin echo (LED-PGSE) NMR technique) in conformational studies of oligopeptides is demonstrated. The title compound shows photoisomerization of the stilbene chromophore, resulting in a change in solution conformation between an unfolded structure and a folded beta-hairpin.

  10. Holographic particle image velocimetry measurements of hairpin vortices in a subcritical air channel flow

    Science.gov (United States)

    Svizher, Alexander; Cohen, Jacob

    2006-01-01

    A holographic particle image velocimetry (HPIV) system is employed to study the evolution of coherent structures artificially generated in a plane Poiseuille air flow. As a first step the hot-wire technique and two-dimensional flow visualization are used to determine the generation conditions and dimensions of the coherent structures, their shedding frequency, trajectory, and convection velocity. Then, the HPIV method is utilized to obtain the instantaneous topology of the hairpin vortex and its associated three-dimensional distribution of the two (streamwise and spanwise) velocity components as well as the corresponding wall-normal vorticity. Finally, the experimental data are compared with results of related experimental and numerical studies. The present experimental results support the view that the generation of hairpins under various base flow conditions is governed by a basic mechanism, the important common elements of which are the shear of the base flow and an initial disturbance having a sufficiently large amplitude.

  11. REVERSION OF MULTIDRUG RESISTANCE IN THE P-GLYCOPROTEIN POSITIVE BREAST CANCER CELL LINE(MCF-7/ADR) BY INTRODUCTION OF HAMMERHEAD RIBOZYME

    Institute of Scientific and Technical Information of China (English)

    袁亚维; 张积仁; K.J.Scanlon; 陆长德; 祁国荣

    1998-01-01

    A hammerhead ribozyme which slte-specifically cleaved the GUC position in codon 880 of the mdrl mRNA was designed. The target site was chosen between the two ATP binding sites, which may be impottant for the function of the P-C-p as an ATPodependent pump. A DNA sequence encoding the riboxyme gene was then incorporated into a eukaryotic expression vector (pH 0 Apt-1 neo) and transfeeted into the breast cancer cell line MCF-7/Adr, which is resistant to adrlamycin and expresses the MDR phenmype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83.5%; and the expressed ribozyme could inhiblte the formation of P-glycoprotein detected by immuno-cytochemistry assay and could reduce the cell''s resistance to adrimycin; this means that the resistant cells were 1 000-fold mcre resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

  12. Effect of hydrophobic interactions on the folding mechanism of β-hairpins.

    Science.gov (United States)

    Popp, Alexander; Wu, Ling; Keiderling, Timothy A; Hauser, Karin

    2014-12-11

    Hydrophobic interactions are essential in stabilizing protein structures. How they affect the folding pathway and kinetics, however, is less clear. We used time-resolved infrared spectroscopy to study the dynamics of hydrophobic interactions of β-hairpin variants of the sequence Trpzip2 (SWTWENGKWTWK-NH2) that is stabilized by two cross-strand Trp-Trp pairs. The hydrophobicity strength was varied by substituting the tryptophans pairwise by either tyrosines or valines. Relaxation dynamics were induced by a laser-excited temperature jump, which separately probed for the loss of the cross-strand β-hairpin interaction and the rise of the disordered structure. All substitutions tested result in reduced thermal stability, lower transition temperatures, and faster dynamics compared to Trpzip2. However, the changes in folding dynamics depend on the amino acid substituted for Trp. The aromatic substitution of Tyr for Trp results in the same kinetics for the unfolding of sheet and growth of disorder, with similar activation energies, independent of the substitution position. Substitution of Trp with a solely hydrophobic Val results in even faster kinetics than substitution with Tyr but is additionally site-dependent. If the hairpin has a Val pair close to its termini, the rate constants for loss of sheet and gain of disorder are the same, but if the pair is close to the turn, the sheet and disorder components show different relaxation kinetics. The Trp → Val substitutions reveal that hydrophobic interactions alone weakly stabilize the hairpin structure, but adding edge-to-face aromatic interaction strengthens it, and both modify the complex folding process.

  13. Diffusion and Surface Reaction in Heterogeneous Catalysis

    Science.gov (United States)

    Baiker, A.; Richarz, W.

    1978-01-01

    Ethylene hydrogenation on a platinum catalyst, electrolytically applied to a tube wall, is a good system for the study of the interactions between diffusion and surface reaction in heterogeneous catalysis. Theoretical background, apparatus, procedure, and student performance of this experiment are discussed. (BB)

  14. Homogeneous Catalysis by Transition Metal Compounds.

    Science.gov (United States)

    Mawby, Roger

    1988-01-01

    Examines four processes involving homogeneous catalysis which highlight the contrast between the simplicity of the overall reaction and the complexity of the catalytic cycle. Describes how catalysts provide circuitous routes in which all energy barriers are relatively low rather than lowering the activation energy for a single step reaction.…

  15. Surface temperature excess in heterogeneous catalysis

    NARCIS (Netherlands)

    Zhu, L.

    2005-01-01

    In this dissertation we study the surface temperature excess in heterogeneous catalysis. For heterogeneous reactions, such as gas-solid catalytic reactions, the reactions take place at the interfaces between the two phases: the gas and the solid catalyst. Large amount of reaction heats are released

  16. Supported Ionic Liquid Phase (SILP) catalysis

    DEFF Research Database (Denmark)

    Riisager, Anders; Fehrmann, Rasmus; Haumann, Marco;

    2006-01-01

    Applications of ionic liquids to replace conventional solvents in homogeneous transition-metal catalysis have increased significantly during the last decade. Biphasic ionic liquid/organic liquid systems offer advantages with regard to product separation, catalyst stability, and recycling but util...

  17. Hydroxide catalysis bonding of silicon carbide

    NARCIS (Netherlands)

    Veggel, A.A. van; Ende, D.A. van den; Bogenstahl, J.; Rowan, S.; Cunningham, W.; Gubbels, G.H.M.; Nijmeijer, H.

    2008-01-01

    For bonding silicon carbide optics, which require extreme stability, hydroxide catalysis bonding is considered [Rowan, S., Hough, J. and Elliffe, E., Silicon carbide bonding. UK Patent 040 7953.9, 2004. Please contact Mr. D. Whiteford for further information: D.Whiteford@admin.gla.ac.uk]. This techn

  18. Supported ionic liquid-phase (SILP) catalysis

    DEFF Research Database (Denmark)

    Riisager, Anders; Fehrmann, Rasmus; Wasserscheid, P.

    2005-01-01

    The concept of supported ionic liquid-phase (SILP) catalysis has been demonstrated for gas- and liquid-phase continuous fixed-bed reactions using rhodium phosphine catalyzed hydroformylation of propene and 1-octene as examples. The nature of the support had important influence on both the catalytic...

  19. Surface temperature excess in heterogeneous catalysis

    NARCIS (Netherlands)

    Zhu, L.

    2005-01-01

    In this dissertation we study the surface temperature excess in heterogeneous catalysis. For heterogeneous reactions, such as gas-solid catalytic reactions, the reactions take place at the interfaces between the two phases: the gas and the solid catalyst. Large amount of reaction heats are released

  20. Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination

    National Research Council Canada - National Science Library

    Washio, T; Sasayama, J; Tomita, M

    1998-01-01

    Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes...

  1. Ultrasensitive detection of nucleic acids by template enhanced hybridization followed by rolling circle amplification and catalytic hairpin assembly.

    Science.gov (United States)

    Song, Weiling; Zhang, Qiao; Sun, Wenbo

    2015-02-11

    An ultrasensitive protocol for fluorescent detection of DNA is designed by combining the template enhanced hybridization process (TEHP) with Rolling Circle Amplification (RCA) and Catalytic Hairpin Assembly (CHA), showing a remarkable amplification efficiency.

  2. Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Singh Upinder

    2009-02-01

    Full Text Available Abstract Background Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica. Results An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%. Conclusion Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

  3. Prediction of the beta-hairpins in proteins using support vector machine.

    Science.gov (United States)

    Hu, Xiu Zhen; Li, Qian Zhong

    2008-02-01

    By using of the composite vector with increment of diversity and scoring function to express the information of sequence, a support vector machine (SVM) algorithm for predicting beta-hairpin motifs is proposed. The prediction is done on a dataset of 3,088 non homologous proteins containing 6,027 beta-hairpins. The overall accuracy of prediction and Matthew's correlation coefficient are 79.9% and 0.59 for the independent testing dataset. In addition, a higher accuracy of 83.3% and Matthew's correlation coefficient of 0.67 in the independent testing dataset are obtained on a dataset previously used by Kumar et al. (Nuclic Acid Res 33:154-159). The performance of the method is also evaluated by predicting the beta-hairpins of in the CASP6 proteins, and the better results are obtained. Moreover, this method is used to predict four kinds of supersecondary structures. The overall accuracy of prediction is 64.5% for the independent testing dataset.

  4. Effectiveness of hairpin probe in increasing the limit of detection for gold nanowire based-biosensor

    Science.gov (United States)

    Tien Cao, Huu; Pham, Xuan Thanh Tung; Linh Ha, Van; Hieu Le, Van

    2014-12-01

    Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless and readily integrated into microelectronics and microfluidics, appear a promising alternative to optical methods for the detection of specific nucleic acid sequences. In keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain, signal-off behavior or instability. To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The observed signal gain is sufficient to achieve a demonstrated detection limit of 25 pM.

  5. "Off-on" electrochemical hairpin-DNA-based genosensor for cancer diagnostics.

    Science.gov (United States)

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

    2011-03-01

    A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

  6. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

    Directory of Open Access Journals (Sweden)

    Das Atze T

    2012-07-01

    Full Text Available Abstract Background The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.

  7. Effect of Polypurine Reverse Hoogsteen Hairpins on Relevant Cancer Target Genes in Different Human Cell Lines.

    Science.gov (United States)

    Villalobos, Xenia; Rodríguez, Laura; Solé, Anna; Lliberós, Carolina; Mencia, Núria; Ciudad, Carlos J; Noé, Véronique

    2015-08-01

    We studied the ability of polypurine reverse Hoogsteen hairpins (PPRHs) to silence a variety of relevant cancer-related genes in several human cell lines. PPRHs are hairpins formed by two antiparallel polypurine strands bound by intramolecular Hoogsteen bonds linked by a pentathymidine loop. These hairpins are able to bind to their target DNA sequence through Watson-Crick bonds producing specific silencing of gene expression. We designed PPRHs against the following genes: BCL2, TOP1, mTOR, MDM2, and MYC and tested them for mRNA levels, cytotoxicity, and apoptosis in prostate, pancreas, colon, and breast cancer cell lines. Even though all PPRHs were effective, the most remarkable results were obtained with those against BCL2 and mammalian target of rapamycin (mTOR) in decreasing cell survival and mRNA levels and increasing apoptosis in prostate, colon, and pancreatic cancer cells. In the case of TOP1, MDM2, and MYC, their corresponding PPRHs produced a strong effect in decreasing cell viability and mRNA levels and increasing apoptosis in breast cancer cells. Thus, we confirm that the PPRH technology is broadly useful to silence the expression of cancer-related genes as demonstrated using target genes involved in metabolism (DHFR), proliferation (mTOR), DNA topology (TOP1), lifespan and senescence (telomerase), apoptosis (survivin, BCL2), transcription factors (MYC), and proto-oncogenes (MDM2).

  8. Cloning of Novel Repeat-associated Small RNAs Derived from Hairpin Precursors in Oryza sativa

    Institute of Scientific and Technical Information of China (English)

    Chengguo YAO; Botao ZHAO; Wei LI; Yang LI; Wenming QIN; Bing HUANG; Youxin JIN

    2007-01-01

    Plant small non-coding RNAs including microRNAs (miRNAs), small interfering RNAs (siRNAs) and trans-acting siRNAs, play important roles in modulating gene expression in cells. Here we isolated 21 novel endogenous small RNA molecules, ranging from 18 to 24 nucleotides, in Oryza sativa that can be mapped to 111 hairpin precursors. Further analysis indicated that most of these hairpin sequences originated from putative miniature inverted-repeat transposable elements, a major type of DNA transposon.Considering that miRNA is characteristic of hairpin-like precursor and plant endogenous siRNAs are often located at transposon regions, we hypothesized that our cloned small RNAs might represent the intermediate product in the evolutionary process between siRNAs and miRNAs. Northern blot analysis indicated that five of them were much more abundantly expressed in flower compared to other tissues, implying their potential function in inflorescence. In conclusion, our results enrich rice small RNA data and provide a meaningful perspective for small RNA annotation in plants.

  9. Metal ion binding and function in natural and artificial small RNA enzymes from a structural perspective.

    Science.gov (United States)

    Wedekind, Joseph E

    2011-01-01

    Ribozymes are often perceived as part of an antiquated catalytic arsenal hearkening back to a pre-biotic RNA World that was eventually supplanted by proteins. However, recent genome-wide searches have revealed a plethora of new catalytic RNA motifs that appear to be variations on well-known themes. This suggests that ribozymes have continued to evolve in order to fulfill specific, RNA-essential biological niches. Although such ribozymes are small and catalyze one-step phosphodiester-bond scission reactions, ongoing structure and function analyses at the lab bench have demonstrated that RNA has the capacity for a diverse number of reactions such as carbon-carbon bond formation, and tRNA aminoacylation. Here we describe the fundamental structure and metal binding properties of four naturally occurring RNA enzymes: the hammerhead, hairpin, hepatitis delta virus, and glmS metabolite sensing ribozyme. In addition, we discuss the fold and ion coordination of three artificial ribozymes developed to probe the boundaries of RNA catalysis; these include the leadzyme, the flexizyme, and the Diels-Alder ribozyme. Our approach is to relate structure to function with the knowledge of ideal metal-ion coordination geometry that we have derived herein from surveys of high-resolution small molecule structures. An emergent theme is that natural and artificial ribozymes that catalyze single-step reactions often possess a pre-formed active site. Multivalent ions facilitate RNA active site formation, but can also provide Lewis acid functionality that is necessary for catalysis. When metal ion binding isn't possible, ribozymes make due by ionizing their bases, or by recruiting cofactors that augment their chemical functionality.

  10. Characterization of a [open quotes]kissing[close quotes] hairpin complex derived from the human immunodeficiency virus genome

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kung-Yao; Tinoco, Ignacio Jr. (Lawrence Berkeley Lab., CA (United States))

    1994-08-30

    Base-pair formation between hairpin loops-a [open quotes]kissing[close quotes] complex-is an RNA-folding motif that links two elements of RNA secondary structure. It is also a unique protein recognition site involved in regulation of ColE1 plasmid DNA replication. The trans-activation response element (TAR), a hairpin and bulge at the 5[prime] end of the untranslated leader region of the human immunodeficiency virus 1 mRNA, enhances the transcription of the virus and is necessary for viral replication. Gel electrophoresis and absorbance melting curves indicate that a synthesized RNA hairpin (Tar*-16) with a loop sequence complementary to the TAR loop sequence (CUGGGA) associates specifically with a 16-nucleotide TAR hairpin (Tar-16) to form a stable complex. RNase T1 probing indicates that the three guanines in the Tar-16 loop become inaccessible in the complex. NMR imino proton spectra reveal that 5 base pairs are formed between the two hairpin loops (Tar-16 and Tar*-16); only the adenine at the 3[prime] terminus of the TAR loop does not form a base pair with the 5[prime]-terminal uracil of the complementary loop. A 14-nucleotide hairpin [CCUA-(UCCCAG)UAGG] with a loop sequence complementary to the TAR loop is conserved within the gag gene of human immunodeficiency virus 1. A synthesized RNA hairpin corresponding to this conserved sequence also binds to the Tar-16 hairpin with high affinity. It is possible that the same RNA loop-loop interaction occurs during the viral life cycle. 40 refs., 7 figs.

  11. Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination.

    Science.gov (United States)

    Washio, T; Sasayama, J; Tomita, M

    1998-12-01

    Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes. Instead of trying to detect each individual hairpin, we averaged the free energy values around the stop codons over the entire genome to predict how extensively the organism relies on hairpin formation in the process of transcription termination. The free energy values of Escherichia coli K-12 shows a sharp drop, as expected, at 30 bp downstream of the stop codon, presumably due to hairpin-forming sequences. Similar drops are observed for Haemophilus influenzae Rd, Bacillus subtilis and Chlamydia trachomatis, suggesting that these organisms also form hairpins at their transcription termination sites. On the other hand, 12 other prokaryotes- Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis PCC6803, Helicobacter pylori, Borrelia burgdorferi, Methanococcus jannaschii, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Aquifex aeolicus, Pyrococcus horikoshii, Mycobacterium tuberculosis and Treponema pallidum -show no apparent decrease in free energy value at the corresponding regions. This result suggests that these prokaryotes, or at least some of them, may never form hairpins at their transcription termination sites.

  12. Limits of neutral drift: lessons from the in vitro evolution of two ribozymes.

    Science.gov (United States)

    Petrie, Katherine L; Joyce, Gerald F

    2014-10-01

    The relative contributions of adaptive selection and neutral drift to genetic change are unknown but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change. The exploration of sequence space was accelerated by increasing the mutation rate using mutagenic nucleotide analogs. Drift was encouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequence used to initiate evolution. This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral drift does, however, provide a repository of genetic variation upon which adaptive selection can act.

  13. Glucosamine and glucosamine-6-phosphate derivatives: catalytic cofactor analogues for the glmS ribozyme.

    Science.gov (United States)

    Posakony, Jeffrey J; Ferré-D'Amaré, Adrian R

    2013-05-17

    Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactors of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogues, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz), and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogues. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection.

  14. Ribozyme-based insulator parts buffer synthetic circuits from genetic context.

    Science.gov (United States)

    Lou, Chunbo; Stanton, Brynne; Chen, Ying-Ja; Munsky, Brian; Voigt, Christopher A

    2012-11-01

    Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as 'insulator parts', is screened in Escherichia coli. We find that ribozymes that cleave the 5' untranslated region (5'-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.

  15. Bimetallic redox synergy in oxidative palladium catalysis.

    Science.gov (United States)

    Powers, David C; Ritter, Tobias

    2012-06-19

    Polynuclear transition metal complexes, which are embedded in the active sites of many metalloenzymes, are responsible for effecting a diverse array of oxidation reactions in nature. The range of chemical transformations remains unparalleled in the laboratory. With few noteworthy exceptions, chemists have primarily focused on mononuclear transition metal complexes in developing homogeneous catalysis. Our group is interested in the development of carbon-heteroatom bond-forming reactions, with a particular focus on identifying reactions that can be applied to the synthesis of complex molecules. In this context, we have hypothesized that bimetallic redox chemistry, in which two metals participate synergistically, may lower the activation barriers to redox transformations relevant to catalysis. In this Account, we discuss redox chemistry of binuclear Pd complexes and examine the role of binuclear intermediates in Pd-catalyzed oxidation reactions. Stoichiometric organometallic studies of the oxidation of binuclear Pd(II) complexes to binuclear Pd(III) complexes and subsequent C-X reductive elimination from the resulting binuclear Pd(III) complexes have confirmed the viability of C-X bond-forming reactions mediated by binuclear Pd(III) complexes. Metal-metal bond formation, which proceeds concurrently with oxidation of binuclear Pd(II) complexes, can lower the activation barrier for oxidation. We also discuss experimental and theoretical work that suggests that C-X reductive elimination is also facilitated by redox cooperation of both metals during reductive elimination. The effect of ligand modification on the structure and reactivity of binuclear Pd(III) complexes will be presented in light of the impact that ligand structure can exert on the structure and reactivity of binuclear Pd(III) complexes. Historically, oxidation reactions similar to those discussed here have been proposed to proceed via mononuclear Pd(IV) intermediates, and the hypothesis of mononuclear Pd

  16. Transition metal catalysis in confined spaces.

    Science.gov (United States)

    Leenders, Stefan H A M; Gramage-Doria, Rafael; de Bruin, Bas; Reek, Joost N H

    2015-01-21

    Transition metal catalysis plays an important role in both industry and in academia where selectivity, activity and stability are crucial parameters to control. Next to changing the structure of the ligand, introducing a confined space as a second coordination sphere around a metal catalyst has recently been shown to be a viable method to induce new selectivity and activity in transition metal catalysis. In this review we focus on supramolecular strategies to encapsulate transition metal complexes with the aim of controlling the selectivity via the second coordination sphere. As we will discuss, catalyst confinement can result in selective processes that are impossible or difficult to achieve by traditional methods. We will describe the template-ligand approach as well as the host-guest approach to arrive at such supramolecular systems and discuss how the performance of the catalyst is enhanced by confining it in a molecular container.

  17. Inverse magnetic catalysis in dense holographic matter

    CERN Document Server

    Preis, Florian; Schmitt, Andreas

    2010-01-01

    We study the chiral phase transition in a magnetic field at finite temperature and chemical potential within the Sakai-Sugimoto model, a holographic top-down approach to (large-N_c) QCD. We consider the limit of a small separation of the flavor D8-branes, which corresponds to a dual field theory comparable to a Nambu-Jona Lasinio (NJL) model. Mapping out the surface of the chiral phase transition in the parameter space of magnetic field strength, quark chemical potential, and temperature, we find that for small temperatures the addition of a magnetic field decreases the critical chemical potential for chiral symmetry restoration - in contrast to the case of vanishing chemical potential where, in accordance with the familiar phenomenon of magnetic catalysis, the magnetic field favors the chirally broken phase. This "inverse magnetic catalysis" (IMC) appears to be associated with a previously found magnetic phase transition within the chirally symmetric phase that shows an intriguing similarity to a transition ...

  18. ELECTROCHEMICAL PROMOTED CATALYSIS: TOWARDS PRACTICAL UTILIZATION

    Directory of Open Access Journals (Sweden)

    DIMITRIOS TSIPLAKIDES

    2008-07-01

    Full Text Available Electrochemical promotion (EP of catalysis has already been recognized as “a valuable development in catalytic research” (J. Pritchard, 1990 and as “one of the most remarkable advances in electrochemistry since 1950” (J. O’M. Bockris, 1996. Laboratory studies have clearly elucidated the phenomenology of electrochemical promotion and have proven that EP is a general phenomenon at the interface of catalysis and electrochemistry. The major progress toward practical utilization of EP is surveyed in this paper. The focus is given on the electropromotion of industrial ammonia synthesis catalyst, the bipolar EP and the development of a novel monolithic electropromoted reactor (MEPR in conjunction with the electropromotion of thin sputtered metal films. Future perspectives of electrochemical promotion applications in the field of hydrogen technologies are discussed.

  19. Tandem Catalysis Utilizing Olefin Metathesis Reactions.

    Science.gov (United States)

    Zieliński, Grzegorz K; Grela, Karol

    2016-07-01

    Since olefin metathesis transformation has become a favored synthetic tool in organic synthesis, more and more distinct non-metathetical reactions of alkylidene ruthenium complexes have been developed. Depending on the conditions applied, the same olefin metathesis catalysts can efficiently promote isomerization reactions, hydrogenation of C=C double bonds, oxidation reactions, and many others. Importantly, these transformations can be carried out in tandem with olefin metathesis reactions. Through addition of one portion of a catalyst, a tandem process provides structurally advanced products from relatively simple substrates without the need for isolation of the intermediates. These aspects not only make tandem catalysis very attractive from a practical point of view, but also open new avenues in (retro)synthetic planning. However, in the literature, the term "tandem process" is sometimes used improperly to describe other types of multi-reaction sequences. In this Concept, a number of examples of tandem catalysis involving olefin metathesis are discussed with an emphasis on their synthetic value.

  20. Heterogenous catalysis mediated by plasmon heating.

    Science.gov (United States)

    Adleman, James R; Boyd, David A; Goodwin, David G; Psaltis, Demetri

    2009-12-01

    We introduce a new method for performing and miniaturizing many types of heterogeneous catalysis involving nanoparticles. The method makes use of the plasmon resonance present in nanoscale metal catalysts to provide the necessary heat of reaction when illuminated with a low-power laser. We demonstrate our approach by reforming a flowing, liquid mixture of ethanol and water over gold nanoparticle catalysts in a microfluidic channel. Plasmon heating of the nanoparticles provides not only the heat of reaction but the means to generate both water and ethanol vapor locally over the catalysts, which in turn allows the chip and the fluid lines to remain at room temperature. The measured products of the reaction, CO(2), CO, and H(2), are consistent with catalytic steam reforming of ethanol. The approach, which we refer to as plasmon-assisted catalysis, is general and can be used with a variety of endothermic catalytic processes involving nanoparticles.

  1. Fluorescence-based characterization of genetically encoded peptides that fold in live cells: progress toward a generic hairpin scaffold

    Science.gov (United States)

    Cheng, Zihao; Campbell, Robert E.

    2007-02-01

    Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold based on a single β-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant β-hairpin peptides to be rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a 16mer tryptophan zipper β-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer β-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the 20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we experimentally demonstrate that the in vitro determined β-hairpin stabilities are well correlated with in vivo β-hairpin stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer β-hairpin peptides through the use of fluorescence-based library screening.

  2. Nanoscale Advances in Catalysis and Energy Applications

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yimin; Somorjai, Gabor A.

    2010-05-12

    In this perspective, we present an overview of nanoscience applications in catalysis, energy conversion, and energy conservation technologies. We discuss how novel physical and chemical properties of nanomaterials can be applied and engineered to meet the advanced material requirements in the new generation of chemical and energy conversion devices. We highlight some of the latest advances in these nanotechnologies and provide an outlook at the major challenges for further developments.

  3. Catalysis in micellar and macromoleular systems

    CERN Document Server

    Fendler, Janos

    1975-01-01

    Catalysis in Micellar and Macromolecular Systems provides a comprehensive monograph on the catalyses elicited by aqueous and nonaqueous micelles, synthetic and naturally occurring polymers, and phase-transfer catalysts. It delineates the principles involved in designing appropriate catalytic systems throughout. Additionally, an attempt has been made to tabulate the available data exhaustively. The book discusses the preparation and purification of surfactants; the physical and chemical properties of surfactants and micelles; solubilization in aqueous micellar systems; and the principles of

  4. Spatially Assisted Schwinger Mechanism and Magnetic Catalysis

    CERN Document Server

    Copinger, Patrick

    2016-01-01

    Using the worldline formalism we compute an effective action for fermions under a temporally modulated electric field and a spatially modulated magnetic field. It is known that the former leads to an enhanced Schwinger Mechanism, while we find that the latter can also result in enhanced particle production and even cause a reorganization of the vacuum to acquire a larger dynamical mass in equilibrium which spatially assists the Magnetic Catalysis.

  5. Spatially Assisted Schwinger Mechanism and Magnetic Catalysis

    Science.gov (United States)

    Copinger, Patrick; Fukushima, Kenji

    2016-08-01

    Using the worldline formalism we compute an effective action for fermions under a temporally modulated electric field and a spatially modulated magnetic field. It is known that the former leads to an enhanced Schwinger mechanism, while we find that the latter can also result in enhanced particle production and even cause a reorganization of the vacuum to acquire a larger dynamical mass in equilibrium which spatially assists the magnetic catalysis.

  6. Spatially Assisted Schwinger Mechanism and Magnetic Catalysis.

    Science.gov (United States)

    Copinger, Patrick; Fukushima, Kenji

    2016-08-19

    Using the worldline formalism we compute an effective action for fermions under a temporally modulated electric field and a spatially modulated magnetic field. It is known that the former leads to an enhanced Schwinger mechanism, while we find that the latter can also result in enhanced particle production and even cause a reorganization of the vacuum to acquire a larger dynamical mass in equilibrium which spatially assists the magnetic catalysis.

  7. USD Catalysis Group for Alternative Energy

    Energy Technology Data Exchange (ETDEWEB)

    Hoefelmeyer, James D.; Koodali, Ranjit; Sereda, Grigoriy; Engebretson, Dan; Fong, Hao; Puszynski, Jan; Shende, Rajesh; Ahrenkiel, Phil

    2012-03-13

    The South Dakota Catalysis Group (SDCG) is a collaborative project with mission to develop advanced catalysts for energy conversion with two primary goals: (1) develop photocatalytic systems in which polyfunctionalized TiO2 are the basis for hydrogen/oxygen synthesis from water and sunlight (solar fuels group), (2) develop new materials for hydrogen utilization in fuel cells (fuel cell group). In tandem, these technologies complete a closed chemical cycle with zero emissions.

  8. Folded biomimetic oligomers for enantioselective catalysis

    OpenAIRE

    Maayan, Galia; Michael D. Ward; Kirshenbaum, Kent

    2009-01-01

    Many naturally occurring biopolymers (i.e., proteins, RNA, DNA) owe their unique properties to their well-defined three-dimensional structures. These attributes have inspired the design and synthesis of folded architectures with functions ranging from molecular recognition to asymmetric catalysis. Among these are synthetic oligomeric peptide (“foldamer”) mimics, which can display conformational ordering at short chain lengths. Foldamers, however, have not been explored as platforms for asymme...

  9. Heterogenous Catalysis Mediated by Plasmon Heating

    OpenAIRE

    Adleman, J.R.; Boyd, D. A.; Goodwin, D. G.; Psaltis, D.

    2009-01-01

    We introduce a new method for performing and miniaturizing many types of heterogeneous catalysis involving nanoparticles. The method makes use of the plasmon resonance present in nanoscale metal catalysts to provide the necessary heat of reaction when illuminated with a low-power laser. We demonstrate our approach by reforming a flowing, liquid mixture of ethanol and water over gold nanoparticle catalysts in a microfluidic channel. Plasmon heating of the nanoparticles provides not only the he...

  10. Heterogeneous Catalysis on a Disordered Surface

    OpenAIRE

    Frachebourg, L.; Krapivsky, P. L.; Redner, S

    1995-01-01

    We introduce a simple model of heterogeneous catalysis on a disordered surface which consists of two types of randomly distributed sites with different adsorption rates. Disorder can create a reactive steady state in situations where the same model on a homogeneous surface exhibits trivial kinetics with no steady state. A rich variety of kinetic behaviors occur for the adsorbate concentrations and catalytic reaction rate as a function of model parameters.

  11. Predictive Modeling in Actinide Chemistry and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Ping [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-05-16

    These are slides from a presentation on predictive modeling in actinide chemistry and catalysis. The following topics are covered in these slides: Structures, bonding, and reactivity (bonding can be quantified by optical probes and theory, and electronic structures and reaction mechanisms of actinide complexes); Magnetic resonance properties (transition metal catalysts with multi-nuclear centers, and NMR/EPR parameters); Moving to more complex systems (surface chemistry of nanomaterials, and interactions of ligands with nanoparticles); Path forward and conclusions.

  12. A Novel Vector for Abundant Expression of Antisense RNA, Triplex-forming RNA and Ribozyme in vivo

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    For abundant expression of antisense RNA, triplex-forming RNA and Ribozyme in vivo, a novel vector pBSKneorU6' was constructed by PCR cloning. This vector contains the intact human snRNA U6 gene expression unit, yet replacing the 61-nt-sequence in the middle of U6 snRNA coding region with three restriction enzyme sites. Hela nuclear extract in vitro transcription experiments demonstrated that this vector can effectively express U6 mutant RNA. Containing neor at the same time, stably transfected pBSKneorU6' can be selected easily.

  13. Shape-controlled nanostructures in heterogeneous catalysis.

    Science.gov (United States)

    Zaera, Francisco

    2013-10-01

    Nanotechnologies have provided new methods for the preparation of nanomaterials with well-defined sizes and shapes, and many of those procedures have been recently implemented for applications in heterogeneous catalysis. The control of nanoparticle shape in particular offers the promise of a better definition of catalytic activity and selectivity through the optimization of the structure of the catalytic active site. This extension of new nanoparticle synthetic procedures to catalysis is in its early stages, but has shown some promising leads already. Here, we survey the major issues associated with this nanotechnology-catalysis synergy. First, we discuss new possibilities associated with distinguishing between the effects originating from nanoparticle size versus those originating from nanoparticle shape. Next, we survey the information available to date on the use of well-shaped metal and non-metal nanoparticles as active phases to control the surface atom ensembles that define the catalytic site in different catalytic applications. We follow with a brief review of the use of well-defined porous materials for the control of the shape of the space around that catalytic site. A specific example is provided to illustrate how new selective catalysts based on shape-defined nanoparticles can be designed from first principles by using fundamental mechanistic information on the reaction of interest obtained from surface-science experiments and quantum-mechanics calculations. Finally, we conclude with some thoughts on the state of the field in terms of the advances already made, the future potentials, and the possible limitations to be overcome.

  14. Hybrid Amyloid Membranes for Continuous Flow Catalysis.

    Science.gov (United States)

    Bolisetty, Sreenath; Arcari, Mario; Adamcik, Jozef; Mezzenga, Raffaele

    2015-12-29

    Amyloid fibrils are promising nanomaterials for technological applications such as biosensors, tissue engineering, drug delivery, and optoelectronics. Here we show that amyloid-metal nanoparticle hybrids can be used both as efficient active materials for wet catalysis and as membranes for continuous flow catalysis applications. Initially, amyloid fibrils generated in vitro from the nontoxic β-lactoglobulin protein act as templates for the synthesis of gold and palladium metal nanoparticles from salt precursors. The resulting hybrids possess catalytic features as demonstrated by evaluating their activity in a model catalytic reaction in water, e.g., the reduction of 4-nitrophenol into 4-aminophenol, with the rate constant of the reduction increasing with the concentration of amyloid-nanoparticle hybrids. Importantly, the same nanoparticles adsorbed onto fibrils surface show improved catalytic efficiency compared to the same unattached particles, pointing at the important role played by the amyloid fibril templates. Then, filter membranes are prepared from the metal nanoparticle-decorated amyloid fibrils by vacuum filtration. The resulting membranes serve as efficient flow catalysis active materials, with a complete catalytic conversion achieved within a single flow passage of a feeding solution through the membrane.

  15. Continuous-variable entanglement via multiphoton catalysis

    Science.gov (United States)

    Hu, Liyun; Liao, Zeyang; Zubairy, M. Suhail

    2017-01-01

    We theoretically investigate the performance of multiphoton catalysis applied on the two-mode squeezed state by examining the entropy of entanglement, logarithmic negativity, Eistein-Podolsky-Rosen (EPR), and Hillery-Zubairy (HZ) correlations, and the fidelity of teleportation. It is found that the entanglement increases with the number of catalysis operations if the squeezing parameter is low initially. Our comparisons show that the HZ correlation presents a better performance than the EPR correlation for detecting the entanglement, and the improvement of HZ correlation definitely results in the improvement of entropy of entanglement rather than negativity; the region of enhanced EPR correlation is a subregion of all other entanglement properties. In addition, we consider the performances of the fidelity by comparing such operations applied before or after the amplitude damping channel. It is shown that the catalysis operation of m =n =1 before the channel presents the best performance in the initial-low squeezing regime. This may provide a useful insight for a long-distance quantum communication.

  16. Computational approaches to homogeneous gold catalysis.

    Science.gov (United States)

    Faza, Olalla Nieto; López, Carlos Silva

    2015-01-01

    Homogenous gold catalysis has been exploding for the last decade at an outstanding pace. The best described reactivity of Au(I) and Au(III) species is based on gold's properties as a soft Lewis acid, but new reactivity patterns have recently emerged which further expand the range of transformations achievable using gold catalysis, with examples of dual gold activation, hydrogenation reactions, or Au(I)/Au(III) catalytic cycles.In this scenario, to develop fully all these new possibilities, the use of computational tools to understand at an atomistic level of detail the complete role of gold as a catalyst is unavoidable. In this work we aim to provide a comprehensive review of the available benchmark works on methodological options to study homogenous gold catalysis in the hope that this effort can help guide the choice of method in future mechanistic studies involving gold complexes. This is relevant because a representative number of current mechanistic studies still use methods which have been reported as inappropriate and dangerously inaccurate for this chemistry.Together with this, we describe a number of recent mechanistic studies where computational chemistry has provided relevant insights into non-conventional reaction paths, unexpected selectivities or novel reactivity, which illustrate the complexity behind gold-mediated organic chemistry.

  17. Characterizing the formation and regeneration of hairpin vortices in a laminar boundary layer

    Science.gov (United States)

    Sabatino, Daniel R.; Maharjan, Rijan

    2015-12-01

    A free surface water channel is used to study hairpin vortex formation created by fluid injection through a narrow slot into a laminar boundary layer. Particle image velocimetry flow-field measurements of injections into quiescent cross-flow conditions confirm that elongated ring vortices are produced with a nondimensionalized circulation strength that is approximately linear with formation time. Unlike circular ring vortices, a limiting strength is not observed at a nondimensional formation time of 4 due to the proximity of the counter-rotating vortex pair. Identical injections are made into a laminar boundary layer at different free-stream velocities and streamwise slot positions (485 ≤ Reδ∗ ≤ 584) with average injection velocity ratios between 0.08 and 0.16. Visualizations indicate that the shear layer between the low x-momentum injected fluid and the boundary layer creates a Kelvin-Helmholtz instability that forms the hairpin vortex head which then monotonically decreases in circulation strength with downstream distance. A similar process can form, or regenerate, a secondary hairpin vortex upstream of the primary vortex with a circulation strength of the head that is comparable to the strength of the primary head at the time of regeneration. However, the legs of the primary vortex continue to strengthen up to regeneration. The peak circulation in the legs is not directly correlated to the strength of the original elongated ring vortex. However, when the circulation is scaled with the injection momentum ratio it is linearly related to scaled injection time. It is proposed that the injection momentum ratio and nondimensionalized injection time based on the wall normal penetration time can be used to identify threshold conditions which produce a secondary vortex. It is suggested that this criterion may be used to identify the minimum strength of flow structures that would be capable of regeneration and thus transition initiation.

  18. Stability of an amphipathic helix-hairpin surfactant peptide in liposomes.

    Science.gov (United States)

    Waring, Alan J; Gupta, Monik; Gordon, Larry M; Fujii, Gary; Walther, Frans J

    2016-12-01

    Surfactant protein B (SP-B; 79 residues) is a member of the saposin superfamily and plays a pivotal role in lung function. The N- and C-terminal regions of SP-B, cross-linked by two disulfides, were theoretically predicted to fold as charged amphipathic helices, suggesting participation in surfactant activities. Previous studies with oxidized Super Mini-B (SMB), a construct based on the N- and C-regions of SP-B (i.e., residues 1-25 and 63-78) joined with a designer turn (-PKGG-) and two disulfides, indicated that freshly prepared SMB in lipids folded as a surface active, α-helix-hairpin. Because other peptides modeled on α-helical SP domains lost helicity and surfactant activity on storage, experiments were here performed on oxidized SMB in surfactant liposomes stored at ~2-8°C for ≤5.5years. Captive bubble surfactometry confirmed low minimum surface tensions for fresh and stored SMB preparations. FTIR spectroscopy of fresh and stored SMB formulations showed secondary structures compatible with the peptide folding as α-helix-hairpin. A homology (I-TASSER) model of oxidized SMB demonstrated a globular protein, exhibiting a core of hydrophobic residues and a surface of polar residues. Since mass spectroscopy indicated that the disulfides were maintained on storage, the stability of SMB may be partly due to the disulfides bringing the N- and C-α-helices closer. Mass spectroscopy of stored SMB preparations showed some methionine oxidation, and also partial deacylation of surfactant phospholipids to form lyso-derivatives. However, the stable conformation and activity of stored SMB surfactant suggest that the active helix-hairpin resists these chemical changes which otherwise may lead to surfactant inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Role of the DIS hairpin in replication of human immunodeficiency virus type 1.

    Science.gov (United States)

    Berkhout, B; van Wamel, J L

    1996-10-01

    The virion-associated genome of human immunodeficiency virus type 1 consists of a noncovalently linked dimer of two identical, unspliced RNA molecules. A hairpin structure within the untranslated leader transcript is postulated to play a role in RNA dimerization through base pairing of the autocomplementary loop sequences. This hairpin motif with the palindromic loop sequence is referred to as the dimer initiation site (DIS), and the type of interaction is termed loop-loop kissing. Detailed phylogenetic analysis of the DIS motifs in different human and simian immunodeficiency viruses revealed conservation of the hairpin structure with a 6-mer palindrome in the loop, despite considerable sequence divergence. This finding supports the loop-loop kissing mechanism. To test this possibility, proviral genomes with mutations in the DIS palindrome were constructed. The appearance of infectious virus upon transfection into SupT1 T cells was delayed for the DIS mutants compared with that obtained by transfection of the wild-type provirus (pLAI), confirming that this RNA motif plays an important role in virus replication. Surprisingly, the RNA genome extracted from mutant virions was found to be fully dimeric and to have a normal thermal stability. These results indicate that the DIS motif is not essential for human immunodeficiency virus type 1 RNA dimerization and suggest that DIS base pairing does not contribute to the stability of the mature RNA dimer. Instead, we measured a reduction in the amount of viral RNA encapsidated in the mutant virions, suggesting a role of the DIS motif in RNA packaging. This result correlates with the idea that the processes of RNA dimerization and packaging are intrinsically linked, and we propose that DIS pairing is a prerequisite for RNA packaging.

  20. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer.

    Science.gov (United States)

    Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa

    2016-09-01

    A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

  1. Numerical simulation of quasi-streamwise hairpin-like vortex generation in turbulent boundary layer

    Institute of Scientific and Technical Information of China (English)

    ZHANG Nan; LU Li-peng; DUAN Zhen-zhen; YUAN Xiang-jiang

    2008-01-01

    A mechanism for generation of near wall quasi-streamwise hairpin-like vortex (QHV) and secondary quasi-streamwise vortices (SQV) is presented. The conceptual model of resonant triad in the theory of hydrodynamic instability and direct numerical simulation of a turbulent boundary layer were applied to reveal the formation of QHV and SQV. The generation procedures and the characteristics of the vortex structures are obtained, which share some similarities with previous numerical simulations. The research using resonant triad conceptual model and numerical simulation provides a possibility for investigating and controling the vortex structures, which play a dominant role in the evolution of coherent structures in the near-wall region.

  2. Formation and evolution of a hairpin vortex induced by subharmonic sinuous low-speed streaks

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jian; Dong, Gang; Lu, Ziheng, E-mail: dgvehicle@yahoo.com [State Key Laboratory of Transient Physics, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu Province, People’s Republic of China (China)

    2014-10-01

    In this paper, a process of the formation and evolution of hairpin vortices, which originated from the interaction between the spanwise-aligned low-speed streaks with a subharmonic sinuous (SS) oscillation mode, is studied using a direct numerical simulation method in a small periodic local region of an incompressible plane channel flow. The initial artificial perturbations are used to excite the SS-mode oscillation of two spanwise-aligned low-speed streaks in such a flow. A new mechanism of formation and decay of the hairpin vortices is proposed in which the shear layer induced by the spanwise collision and merging between the low-speed streaks is emphasized. Our results show that the streamwise vortices can be induced by the SS-mode streaks and then developed into an X-like pattern at the initial stage due to the mutual induction effect. The X-like vortices further enhance the spanwise oscillation and lift-up of the two streaks that thus lead to the spanwise collision and merging of the low-speed streaks and produce a low-speed region in high-speed fluid. The strong shear layer between the high- and low-speed fluids gives rise to the spanwise vorticity that connects the X-like streamwise vortices and forms the Λ-like vortex. Once the low-speed region entirely enters the high-speed fluid, the shear layer shows the ring shape and results in the transition from a Λ-like vortex to Ω-like one. After that, the viscous diffusion of the low-speed region in the high-speed fluid leads to the decay of the Ω-like vortex; the collision and merging of the low-speed streaks simultaneously reoccur upstream and give birth to a secondary Λ-like vortex, which exhibits behavior that is nearly similar with that of the primary one. Although the hairpin vortex packet is not observed in the present plane channel flow, the regeneration of the hairpin vortex suggests that this type of vortical structure plays an important role in the wall-bounded flow. (paper)

  3. A β-hairpin-binding protein for three different disease-related amyloidogenic proteins.

    Science.gov (United States)

    Shaykhalishahi, Hamed; Mirecka, Ewa A; Gauhar, Aziz; Grüning, Clara S R; Willbold, Dieter; Härd, Torleif; Stoldt, Matthias; Hoyer, Wolfgang

    2015-02-01

    Amyloidogenic proteins share a propensity to convert to the β-structure-rich amyloid state that is associated with the progression of several protein-misfolding disorders. Here we show that a single engineered β-hairpin-binding protein, the β-wrapin AS10, binds monomers of three different amyloidogenic proteins, that is, amyloid-β peptide, α-synuclein, and islet amyloid polypeptide, with sub-micromolar affinity. AS10 binding inhibits the aggregation and toxicity of all three proteins. The results demonstrate common conformational preferences and related binding sites in a subset of the amyloidogenic proteins. These commonalities enable the generation of multispecific monomer-binding agents.

  4. Photon-Ion Catalysis Synergy Material and Its Application

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The co-operation action mechanism and model of photon-ion catalysis synergy material composed of thallium and valency-variable rare earth elements and semiconductor oxide were proposed. The radiation catalysis reactions of water and oxygen assisted by the synergy material that could largely increase electron, free radical and negative ion products were discussed. The applications of photon-ion catalysis synergy material in areas of air cleaning material, antibacterial material, healthy material and energy resource material were suggested.

  5. A new era of catalysis: efficiency, value, and sustainability.

    Science.gov (United States)

    Cheng, Soofin; Lin, Shawn D

    2014-06-01

    Value proposition: Global warming and climate change urge the chemical industry to develop new processes, in which sustainability is a necessity and requirement. Catalysis is recognized to be one of the key technologies in enabling sustainability. This special issue, assembled by guest editors Soofing Chen and Shawn D. Lin, highlights some of the best work presented at "The 6th Asia-Pacific Congress on Catalysis (APCAT-6)", with as major theme "New Era of Catalysis: Efficiency, Value, and Sustainability".

  6. Special Issue: Coinage Metal (Copper, Silver, and Gold Catalysis

    Directory of Open Access Journals (Sweden)

    Sónia Alexandra Correia Carabineiro

    2016-06-01

    Full Text Available The subject of catalysis by coinage metals (copper, silver, and gold comes up increasingly day-by-day. This Special Issue aims to cover the numerous aspects of the use of these metals as catalysts for several reactions. It deals with synthesis and characterization of copper, silver and gold based catalysis, their characterization and use, both for heterogeneous and homogeneous catalysis, and some of their potential applications.

  7. Density functional theory studies of transition metal nanoparticles in catalysis

    DEFF Research Database (Denmark)

    Greeley, Jeffrey Philip; Rankin, Rees; Zeng, Zhenhua

    2013-01-01

    Periodic Density Functional Theory calculations are capable of providing powerful insights into the structural, energetics, and electronic phenomena that underlie heterogeneous catalysis on transition metal nanoparticles. Such calculations are now routinely applied to single crystal metal surfaces...... and to subnanometer metal clusters. Descriptions of catalysis on truly nanosized structures, however, are generally not as well developed. In this talk, I will illustrate different approaches to analyzing nanocatalytic phenomena with DFT calculations. I will describe case studies from heterogeneous catalysis...

  8. Neutrons for Catalysis: A Workshop on Neutron Scattering Techniques for Studies in Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Overbury, Steven {Steve} H [ORNL; Coates, Leighton [ORNL; Herwig, Kenneth W [ORNL; Kidder, Michelle [ORNL

    2011-10-01

    This report summarizes the Workshop on Neutron Scattering Techniques for Studies in Catalysis, held at the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory (ORNL) on September 16 and 17, 2010. The goal of the Workshop was to bring experts in heterogeneous catalysis and biocatalysis together with neutron scattering experimenters to identify ways to attack new problems, especially Grand Challenge problems in catalysis, using neutron scattering. The Workshop locale was motivated by the neutron capabilities at ORNL, including the High Flux Isotope Reactor (HFIR) and the new and developing instrumentation at the SNS. Approximately 90 researchers met for 1 1/2 days with oral presentations and breakout sessions. Oral presentations were divided into five topical sessions aimed at a discussion of Grand Challenge problems in catalysis, dynamics studies, structure characterization, biocatalysis, and computational methods. Eleven internationally known invited experts spoke in these sessions. The Workshop was intended both to educate catalyst experts about the methods and possibilities of neutron methods and to educate the neutron community about the methods and scientific challenges in catalysis. Above all, it was intended to inspire new research ideas among the attendees. All attendees were asked to participate in one or more of three breakout sessions to share ideas and propose new experiments that could be performed using the ORNL neutron facilities. The Workshop was expected to lead to proposals for beam time at either the HFIR or the SNS; therefore, it was expected that each breakout session would identify a few experiments or proof-of-principle experiments and a leader who would pursue a proposal after the Workshop. Also, a refereed review article will be submitted to a prominent journal to present research and ideas illustrating the benefits and possibilities of neutron methods for catalysis research.

  9. Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes

    Directory of Open Access Journals (Sweden)

    Priya Mishra

    2016-06-01

    Full Text Available The chikungunya virus (CHIKV is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNAval Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR, comparative TCID50-IFA analysis, in situ Immuno-fluorescent Assays (IFA and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies

  10. Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes

    Science.gov (United States)

    Mishra, Priya; Furey, Colleen; Balaraman, Velmurugan; Fraser, Malcolm J.

    2016-01-01

    The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs) targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNAval Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE) Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR), comparative TCID50-IFA analysis, in situ Immuno-fluorescent Assays (IFA) and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies PMID:27294950

  11. Magnetic catalysis and inverse magnetic catalysis in nonlocal chiral quark models

    Science.gov (United States)

    Pagura, V. P.; Gómez Dumm, D.; Noguera, S.; Scoccola, N. N.

    2017-02-01

    We study the behavior of strongly interacting matter under an external constant magnetic field in the context of nonlocal chiral quark models within the mean field approximation. We find that at zero temperature the behavior of the quark condensates shows the expected magnetic catalysis effect, our predictions being in good quantitative agreement with lattice QCD results. On the other hand, in contrast to what happens in the standard local Nambu-Jona-Lasinio model, when the analysis is extended to the case of finite temperature, our results show that nonlocal models naturally lead to the inverse magnetic catalysis effect.

  12. Magnetic catalysis and inverse magnetic catalysis in nonlocal chiral quark models

    CERN Document Server

    Pagura, V P; Noguera, S; Scoccola, N N

    2016-01-01

    We study the behavior of strongly interacting matter under an external constant magnetic field in the context of nonlocal chiral quark models within the mean field approximation. We find that at zero temperature the behavior of the quark condensates shows the expected magnetic catalysis effect, our predictions being in good quantitative agreement with lattice QCD results. On the other hand, in contrast to what happens in the standard local Nambu-Jona-Lasinio model, when the analysis is extended to the case of finite temperature our results show that nonlocal models naturally lead to the Inverse Magnetic Catalysis effect.

  13. Controllable Catalysis with Nanoparticles: Bimetallic Alloy Systems and Surface Adsorbates

    KAUST Repository

    Chen, Tianyou

    2016-05-16

    Transition metal nanoparticles are privileged materials in catalysis due to their high specific surface areas and abundance of active catalytic sites. While many of these catalysts are quite useful, we are only beginning to understand the underlying catalytic mechanisms. Opening the “black box” of nanoparticle catalysis is essential to achieve the ultimate goal of catalysis by design. In this Perspective we highlight recent work addressing the topic of controlled catalysis with bimetallic alloy and “designer” adsorbate-stabilized metal nanoparticles.

  14. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    Science.gov (United States)

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay.

  15. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers.

    Science.gov (United States)

    Durand, Guillaume; Dausse, Eric; Goux, Emma; Fiore, Emmanuelle; Peyrin, Eric; Ravelet, Corinne; Toulmé, Jean-Jacques

    2016-05-19

    Loop-loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop-loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 10(6) RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5'CCNY and 5'RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch-ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop-loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch-aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. In Vitro Biological Activity of Anti-C Ⅱ TA Hammerhead Ribozyme--A Novel Approach for Autoimmune Diseases

    Institute of Scientific and Technical Information of China (English)

    刘芳; 邹萍; 郭荣; 陆华中; 范华骅

    2003-01-01

    This study investigated the feasibility of using an hammerhead ribozyme against C Ⅱ TA, a major regulator of MHC Ⅱ antigens, to repress the expression of MHC Ⅱ molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463-465 GUC triplet of C Ⅱ TA and its target gene were transcribed, then mixed up and incubated in vitro. The cleavage products were analyzed by PAGE and silver-staining. Rz464 was then inserted into the pIRES2-EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class Ⅱ MHC induction by recombinant human interferon-gamma (IFN-γ). mRNA of C Ⅱ TA was measured by RT-PCR. Our results showed that Rz464 could exclusively cleave C Ⅱ TA RNA. When induced with IFN-γ, the expression of HLA-DR, -DP, -DQ on pRz464+ Hela was induced, and the mRNA content of C ⅡTA decreased too. It is concluded that Rz464 could inhibit C Ⅱ TA and thus the family of genes was regulated by C Ⅱ TA:MHC Ⅱ molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto-immune diseases.

  17. A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus.

    Science.gov (United States)

    Chen, Huixin; Takei, Fumie; Koay, Evelyn Siew-Chuan; Nakatani, Kazuhiko; Chu, Justin Jang Hann

    2013-03-01

    Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples.

  18. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening.

    Directory of Open Access Journals (Sweden)

    Kimberly Brown Dahlman

    Full Text Available There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  19. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening.

    Science.gov (United States)

    Dahlman, Kimberly Brown; Parker, Joel S; Shamu, Tambudzai; Hieronymus, Haley; Chapinski, Caren; Carver, Brett; Chang, Kenneth; Hannon, Gregory J; Sawyers, Charles L

    2012-01-01

    There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  20. Ultrasensitive detection of microRNAs based on hairpin fluorescence probe assisted isothermal amplification.

    Science.gov (United States)

    Ma, Cuiping; Liu, Sen; Shi, Chao

    2014-08-15

    A hairpin fluorescence probe assisted isothermal amplification strategy was used for microRNAs (miRNAs) detection. The fluorescence hairpin probe was rationally designed by software NUPACK to reduce background signal. This isothermal amplification method consisted of two circuits. The amplification strategy not only could detect miRNA, but also amplified and reversely transcribed miRNA into DNA to enhance the stability of the target. The approach was ultrasensitive and as low as 8.5×10(-15)mol/L miR-Let-7a, corresponding to 8.5×10(-20)mol miR-Let-7a in 10µL, was able to be detected within 20min at 37°C. Moreover, successful detection of miR-Let-7a in a total RNA sample was also achieved. Thus, the rapid, simple, isothermal, and highly sensitive approach should be a promising tool for on-the-spot detection.

  1. Grooved nanowires from self-assembling hairpin molecules for solar cells.

    Science.gov (United States)

    Tevis, Ian D; Tsai, Wei-Wen; Palmer, Liam C; Aytun, Taner; Stupp, Samuel I

    2012-03-27

    One of the challenges facing bulk heterojunction organic solar cells is obtaining organized films during the phase separation of intimately mixed donor and acceptor components. We report here on the use of hairpin-shaped sexithiophene molecules to generate by self-assembly grooved nanowires as the donor component in bulk heterojunction solar cells. Photovoltaic devices were fabricated via spin-casting to produce by solvent evaporation a percolating network of self-assembled nanowires and fullerene acceptors. Thermal annealing was found to increase power conversion efficiencies by promoting domain growth while still maintaining this percolating network of nanostructures. The benefits of self-assembly and grooved nanowires were examined by building devices from a soluble sexithiophene derivative that does not form one-dimensional structures. In these systems, excessive phase separation caused by thermal annealing leads to the formation of defects and lower device efficiencies. We propose that the unique hairpin shape of the self-assembling molecules allows the nanowires as they form to interact well with the fullerenes in receptor-ligand type configurations at the heterojunction of the two domains, thus enhancing device efficiencies by 23%.

  2. Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids.

    Science.gov (United States)

    Lv, Yifan; Cui, Liang; Peng, Ruizi; Zhao, Zilong; Qiu, Liping; Chen, Huapei; Jin, Cheng; Zhang, Xiao-Bing; Tan, Weihong

    2015-12-01

    Here, we propose an efficient strategy for enzyme- and hairpin-free nucleic acid detection called an entropy beacon (abbreviated as Ebeacon). Different from previously reported DNA hybridization/displacement-based strategies, Ebeacon is driven forward by increases in the entropy of the system, instead of free energy released from new base-pair formation. Ebeacon shows high sensitivity, with a detection limit of 5 pM target DNA in buffer and 50 pM in cellular homogenate. Ebeacon also benefits from the hairpin-free amplification strategy and zero-background, excellent thermostability from 20 °C to 50 °C, as well as good resistance to complex environments. In particular, based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs, Ebeacon also shows high selectivity toward base mutations, such as substitution, insertion, and deletion and, therefore, is an efficient nucleic acid detection method, comparable to most reported enzyme-free strategies.

  3. Predicting 3D structure, flexibility and stability of RNA hairpins in monovalent and divalent ion solutions

    CERN Document Server

    Shi, Ya-Zhou; Wang, Feng-Hua; Zhu, Xiao-Long; Tan, Zhi-Jie

    2015-01-01

    A full understanding of RNA-mediated biology would require the knowledge of three-dimensional (3D) structures, structural flexibility and stability of RNAs. To predict RNA 3D structures and stability, we have previously proposed a three-bead coarse-grained predictive model with implicit salt/solvent potentials. In this study, we will further develop the model by improving the implicit-salt electrostatic potential and involving a sequence-dependent coaxial stacking potential to enable the model to simulate RNA 3D structure folding in divalent/monovalent ion solutions. As compared with the experimental data, the present model can predict 3D structures of RNA hairpins with bulge/internal loops (<77nt) from their sequences at the corresponding experimental ion conditions with an overall improved accuracy, and the model also makes reliable predictions for the flexibility of RNA hairpins with bulge loops of different length at extensive divalent/monovalent ion conditions. In addition, the model successfully pred...

  4. 抗HPV11 E2核酶的计算机设计%Computer design of anti-HPV11 E2 ribozyme

    Institute of Scientific and Technical Information of China (English)

    侯化; 王曙; 杨光彩

    2001-01-01

    HPV11 E2 mRNA was taken as the target RNA, ribozyme were designed accounting to the hammerhead structure described by Symons. Computer was used to analyze the possible secondary cleavage sites on HPV11 E2 mRNA and to predict the secondary structures of substrate and ribozymes. According to the theory of Symons headhammer ribozyme, there are 32 sites to targetting sequences. The secondary structures of HPV11 mRNA from nt2510 to nt3518 were relatively stable. The region was of important biological function and was the ideal attacking region for ribozyme. Two ribozymes targeting nt2777 and nt3281 on the HPV11 mRNA were designed and were named RZ2777 and RZ3281 respectively. No analogous sequence of substrate which combined with ribozyme was found in the mRNA of others genes in known Human Gene Bank through computer probe. Not all GUX or CUX could be taken as the cleavage site of ribozyme. The nt2777 and nt3281 onr HPV11 mRNA are the most ideal attacking sites for ribozymes. So computer analysis was used to select the optium ribozyme as soon as possible and to lay a solid foundation for using ribozyme in vivo.%借助计算机软件分析,设计出能特异性切割HPV11型644nt E2 mRNA的核酶。遵循Symons锤头状核酶结构和GUX剪切位点原则,靶序列存在32个剪切位点,通过计算机软件分析核酶的最佳剪切位点,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析,筛选出2个锤头结构核酶。针对这两位点设计的核酶分别命名为RZ2777和RZ3281。计算机分析显示,两核酶与底物切点两翼碱基形成锤头状结构,切点所在基因序列具有相对松弛的二级结构,位于该基因重要生物功能区内,是核酶的理想攻击区域,通过基因库检索,在已知人类基因中排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性。并非所有的GUX位点(X:C、U、A)或CUX均可作为核酶的最

  5. Supramolecular control of selectivity in transition metal catalysis: Substrate preorganization & cofactor-steered catalysis

    NARCIS (Netherlands)

    Dydio, P.F.

    2013-01-01

    The selectivity displayed by transition metal catalysts is one of the key elements in catalysis, and various tools to control this by ligand modification have been reported. Some selectivity issues are, however, difficult to solve using the traditional methods. Therefore we have an interest in the

  6. Next-Generation Catalysis for Renewables: Combining Enzymatic with Inorganic Heterogeneous Catalysis for Bulk Chemical Production

    DEFF Research Database (Denmark)

    Vennestrøm, Peter Nicolai Ravnborg; Christensen, C.H.; Pedersen, S.

    2010-01-01

    of combination involves one-pot cascade catalysis with active sites from bio- and inorganic catalysts. In this article the emphasis is placed specifically on oxidase systems involving the coproduction of hydrogen peroxide, which can be used to create new in situ collaborative oxidation reactions for bulk...

  7. Transient β-hairpin formation in α-synuclein monomer revealed by coarse-grained molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hang; Ma, Wen [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Han, Wei [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Schulten, Klaus, E-mail: kschulte@ks.uiuc.edu [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States)

    2015-12-28

    Parkinson’s disease, originating from the intrinsically disordered peptide α-synuclein, is a common neurodegenerative disorder that affects more than 5% of the population above age 85. It remains unclear how α-synuclein monomers undergo conformational changes leading to aggregation and formation of fibrils characteristic for the disease. In the present study, we perform molecular dynamics simulations (over 180 μs in aggregated time) using a hybrid-resolution model, Proteins with Atomic details in Coarse-grained Environment (PACE), to characterize in atomic detail structural ensembles of wild type and mutant monomeric α-synuclein in aqueous solution. The simulations reproduce structural properties of α-synuclein characterized in experiments, such as secondary structure content, long-range contacts, chemical shifts, and {sup 3}J(H{sub N}H{sub C{sub α}})-coupling constants. Most notably, the simulations reveal that a short fragment encompassing region 38-53, adjacent to the non-amyloid-β component region, exhibits a high probability of forming a β-hairpin; this fragment, when isolated from the remainder of α-synuclein, fluctuates frequently into its β-hairpin conformation. Two disease-prone mutations, namely, A30P and A53T, significantly accelerate the formation of a β-hairpin in the stated fragment. We conclude that the formation of a β-hairpin in region 38-53 is a key event during α-synuclein aggregation. We predict further that the G47V mutation impedes the formation of a turn in the β-hairpin and slows down β-hairpin formation, thereby retarding α-synuclein aggregation.

  8. Phylogenetic study on structural elements of HIV-1 poly(A region. 2. USE domain and TAR hairpin

    Directory of Open Access Journals (Sweden)

    Zarudnaya M. I.

    2014-01-01

    Full Text Available Aim Phylogenetic study on structural elements in the poly(A region of human immunodeficiency virus type 1 (HIV-1, in particular the major upstream sequence element (USE, which stimulates polyadenylation of HIV-1 transcript, and the TAR (trans-activation response hairpin, which juxtaposes spatially the AAUAAA and USE signals. Methods. The secondary structure of these elements has been predicted by UNA Fold program. Results. The structure of USE domain and TAR hairpin has been analysed in 1679 HIV-1 genomes and 17 genomes of simian immunodeficiency virus SIVcpzPtt. We found 376 and 588 different sequences for these elements, respectively, and revealed the most frequent base changes and subtypeand country-specific mutations. Only 43 % of HIV-1 isolates contain variants of the USE domain which occur with a frequency 5 % (the main variants and 35 % of isolates contain main variants of the TAR hairpin. We found that the SIV USE domain and TAR hairpin most closely resemble those found in HIV-1 genomes of A/G-containing subtypes. Conclusions. The results of our large-scale phylogenetic study support a hypothesis on the interaction between tRNA3Lys and the 3' end of HIV-1 genomic RNA and a controversial supposition of HIV-1 genome dimerization by the TAR-TAR kissing mechanism. Since the TAR hairpin is a target for developing antiviral drugs based on the inhibition of signal elements, the data on specific structural features of this hairpin may be useful for new antivirals design.

  9. Molecular catalysis of rare-earth elements

    Energy Technology Data Exchange (ETDEWEB)

    Roesky, Peter W. (ed.) [Karlsruhe Institute of Technology (KIT) (Germany). Inst. of Inorganic Chemistry

    2010-07-01

    This volume reviews the recent developments in the use of molecular rare-earth metal compounds in catalysis. Most of the applications deal with homogenous catalysis but in some cases, heterogeneous systems are also mentioned. The rare-earth elements, which are the lanthanides and their close relatives - scandium and yttrium - have not been in the focus of molecular chemistry for a long time and therefore have also not been considered as homogenous catalysts. Although the first organometallic compounds of the lanthanides, which are tris(cyclopentadienyl) lanthanide complexes, were already prepared in the 1950s, it was only in the late 1970s and early 1980s when a number of research groups began to focus on this class of compounds. One reason for the development was the availability of single crystal X-ray diffraction techniques, which made it possible to characterize these compounds.Moreover, new laboratory techniques to handle highly air and moisture sensitive compounds were developed at the same time. Concomitant with the accessibility of this new class of compounds, the application in homogenous catalysis was investigated. One of the first applications in this field was the use of lanthanide metallocenes for the catalytic polymerization of ethylene in the early 1980s. In the last two or three decades, a huge number of inorganic and organometallic compounds of the rare-earth elements were synthesized and some of them were also used as catalysts. Although early work in homogenous catalysis basically focused only on the hydrogenation and polymerization of olefins, the scope for catalytic application today is much broader. Thus, a large number of catalytic {sigma}-bond metathesis reactions, e.g. hydroamination, have been reported in the recent years. This book contains four chapters in which part of the recent development of the use of molecular rare-earth metal compounds in catalysis is covered. To keep the book within the given page limit, not all aspects could be

  10. Catalysis of Schwinger Vacuum Pair Production

    CERN Document Server

    Dunne, Gerald V; Schützhold, Ralf

    2009-01-01

    We propose a new catalysis mechanism for non-perturbative vacuum electron-positron pair production, by superimposing a plane-wave X-ray probe beam with a strongly focused optical laser pulse, such as is planned at the Extreme Light Infrastructure (ELI) facility. We compute the absorption coefficient arising from vacuum polarization effects for photons below threshold in a strong electric field. This set-up should facilitate the (first) observation of this non-perturbative QED effect with planned light sources such as ELI yielding an envisioned intensity of order 10^{26}W/cm^2.

  11. New strategies in chemical synthesis and catalysis

    CERN Document Server

    Pignataro, Bruno

    2012-01-01

    Providing a comprehensive overview of the essential topics, this book covers the core areas of organic, inorganic, organometallic, biochemical synthesis and catalysis.The authors are among the rising stars in European chemistry, a selection of participants in the 2010 European Young Chemists Award competition, and their contributions deal with most of the frontier issues in chemical synthesis. They give an account of the latest research results in chemistry in Europe, as well as the state of the art in their field of research and the outlook for the future.

  12. Surface Science Foundations of Catalysis and Nanoscience

    CERN Document Server

    Kolasinski, Kurt K

    2012-01-01

    Surface science has evolved from being a sub-field of chemistry or physics, and has now established itself as an interdisciplinary topic. Knowledge has developed sufficiently that we can now understand catalysis from a surface science perspective. No-where is the underpinning nature of surface science better illustrated than with nanoscience. Now in its third edition, this successful textbook aims to provide students with an understanding of chemical transformations and the formation of structures at surfaces. The chapters build from simple to more advanced principles with each featuring exerc

  13. Concepts of Modern Catalysis and Kinetics

    CERN Document Server

    Chorkendorff, I

    2003-01-01

    Until now, the literature has offered a rather limited approach to the use of fundamental kinetics and their application to catalytic reactions. Subsequently, this book spans the full range from fundamentals of kinetics and heterogeneous catalysis via modern experimental and theoretical results of model studies to their equivalent large-scale industrial production processes. The result is key knowledge for students at technical universities and professionals already working in industry. "...such an enterprise will be of great value to the community, to professionals as well as graduate an

  14. Relativistic effects in homogeneous gold catalysis.

    Science.gov (United States)

    Gorin, David J; Toste, F Dean

    2007-03-22

    Transition-metal catalysts containing gold present new opportunities for chemical synthesis, and it is therefore not surprising that these complexes are beginning to capture the attention of the chemical community. Cationic phosphine-gold(i) complexes are especially versatile and selective catalysts for a growing number of synthetic transformations. The reactivity of these species can be understood in the context of theoretical studies on gold; relativistic effects are especially helpful in rationalizing the reaction manifolds available to gold catalysts. This Review draws on experimental and computational data to present our current understanding of homogeneous gold catalysis, focusing on previously unexplored reactivity and its application to the development of new methodology.

  15. Catalysis on cobalt oxide-based nanocatalysts

    Science.gov (United States)

    Zhang, Shiran

    Heterogeneous catalysis, being the focus of attention in the realm of catalysis, plays a vital role in modern chemical and energy industries. A prototype of heterogeneous catalyst consists of metal nanoparticles dispersed and supported on a substrate. Transition metal oxide is one of the key components of heterogeneous catalyst and is frequently used as catalyst support for noble metal nanoparticle catalysts due to low cost. As a result of the high cost of noble metal elements, it is particularly favorable to design and develop transition metal oxide-based nanocatalysts mainly made of earthabundant elements with no or less noble metal with comparable or better catalytic performance than noble metal-based nanocatalysts in a catalytic reaction. In some cases, surface chemistry and structure of nanocatalysts are not invariable during catalysis. They evolve in terms of surface restructuring or phase change, which contributes to the complexity of catalyst surface under different catalytic conditions. Transition metal oxides, especially reducible transition metal oxides, have multiple cationic valence states and crystallographic structures. New catalytic active phases or sites could be formed upon surface restructuring under certain catalytic conditions while they may not be preserved if exposed to ambient conditions. Thus, it is essential to characterize catalyst surface under reaction conditions so that chemistry and structure of catalyst surface could be correlated with the corresponding catalytic performance. It also suggests a new route to design nanocatalysts through restructuring catalyst precursor under certain catalytic conditions tracked with in-situ analytical techniques. Catalysis occurs on catalyst surface. For noble metal nanoparticle catalysts, only atoms exposed on surface participate in catalytic processes, while atoms in bulk do not. In order to make full use of noble metal atoms, it is crucial to maximize the dispersion. A configuration of noble metal

  16. LI Can elected president of int'l catalysis association

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ Prof.LI Can,vice directorgeneral of the CAS Dalian Institute of Chemical Physics,was elected new president of the Executive Committee of the International Association for Catalysis Societies (IACS) at the 14th International Congress on Catalysis held from 13 to 18 July in Seoul,ROK.It is the first time for a Chinese scientist to serve the post.

  17. Heterogeneous Catalysis with Renewed Attention: Principles, Theories, and Concepts

    Science.gov (United States)

    Dumeignil, Franck; Paul, Jean-Francois; Paul, Sebastien

    2017-01-01

    With the development of a strong bioeconomy sector related to the creation of next-generation biorefineries, heterogeneous catalysis is receiving renewed attention. Indeed, catalysis is at the core of biorefinery design, and many new catalysts and catalytic processes are being developed. On the one hand, they are based on knowledge acquired during…

  18. The nature of the active site in heterogeneous metal catalysis

    DEFF Research Database (Denmark)

    Nørskov, Jens Kehlet; Bligaard, Thomas; Larsen, Britt Hvolbæk

    2008-01-01

    This tutorial review, of relevance for the surface science and heterogeneous catalysis communities, provides a molecular-level discussion of the nature of the active sites in metal catalysis. Fundamental concepts such as "Bronsted-Evans-Polanyi relations'' and "volcano curves'' are introduced...

  19. Factors Affecting the Relative Efficiency of General Acid Catalysis

    Science.gov (United States)

    Kwan, Eugene E.

    2005-01-01

    A simple framework for evaluating experimental kinetic data to provide support for Specific Acid Catalysis (SAC) and General Acid Catalysis (GAC) is described based on the factors affecting their relative efficiency. Observations reveal that increasing the SAC-to-GAC rate constant ratio reduces the effective pH range for GAC.

  20. Chloromethylation of 2-chloroethylbenzene catalyzed by micellar catalysis

    Institute of Scientific and Technical Information of China (English)

    LIU QiFa; LU Ming; WEI Wei

    2009-01-01

    The chloromethylation reaction of 2-chloroethylbenzene was performed successfully by micellar catalysis in the biphasic oil/water system.The effects of anionic,cationic and non-ionic surfactants on the reaction were compared.The mechanism of chloromethyiation reaction and the mechanism of micellar catalysis were investigated.The results show that the micellar catalysis is an effective way to realize the chloromethylation of 2-chloroethylbenzene,and the cationic surfactant shows the most effectiveness.The longer the hydrophobic chain of the cationic surfactant is,the better the catalysis effect will be,and the addition of inorganic electrolyte into the aqueous phase can markedly promote the catalysis effect.

  1. Ribozyme对癌基因ki-rasG12V mRNA的剪切及其特异性%Cleavage of Oncogene ki-rasG12V mRNA by Ribozyme and It' s Specificity

    Institute of Scientific and Technical Information of China (English)

    吴国祥; 方裕强; 许国铭; 李兆申; 陆德如

    2000-01-01

    目的:设计切割ki-rasG12vmRNA的特异性ribozyme(Rz217),明确其对癌基因ki-rasG12VmRNA的细胞内外切割活性,为以ki-rasG12VmRNA为特异性靶分子的基因治疗及癌基因ki-ras的功能研究提拱一种新的途径。方法:依Symons总结的"锤头结构"原理,设计一种能特异性切割ki-rasG12VmRNA的ribozyme,利用DNA重组技术构建ki-rasG12V外显子1和ri-bozyme Rz217的体外转录质粒及ribozyme Rz217的真核表达质粒,体外转录获得ribozyme Rz217及ki-rasG12V外显子1 mRNA,在含Mg2+溶液中ribozyme Rz217对其靶RNA分子进行切割。以RT-PCR对转染ribozyme Rz217真核表达质粒的细胞ki-rasG12VmRNA进行半定量分析。结果:ki-rasG12V外显子1体外转录mRNA分子,能被ribozyme Rz217定点切割而野生型ki-ras外显子1体外转录mRNA则不被切割;转染ribozyme Rz217的胰癌细胞ki-rasG12VmRNA含量减少,而转染ribozyme Re217的肝癌细胞其内源性ki-ras mRNA含量无明显变化。结论:ribozyme Rz217无论在细胞内外均能剪切突变型ki-ras mRNA(G12V)而且其切割作用为突变型ki-rasG12VmRNA特异性的。%Objective: To design and confirm the cleavage activity of ribozyme Rz217 to oncogene ki-rasG12V messenger RNA and search for a new method for gene therapy targeting oncogene ki-ras. Methods: According to Symon' s principle,design an ribozyme specific for ki-rasc12v mRNA, both the constructs for transcription in vitro of ribozyme Rz217 and ki-ras exonl and the mammalian expression constructs of ribozyme Rz217 were constructed by DNA recombinant technique,ribozyme Rz217 and ki-ras exonl mRNA was obtained by transcription in vitro with T7 and SP6 RNA polymerase. Pancre atic carcinoma cell line PaTu8988 and human hepatocellular carcinomacell line BEL7404 were transfected with Rz217 mammalian expression constructs and the level of endogenous ki-rasG12V mRNA or ki-ras mRNA was determined by semiquantitative RT-PCR. Results: Not only in vitro

  2. Mechanical catalysis on the centimetre scale.

    Science.gov (United States)

    Miyashita, Shuhei; Audretsch, Christof; Nagy, Zoltán; Füchslin, Rudolf M; Pfeifer, Rolf

    2015-03-06

    Enzymes play important roles in catalysing biochemical transaction paths, acting as logical machines through the morphology of the processes. A key challenge in elucidating the nature of these systems, and for engineering manufacturing methods inspired by biochemical reactions, is to attain a comprehensive understanding of the stereochemical ground rules of enzymatic reactions. Here, we present a model of catalysis that can be performed magnetically by centimetre-sized passive floating units. The designed system, which is equipped with permanent magnets only, passively obeys the local causalities imposed by magnetic interactions, albeit it shows a spatial behaviour and an energy profile analogous to those of biochemical enzymes. In this process, the enzyme units trigger physical conformation changes of the target by levelling out the magnetic potential barrier (activation potential) to a funnel type and, thus, induce cascading conformation changes of the targeted substrate units reacting in parallel. The inhibitor units, conversely, suppress such changes by increasing the potential. Because the model is purely mechanical and established on a physics basis in the absence of turbulence, each performance can be explained by the morphology of the unit, extending the definition of catalysis to systems of alternative scales.

  3. Transition Metal Catalysis Using Functionalized Dendrimers.

    Science.gov (United States)

    Oosterom, G. Eric; Reek, Joost N. H.; Kamer, Paul C. J.; van Leeuwen, Piet W. N. M.

    2001-05-18

    Dendrimers are well-defined hyperbranched macromolecules with characteristic globular structures for the larger systems. These novel polymers have inspired many chemists to develop new materials and several applications have been explored, catalysis being one of them. The recent impressive strides in synthetic procedures increased the accessibility of functionalized dendrimers, resulting in a rapid development of dendrimer chemistry. The position of the catalytic site(s) as well as the spatial separation of the catalysts appears to be of crucial importance. Dendrimers that are functionalized with transition metals in the core potentially can mimic the properties of enzymes, their efficient natural counterparts, whereas the surface-functionalized systems have been proposed to fill the gap between homogeneous and heterogeneous catalysis. This might yield superior catalysts with novel properties, that is, special reactivity or stability. Both the core and periphery strategies lead to catalysts that are sufficiently larger than most substrates and products, thus separation by modern membrane separation techniques can be applied. These novel homogeneous catalysts can be used in continuous membrane reactors, which will have major advantages particularly for reactions that benefit from low substrate concentrations or suffer from side reactions of the product. Here we review the recent progress and breakthroughs made with these promising novel transition metal functionalized dendrimers that are used as catalysts, and we will discuss the architectural concepts that have been applied.

  4. Cisplatin sensitivity and mechanisms of anti-HPV16 E6-ribozyme on cervical carcinoma CaSKi cell line

    Institute of Scientific and Technical Information of China (English)

    Zhiguo Rao; Jianfei Gao; Bicheng Zhang; Bo Yang; Jiren Zhang

    2012-01-01

    Objective: The aim of this study was to study the cisplatin sensitizing effect and mechanism of anti-HPV16 E6- ribozyme on cervical carcinoma cell line.Methods: The anti-HPV16E6-ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell, which named as CaSKi-R, CaSKi-P respectively.E6 mRNA, the sensitivity to cisplatin, apoptosis rates, expression of p53, Bcl-2, Bax and C-myc proteins and mRNA were examined by Northern blot, MTT colorimetric assay, PI/Annexin V stained methods, flow cytometry anslysis and RT-PCR, respectively.Results: E6 mRNA was less in CaSKi-R than in CaSKi.The sensitivity of CaSKi-R cells to cisplatin was 2.28 and 2.21 times than that of CaSKi and CaSKi-P cells.The apoptotic rates in CaSKi, CaSKi-P and CaSKi-R cells was (18.9 ± 3.5)%, (19.7 ± 4.8)% and (40.4 ± 4.5)%.The apoptotic rates was increased in CaSKi-R than that of CaSKi cells treated with cisplatin (P = 0.003).Comapred with CaSKi cell, the expression of p53 (P = 0.000), Bax protein (P = 0.002) was significantly higher and the expression of Bcl-2 protein (P = 0.005), C-myc protein (P = 0.005) was significantly lower in CaSKi-R than that of CaSKi cell treated with cisplatin.Comapred with CaSKi cell, the expression of p53, Bax mRNA in CaSKi-R cell treated with cisplatin increased, while Bcl-2, C-myc mRNA decreased.Conclusion: CaSKi-R cells transfected by anti-HPVE6-ribozyme increased the sensitivity to cisplatin.The increase of sensitivity to cisplatin in CaSKi-R cells may be associated with increasing expression of p53, Bax protein, and decreasing expression of C-myc, Bcl-2 proteins.

  5. Microsecond resolved electron density measurements with a hairpin resonator probe in a pulsed ICP discharge

    CERN Document Server

    Peterson, David; Larson, Lynda; Shannon, Steven

    2016-01-01

    Time resolved electron density measurements in pulsed RF discharges are shown using a hairpin resonance probe using low cost electronics, on par with normal Langmuir probe boxcar mode operation. Time resolution of less than one microsecond has been demonstrated. A signal generator produces the applied microwave frequency; the reflected waveform is passed through a directional coupler and filtered to remove the RF component. The signal is heterodyned with a frequency mixer and read by an oscilloscope. At certain points during the pulse, the plasma density is such that the applied frequency is the same as the resonance frequency of the probe/plasma system, creating a dip in the reflected signal. The applied microwave frequency is shifted in small increments in a frequency boxcar routine to determine the density as a function of time. The system uses a grounded probe to produce low cost, high fidelity, and highly reproducible electron density measurements that can work in harsh chemical environments. Measurement...

  6. Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures

    Energy Technology Data Exchange (ETDEWEB)

    Nandi, Chayan K.; Parui, Partha P.; Brutschy, Bernhard [University of Frankfurt, Institute for Physical and Theoretical Chemistry, Frankfurt (Germany); Schmidt, Thorsten L.; Heckel, Alexander [University of Frankfurt, Cluster of Excellence Macromolecular Complexes, c/o Institute for Organic Chemistry and Chemical Biology, Frankfurt (Germany)

    2008-03-15

    We have recently constructed a ''DNA strut'' consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson-Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions. (orig.)

  7. Nanoplasmonic Tweezers Visualize Protein p53 Suppressing Unzipping of Single DNA-Hairpins

    CERN Document Server

    Kotnala, Abhay

    2014-01-01

    Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein-DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be $2\\times 10^{-20}$ J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein-DNA interactions to revolutionize the fight against genetic diseases.

  8. Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

    Science.gov (United States)

    Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

    2012-09-15

    A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Double nanohole optical tweezers visualize protein p53 suppressing unzipping of single DNA-hairpins.

    Science.gov (United States)

    Kotnala, Abhay; Gordon, Reuven

    2014-06-01

    Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein-DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be 2 × 10(-20) J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein-DNA interactions to revolutionize the fight against genetic diseases.

  10. Structure of the antimicrobial beta-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Kaznessis, Yiannis N

    2007-01-01

    All atom molecular dynamics simulations of the 18-residue beta-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH(2)) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure of t...

  11. Long inverted repeat transiently stalls DNA replication by forming hairpin structures on both leading and lagging strands.

    Science.gov (United States)

    Lai, Pey Jiun; Lim, Chew Theng; Le, Hang Phuong; Katayama, Tsutomu; Leach, David R F; Furukohri, Asako; Maki, Hisaji

    2016-02-01

    Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading- and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCD-sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.

  12. Structural classification of the amide I sites of a beta-hairpin with isotope label 2DIR spectroscopy.

    NARCIS (Netherlands)

    Roy, S.; Jansen, T.L.Th.A.; Knoester, J.

    2010-01-01

    We present a theoretical study of the possibility to use isotope label two-dimensional infrared (2DIR) spectroscopy to obtain site specific structural information in trpzip2. This small beta-hairpin peptide was designed as a model system for studying protein folding in beta-sheet structures. In

  13. Dual hairpin-like molecular beacon based on coralyne-adenosine interaction for sensing melamine in dairy products.

    Science.gov (United States)

    Wang, Guangfeng; Zhu, Yanhong; Chen, Ling; Zhang, Xiaojun

    2014-11-01

    This study presents a novel dual hairpin-like molecular beacon (MB) for the selective and sensitive detection of melamine (MA) based on the conjugation of MA and thymine. In this protocol, the coordination between coralyne and adenosine (A) leaded a dual hairpin-like MB and the fluorophore-quencher pair is close proximity resulting in the fluorescence quenching. With the addition of MA, it conjugated with thymine in the loop part of dual hairpin-like MB by triple H-bonds, triggering the dissociation of the dual hairpin-like MB. The resulting spatial separation of the fluorophore from quencher induced the enhancement in fluorescence emission. Under the optimized conditions, the sensor exhibited a wide linear range of 8×10(-9)-1.6×10(-5) M (R(2)=0.9969) towards MA, with a low detection limit of 5 nM, approximately 4000 times lower than the Drug Administration and the US Food estimated MA safety limit. The real milk samples were also investigated with a satisfying result.

  14. A Comparison of Lu You’s Phoenix Hairpin and Elizabeth Barrett Browning’s Sonnets from the Portuguese (43)

    Institute of Scientific and Technical Information of China (English)

    雷华

    2010-01-01

    Both Lu You and Elizabeth Barrett Browning enjoy great popularity as a poet.In this paper I compare and contract Lu You’s well-known love ci-poem Phoenix Hairpin with Elizabeth Barrett Browning’s famous Sonnets from the Portuguese (43) in respect of theme,style,and way of expression.

  15. Combinatorial pattern discovery approach for the folding trajectory analysis of a beta-hairpin.

    Directory of Open Access Journals (Sweden)

    Laxmi Parida

    2005-06-01

    Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity c in RO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the

  16. Combinatorial Pattern Discovery Approach for the Folding Trajectory Analysis of a beta-Hairpin.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity cinRO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the

  17. Water isotope effect on the thermostability of a polio viral RNA hairpin: A metadynamics study

    Science.gov (United States)

    Pathak, Arup K.; Bandyopadhyay, Tusar

    2017-04-01

    Oral polio vaccine is considered to be the most thermolabile of all the common childhood vaccines. Despite heavy water (D2O) having been known for a long time to stabilise attenuated viral RNA against thermodegradation, the molecular underpinnings of its mechanism of action are still lacking. Whereas, understanding the basis of D2O action is an important step that might reform the way other thermolabile drugs are stored and could possibly minimize the cold chain problem. Here using a combination of parallel tempering and well-tempered metadynamics simulation in light water (H2O) and in D2O, we have fully described the free energy surface associated with the folding/unfolding of a RNA hairpin containing a non-canonical basepair motif, which is conserved within the 3'-untranslated region of poliovirus-like enteroviruses. Simulations reveal that in heavy water (D2O) there is a considerable increase of the stability of the folded basin as monitored through an intramolecular hydrogen bond (HB), size, shape, and flexibility of RNA structures. This translates into a higher melting temperature in D2O by 41 K when compared with light water (H2O). We have explored the hydration dynamics of the RNA, hydration shell around the RNA surface, and spatial dependence of RNA-solvent collective HB dynamics in the two water systems. Simulation in heavy water clearly showed that D2O strengthens the HB network in the solvent, lengthens inter-residue water-bridge lifetime, and weakens dynamical coupling of the hairpin to its solvation environment, which enhances the rigidity of solvent exposed sites of the native configurations. The results might suggest that like other added osmoprotectants, D2O can act as a thermostabilizer when used as a solvent.

  18. Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance.

    Science.gov (United States)

    Chou, Shuli; Shao, Changxuan; Wang, Jiajun; Shan, Anshan; Xu, Lin; Dong, Na; Li, Zhongyu

    2016-01-01

    The β-hairpin structure has been proposed to exhibit potent antimicrobial properties with low cytotoxicity, thus, multiple β-hairpin structures have been proved to be highly stable in structures containing tightly packed hydrophobic cores. The aim of this study was to develop peptide-based synthetic strategies for generating short, but effective AMPs as inexpensive antimicrobial agents. Multiple-stranded β-hairpin peptides with the same β-hairpin unit, (WRXxRW)n where n=1, 2, 3, or 4 and Xx represent the turn sequence, were synthesized, and their potential as antimicrobial agents was evaluated. Owning to the tightly packed hydrophobic core and paired Trp of this multiple-stranded β-hairpin structure, all the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells (hRBCs), and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8μM against various tested bacteria. Not only that, but W2 also showed obvious synergy with streptomycin and chloramphenicol against Escherichia coli, and displayed synergy with ciprofloxacin against Staphylococcus aureus with the FICI values ⩽0.5. Fluorescence spectroscopy and electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Collectively, based on the multiple β-hairpin peptides, the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents. We successfully screened a peptide W2 ((WRPGRW)2) from a series of multiple-stranded β-hairpin antimicrobial peptides based on the "S-shaped" motif that induced the formation of a globular structure, and Trp zipper was used to replace the disulfide bonds to reduce the cost of production. This novel structure applied to AMPs improved cell selectivity and salt stability. The findings of this study will promote the development of peptide

  19. Magnetic catalysis (and inverse catalysis) at finite temperature in two-color lattice QCD

    CERN Document Server

    Ilgenfritz, E -M; Petersson, B; Schreiber, A

    2013-01-01

    Two-color lattice QCD with N_f=4 staggered fermion degrees of freedom (no rooting trick is applied) with equal electric charge q is studied in a homogeneous magnetic background field B and at non-zero temperature T. In order to circumvent renormalization as a function of the bare coupling we apply a fixed-scale approach. We study the influence of the magnetic field on the critical temperature. At rather small pseudo-scalar meson mass (m_pi \\approx 175 MeV \\approx T_c(B=0)) we confirm magnetic catalysis for sufficiently strong magnetic field strength, while at T=195 MeV and weak magnetic field (qB {\\lesssim} 0.8 GeV^2) we find a rise of the Polyakov loop with qB and thus, indications for an inverse magnetic catalysis.

  20. A regenerative electrochemical biosensor for mercury(II) by using the insertion approach and dual-hairpin-based amplification

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Jing; Ling, Yu; Gao, Zhong Feng [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: luohq@swu.edu.cn [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: linb@swu.edu.cn [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2015-09-15

    Highlights: • The dual-hairpin structure as a signal amplifier is label-free and handy. • The strategy uses the insertion approach to improve the hybridization efficiency. • This biosensor has a low detection limit (28 pM) for detection of Hg{sup 2+}. • This biosensor can be easily regenerated by using L-cysteine. - Abstract: A simple and effective biosensor for Hg{sup 2+} determination was investigated. The novel biosensor was prepared by the insertion approach that the moiety-labeled DNA inserted into a loosely packed cyclic-dithiothreitol (DTT) monolayer, improving the hybridization efficiency. Electrochemical impedance spectroscopy studies of two biosensors (single-hairpin and dual-hairpin structure DNA modified electrodes) used for Hg{sup 2+} detection indicated that the dual-hairpin modified electrode had a larger electron transfer resistance change (ΔR{sub ct}). Consequently, the dual-hairpin structure was used as a signal amplifier for the preparation of a selective Hg{sup 2+} biosensor. This biosensor exhibited an excellent selectivity toward Hg{sup 2+} over Cd{sup 2+}, Pd{sup 2+}, Co{sup 2+} etc. Also, a linear relation was observed between the ΔR{sub ct} and Hg{sup 2+} concentrations in a range from 0.1 nM to 5 μM with a detection limit of 28 pM under optimum conditions. Moreover, the biosensor can be reused by using L-cysteine and successfully applied for detecting Hg{sup 2+} in real samples.

  1. Interstrand side chain--side chain interactions in a designed beta-hairpin: significance of both lateral and diagonal pairings.

    Science.gov (United States)

    Syud, F A; Stanger, H E; Gellman, S H

    2001-09-12

    The contributions of interstrand side chain-side chain contacts to beta-sheet stability have been examined with an autonomously folding beta-hairpin model system. RYVEV(D)PGOKILQ-NH2 ((D)P = D-proline, O = ornithine) has previously been shown to adopt a beta-hairpin conformation in aqueous solution, with a two-residue loop at D-Pro-Gly. In the present study, side chains that display interstrand NOEs (Tyr-2, Lys-9, and Leu-11) are mutated to alanine or serine, and the conformational impact of the mutations is assessed. In the beta-hairpin conformation Tyr-2 and Leu-11 are directly across from one another (non-hydrogen bonded pair). This "lateral" juxtaposition of two hydrophobic side chains appears to contribute to beta-hairpin conformational stability, which is consistent with results from other beta-sheet model studies and with statistical analyses of interstrand residue contacts in protein crystal structures. Interaction between the side chains of Tyr-2 and Lys-9 also stabilizes the beta-hairpin conformation. Tyr-2/Lys-9 is a "diagonal" interstrand juxtaposition because these residues are not directly across from one another in terms of the hydrogen bonding registry between the strands. This diagonal interaction arises from the right-handed twist that is commonly observed among beta-sheets. Evidence of diagonal side chain-side chain contacts has been observed in other autonomously folding beta-sheet model systems, but we are not aware of other efforts to determine whether a diagonal interaction contributes to beta-sheet stability.

  2. Organic photoredox catalysis for the oxidation of silicates: applications in radical synthesis and dual catalysis.

    Science.gov (United States)

    Lévêque, Christophe; Chenneberg, Ludwig; Corcé, Vincent; Ollivier, Cyril; Fensterbank, Louis

    2016-08-01

    Metal free photooxidation of alkyl bis(catecholato)silicates with the organic dye 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyano-benzene (4CzIPN) allows the smooth formation of alkyl radicals. The latter can be efficiently engaged either with radical acceptors to provide homolytic addition products or in photoredox/nickel dual catalysis reactions to obtain cross-coupling products.

  3. Mechanistic studies of copper(II)-aminoglycoside mediated DNA damage and magnesium catalyzed nuclease activity of hammerhead ribozyme

    Science.gov (United States)

    Patwardhan, Anjali A.

    The antibacterial activity of aminoglycosides stems from their high affinity binding to the 16S rRNA in bacteria resulting in inhibition of protein synthesis. Used to treat acute bacterial infections these antibiotics have limited applications due to their high dosage requirements and the emergence of resistant strains. We have synthesized and characterized Cu(II) derivatives of the aminoglycosides, kanamycin A, tobramycin, neamine, kanamycin B, neomycin B, and paromomycin. The first three exhibit preferential and tight binding to Cu(II) as against neomycin B and kanamycin B and paromomycin. EPR of frozen solutions and UV-visible spectroscopy suggest a change in geometry around the Cu(II) but the stabilities of the complexes in water differ. These copper derivatives efficiently cleave plasmid DNA at micromolar concentrations (hydrolytic) and at nanomolar concentrations in the presence co-reactants like hydrogen peroxide or ascorbic acid. Hydrolysis is multi turnover and exhibits Michelis-Menten kinetics with enzyme-like behavior whereas oxidative cleavage is highly specific with C-4' H abstraction resulting in characteristic base propenal and nucleotide base products. Hydroxyl radicals generated are copper based and are generated in close proximity of the substrate. Hammerhead ribozymes are selectively hydrolyzed in the presence of divalent ions with Mg2+ being the metal ion of choice in vivo . Our studies with complex ions like cobalt hexaammine and fac-triamminetriaquochromium(III) establish outer sphere interactions of Mg2+ with the hammerhead in the catalytic site. There are two sets of sites, one structural and one catalytic. Complex ions in the catalytic site and divalent ions in the structural site result in a slow but active hammerhead ribozyme suggesting that the complex ions are not inhibitory, contrary to what was suggested previously.

  4. Electronic Structure and Catalysis on Metal Surfaces

    Science.gov (United States)

    Greeley, Jeff; Norskov, Jens K.; Mavrikakis, Manos

    2002-10-01

    The powerful computational resources available to scientists today, together with recent improvements in electronic structure calculation algorithms, are providing important new tools for researchers in the fields of surface science and catalysis. In this review, we discuss first principles calculations that are now capable of providing qualitative and, in many cases, quantitative insights into surface chemistry. The calculations can aid in the establishment of chemisorption trends across the transition metals, in the characterization of reaction pathways on individual metals, and in the design of novel catalysts. First principles studies provide an excellent fundamental complement to experimental investigations of the above phenomena and can often allow the elucidation of important mechanistic details that would be difficult, if not impossible, to determine from experiments alone.

  5. Magnetic Catalysis in Graphene Effective Field Theory

    CERN Document Server

    DeTar, Carleton; Zafeiropoulos, Savvas

    2016-01-01

    We report on the first observation of magnetic catalysis at zero temperature in a fully nonperturbative simulation of the graphene effective field theory. Using lattice gauge theory, a nonperturbative analysis of the theory of strongly-interacting, massless, (2+1)-dimensional Dirac fermions in the presence of an external magnetic field is performed. We show that in the zero-temperature limit, a nonzero value for the chiral condensate is obtained which signals the spontaneous breaking of chiral symmetry. This result implies a nonzero value for the dynamical mass of the Dirac quasiparticle. This in turn has been posited to account for the quantum-Hall plateaus that are observed at large magnetic fields.

  6. A simplified electrostatic model for hydrolase catalysis.

    Science.gov (United States)

    Pessoa Filho, Pedro de Alcantara; Prausnitz, John M

    2015-07-01

    Toward the development of an electrostatic model for enzyme catalysis, the active site of the enzyme is represented by a cavity whose surface (and beyond) is populated by electric charges as determined by pH and the enzyme's structure. The electric field in the cavity is obtained from electrostatics and a suitable computer program. The key chemical bond in the substrate, at its ends, has partial charges with opposite signs determined from published force-field parameters. The electric field attracts one end of the bond and repels the other, causing bond tension. If that tension exceeds the attractive force between the atoms, the bond breaks; the enzyme is then a successful catalyst. To illustrate this very simple model, based on numerous assumptions, some results are presented for three hydrolases: hen-egg white lysozyme, bovine trypsin and bovine ribonuclease. Attention is given to the effect of pH.

  7. Asymmetric Aminalization via Cation-Binding Catalysis

    DEFF Research Database (Denmark)

    Park, Sang Yeon; Liu, Yidong; Oh, Joong Suk

    2017-01-01

    Asymmetric cation-binding catalysis, in principle, can generate "chiral" anionic nucleophiles, where the counter cations are coordinated within chiral environments. Nitrogen-nucleophiles are intrinsically basic, therefore, its use as nucleophiles is often challenging and limiting the scope...... of the reaction. Particularly, a formation of configurationally labile aminal centers with alkyl substituents has been a formidable challenge due to the enamine/imine equilibrium of electrophilic substrates. Herein, we report enantioselective nucleophilic addition reactions of potassium phthalimides to Boc......-protected alkyl- and aryl-substituted α-amido sulfones. In-situ generated imines smoothly reacted with the nitrogen nucleophiles to corresponding aminals with good to excellent enantioselectivitiy under mild reaction conditions. In addition, transformation of aminal products gave biologically relevant...

  8. Relation between Hydrogen Evolution and Hydrodesulfurization Catalysis

    DEFF Research Database (Denmark)

    Šaric, Manuel; Moses, Poul Georg; Rossmeisl, Jan

    2016-01-01

    A relation between hydrogen evolution and hydrodesulfurization catalysis was found by density functional theory calculations. The hydrogen evolution reaction and the hydrogenation reaction in hydrodesulfurization share hydrogen as a surface intermediate and, thus, have a common elementary step......, which indicates that the same catalyst should perform well for both hydrogen evolution and hydrogenation. If that catalyst also fulfills additional criteria for breaking carbon–sulfur bonds and releasing hydrogen sulfide, it will be a good hydrodesulfurization catalyst. The hydrogen evolution reaction...... is normally performed at room temperature and standard pressure, whereas the hydrodesulfurization reaction is driven by high temperature and pressure. Owing to the very different operating conditions, the adsorption free energy of hydrogen differs between hydrodesulfurization and the hydrogen evolution...

  9. Heterogeneous catalysis in highly sensitive microreactors

    DEFF Research Database (Denmark)

    Olsen, Jakob Lind

    This thesis present a highly sensitive silicon microreactor and examples of its use in studying catalysis. The experimental setup built for gas handling and temperature control for the microreactor is described. The implementation of LabVIEW interfacing for all the experimental parts makes...... automated experiments and data collection possible. An argon ush at the O-rings (used to interface the silicon microreactor with the gas system), which was developed, is presented. It enables experiments with temperatures up to 400., and up to 500. for short periods of time. The CO oxidation reaction...... of adsorbates readily converted to methanol as the source of the transient increase in methanol production, is eliminated. A study of mass selected ruthenium nanoparticles from a magnetron-sputter gas-aggregation source, deposited in microreactors, is presented. It is, shown that CO methanation can be measured...

  10. Catalysis in solid oxide fuel cells.

    Science.gov (United States)

    Gorte, R J; Vohs, J M

    2011-01-01

    Solid oxide fuel cells (SOFCs) and solid oxide electrolyzers (SOEs) hold much promise as highly efficient devices for the direct interconversion of chemical and electrical energy. Commercial application of these devices, however, requires further improvements in their performance and stability. Because the performance of SOFC and SOE electrodes depends on their microstructures, electronic and ionic conductivities, and chemical reactivities, the needed improvements require the expertise of various disciplines, with catalytic science playing an important role. Highly active and thermally stable catalysts are required to limit the internal losses in the devices, increase the range of fuels they can use, and decrease the temperatures at which they operate. In this article we review some of the most important recent advances in catalysis for SOFC and SOE electrodes and highlight additional improvements that are needed.

  11. Prebiotic RNA Synthesis by Montmorillonite Catalysis

    Science.gov (United States)

    Jheeta, Sohan; Joshi, Prakash C.

    2014-08-01

    This review summarizes our recent findings on the role of mineral salts in prebiotic RNA synthesis, which is catalyzed by montmorillonite clay minerals. The clay minerals not only catalyze the synthesis of RNA but also facilitate homochiral selection. Preliminary data of these findings have been presented at the "Horizontal Gene Transfer and the Last Universal Common Ancestor (LUCA)" conference at the Open University, Milton Keynes, UK, 5-6 September 2013. The objective of this meeting was to recognize the significance of RNA in LUCA. We believe that the prebiotic RNA synthesis from its monomers must have been a simple process. As a first step, it may have required activation of the 5'-end of the mononucleotide with a leaving group, e.g., imidazole in our model reaction (Figure 1). Wide ranges of activating groups are produced from HCN under plausible prebiotic Earth conditions. The final step is clay mineral catalysis in the presence of mineral salts to facilitate selective production of functional RNA. Both the clay minerals and mineral salts would have been abundant on early Earth. We have demonstrated that while montmorillonite (pH 7) produced only dimers from its monomers in water, addition of sodium chloride (1 M) enhanced the chain length multifold, as detected by HPLC. The effect of monovalent cations on RNA synthesis was of the following order: Li+ > Na+ > K+. A similar effect was observed with the anions, enhancing catalysis in the following order: Cl- > Br- > I-. The montmorillonite-catalyzed RNA synthesis was not affected by hydrophobic or hydrophilic interactions. We thus show that prebiotic synthesis of RNA from its monomers was a simple process requiring only clay minerals and a small amount of salt.

  12. Prebiotic RNA Synthesis by Montmorillonite Catalysis

    Directory of Open Access Journals (Sweden)

    Sohan Jheeta

    2014-08-01

    Full Text Available This review summarizes our recent findings on the role of mineral salts in prebiotic RNA synthesis, which is catalyzed by montmorillonite clay minerals. The clay minerals not only catalyze the synthesis of RNA but also facilitate homochiral selection. Preliminary data of these findings have been presented at the “Horizontal Gene Transfer and the Last Universal Common Ancestor (LUCA” conference at the Open University, Milton Keynes, UK, 5–6 September 2013. The objective of this meeting was to recognize the significance of RNA in LUCA. We believe that the prebiotic RNA synthesis from its monomers must have been a simple process. As a first step, it may have required activation of the 5'-end of the mononucleotide with a leaving group, e.g., imidazole in our model reaction (Figure 1. Wide ranges of activating groups are produced from HCN under plausible prebiotic Earth conditions. The final step is clay mineral catalysis in the presence of mineral salts to facilitate selective production of functional RNA. Both the clay minerals and mineral salts would have been abundant on early Earth. We have demonstrated that while montmorillonite (pH 7 produced only dimers from its monomers in water, addition of sodium chloride (1 M enhanced the chain length multifold, as detected by HPLC. The effect of monovalent cations on RNA synthesis was of the following order: Li+ > Na+ > K+. A similar effect was observed with the anions, enhancing catalysis in the following order: Cl− > Br− > I−. The montmorillonite-catalyzed RNA synthesis was not affected by hydrophobic or hydrophilic interactions. We thus show that prebiotic synthesis of RNA from its monomers was a simple process requiring only clay minerals and a small amount of salt.

  13. An electrochemical biosensor for sensitive detection of microRNA-155: combining target recycling with cascade catalysis for signal amplification.

    Science.gov (United States)

    Wu, Xiaoyan; Chai, Yaqin; Zhang, Pu; Yuan, Ruo

    2015-01-14

    In this work, a new electrochemical biosensor based on catalyzed hairpin assembly target recycling and cascade electrocatalysis (cytochrome c (Cyt c) and alcohol oxidase (AOx)) for signal amplification was constructed for highly sensitive detection of microRNA (miRNA). It is worth pointing out that target recycling was achieved only based on strand displacement process without the help of nuclease. Moreover, porous TiO2 nanosphere was synthesized, which could offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA strand 1 (S1) and Cyt c accordingly. With the mimicking sandwich-type reaction, the cascade catalysis amplification strategy was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H2O2, which was further electrocatalyzed by PtNPs and Cyt c. This newly designed biosensor provided a sensitive detection of miRNA-155 from 0.8 fM to 1 nM with a relatively low detection limit of 0.35 fM.

  14. Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

    OpenAIRE

    Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

    2014-01-01

    Adenosine-to-inosine RNA editing is an endogenous mechanism for regulating various biological processes. A method for site-specific and editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. In this paper, we describe a strategy for constructing a trans-acting hammerhead ribozyme that specifically cleaves target RNA dependent on the editing state at the specific site.

  15. Lentiviral transduction of Tar Decoy and CCR5 ribozyme into CD34+ progenitor cells and derivation of HIV-1 resistant T cells and macrophages

    Directory of Open Access Journals (Sweden)

    Rossi John

    2004-12-01

    Full Text Available Abstract Background RNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys, and small interfering RNAs (siRNAs. Lentiviral vectors are ideal for transduction of such inhibitory RNAs into hematopoietic stem cells due to their ability to transduce non-dividing cells and their relative refractiveness to gene silencing. The objective of this study is to introduce an HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme into CD34+ hematopoietic progenitor cells via an HIV-based lentiviral vector to derive viral resistant progeny T cells and macrophages. Results High efficiency and sustained gene transfer into CD34+ cells were achieved with lentiviral vector constructs harboring either Tar decoy or Tar decoy in combination with CCR5 ribozyme. Cells transduced with these constructs differentiated normally into T-lymphocytes in vivo in thy/liv grafts of SCID-hu mice, and into macrophages in vitro in the presence of appropriate growth factors. When challenged in vitro, the differentiated T lymphocytes and macrophages showed marked resistance against HIV-1 infection. Conclusions Viral resistant transgenic T cells and macrophages that express HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme could be obtained by lentiviral gene transduction of CD34+ progenitor cells. These results showed for the first time that expression of these anti-HIV-1 transgenes in combination do not interfere with normal thymopoiesis and thus have set the stage for their application in stem cell based gene therapy for HIV/AIDS.

  16. Structure et réarrangements conformationnels au cours de l’épissage du composant ribozyme d’un intron de groupe II

    OpenAIRE

    Li, Cheng-Fang

    2011-01-01

    Group II introns are a class of RNAs best known for their ribozyme-catalyzed, self-splicing reaction. Under certain conditions, the introns can excise themselves from precursor mRNAs and ligate together their flanking exons, without the aid of proteins. Group II introns generally excise from pre-mRNA as a lariat, like the one formed by spliceosomal introns, similarities in the splicing mechanism suggest that group II introns and nuclear spliceosomal introns may share a common evolutionary anc...

  17. Catalysis of Radical Reactions: A Radical Chemistry Perspective.

    Science.gov (United States)

    Studer, Armido; Curran, Dennis P

    2016-01-04

    The area of catalysis of radical reactions has recently flourished. Various reaction conditions have been discovered and explained in terms of catalytic cycles. These cycles rarely stand alone as unique paths from substrates to products. Instead, most radical reactions have innate chains which form products without any catalyst. How do we know if a species added in "catalytic amounts" is a catalyst, an initiator, or something else? Herein we critically address both catalyst-free and catalytic radical reactions through the lens of radical chemistry. Basic principles of kinetics and thermodynamics are used to address problems of initiation, propagation, and inhibition of radical chains. The catalysis of radical reactions differs from other areas of catalysis. Whereas efficient innate chain reactions are difficult to catalyze because individual steps are fast, both inefficient chain processes and non-chain processes afford diverse opportunities for catalysis, as illustrated with selected examples.

  18. Applications of metal-organic frameworks in heterogeneous supramolecular catalysis.

    Science.gov (United States)

    Liu, Jiewei; Chen, Lianfen; Cui, Hao; Zhang, Jianyong; Zhang, Li; Su, Cheng-Yong

    2014-08-21

    This review summarizes the use of metal-organic frameworks (MOFs) as a versatile supramolecular platform to develop heterogeneous catalysts for a variety of organic reactions, especially for liquid-phase reactions. Following a background introduction about catalytic relevance to various metal-organic materials, crystal engineering of MOFs, characterization and evaluation methods of MOF catalysis, we categorize catalytic MOFs based on the types of active sites, including coordinatively unsaturated metal sites (CUMs), metalloligands, functional organic sites (FOS), as well as metal nanoparticles (MNPs) embedded in the cavities. Throughout the review, we emphasize the incidental or deliberate formation of active sites, the stability, heterogeneity and shape/size selectivity for MOF catalysis. Finally, we briefly introduce their relevance into photo- and biomimetic catalysis, and compare MOFs with other typical porous solids such as zeolites and mesoporous silica with regard to their different attributes, and provide our view on future trends and developments in MOF-based catalysis.

  19. Nanostructured Membranes for Enzyme Catalysis and Green Synthesis of Nanoparticles

    Science.gov (United States)

    Macroporous membranes functionalized with ionizable macromolecules provide promising applications in toxic metal capture at high capacity, nanoparticle synthesis, and catalysis. Our low-pressure membrane approach is marked by reaction and separation selectivity and their tunabil...

  20. Bridging heterogeneous and homogeneous catalysis concepts, strategies, and applications

    CERN Document Server

    Li, Can

    2014-01-01

    This unique handbook fills the gap in the market for an up-to-date work that links both homogeneous catalysis applied to organic reactions and catalytic reactions on surfaces of heterogeneous catalysts.

  1. Nanostructured Membranes for Enzyme Catalysis and Green Synthesis of Nanoparticles

    Science.gov (United States)

    Macroporous membranes functionalized with ionizable macromolecules provide promising applications in toxic metal capture at high capacity, nanoparticle synthesis, and catalysis. Our low-pressure membrane approach is marked by reaction and separation selectivity and their tunabil...

  2. Selective Oxidation and Ammoxidation of Olefins by Heterogeneous Catalysis.

    Science.gov (United States)

    Grasselli, Robert K.

    1986-01-01

    Shows how the ammoxidation of olefins can be understood in terms of free radicals and surface bound organometallic intermediates. Also illustrates the close intellectual relationships between heterogeneous catalysis and organometallic chemistry. (JN)

  3. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2015-04-03

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

  4. 3. International conference on catalysis in membrane reactors

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-09-01

    The 3. International Conference on Catalysis in Membrane Reactors, Copenhagen, Denmark, is a continuation of the previous conferences held in Villeurbanne 1994 and Moscow 1996 and will deal with the rapid developments taking place within membranes with emphasis on membrane catalysis. The approx. 80 contributions in form of plenary lectures and posters discuss hydrogen production, methane reforming into syngas, selectivity and specificity of various membranes etc. The conference is organised by the Danish Catalytic Society under the Danish Society for Chemical Engineering. (EG)

  5. A modified group I intron can function as both a ribozyme and a 5' exon in a trans-exon ligation reaction.

    Science.gov (United States)

    Tasiouka, K I; Burke, J M

    1994-06-24

    Here, we show that a single RNA molecule derived from a group-I intron can provide the catalytic activity, the substrate recognition domain and the attacking nucleophile in a reaction that mimics the exon ligation step of splicing. To accomplish this reaction, we have linked a 5' exon sequence to the 3' end of an attenuated form of the self-splicing Tetrahymena rRNA intron. The ribozyme (I-E1) attacks an oligoribonucleotide analog of the 3' splice site (I'-E2) to generate a product containing ligated exons (I-E1-E2) and a small intron fragment (I'). Two modified introns were constructed and tested for activity. A construct designed to interact with the 3' splice site through intermolecular P9.0 and P10 helices was found to be inactive due to failure to form a stable ribozyme-substrate complex. A second modified intron and substrate combination was engineered, in which the complex was further stabilized by an intermolecular P9.2 helix. In this case, stable complexes and reaction products were identified. The reaction efficiency was low compared to splicing of the unmodified intron-containing precursor, and will be optimized in future experiments. Following optimization, we believe that this system may be exploited to examine the functional consequences of a wide variety of 3' splice-site modifications, and may provide the basis for development of highly selective trans-acting ribozymes.

  6. Non-Watson Crick base pairs might stabilize RNA structural motifs in ribozymes – A comparative study of group-I intron structures

    Indian Academy of Sciences (India)

    K Chandrasekhar; R Malathi

    2003-09-01

    In recent decades studies on RNA structure and function have gained significance due to discoveries on diversified functions of RNA. A common element for RNA secondary structure formed by series of non-Watson/Watson Crick base pairs, internal loops and pseudoknots have been the highlighting feature of recent structural determination of RNAs. The recent crystal structure of group-I introns has demonstrated that these might constitute RNA structural motifs in ribozymes, playing a crucial role in their enzymatic activity. To understand the functional significance of these non-canonical base pairs in catalytic RNA, we analysed the sequences of group-I introns from nuclear genes. The results suggest that they might form the building blocks of folded RNA motifs which are crucial to the catalytic activity of the ribozyme. The conservation of these, as observed from divergent organisms, argues for the presence of non-canonical base pairs as an important requisite for the structure and enzymatic property of ribozymes by enabling them to carry out functions such as replication, polymerase activity etc. in primordial conditions in the absence of proteins.

  7. Novel guanidinylated bioresponsive poly(amidoamines designed for short hairpin RNA delivery

    Directory of Open Access Journals (Sweden)

    Yu J

    2016-12-01

    Full Text Available Jiankun Yu,1 Jinmin Zhang,1 Haonan Xing,1 Yanping Sun,1 Zhen Yang,1 Tianzhi Yang,2 Cuifang Cai,1 Xiaoyun Zhao,3 Li Yang,1 Pingtian Ding1 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China; 2Department of Basic Pharmaceutical Sciences, School of Pharmacy, Husson University, Bangor, ME, USA; 3Department of Microbiology and Cell Biology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, China Abstract: Two different disulfide (SS-containing poly(amidoamine (PAA polymers were constructed using guanidino (Gua-containing monomers (ie, arginine [Arg] and agmatine [Agm] and N,N'-cystamine bisacrylamide (CBA by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the

  8. Hydrogels constructed via self-assembly of beta-hairpin molecules

    Science.gov (United States)

    Ozbas, Bulent

    There is a recent and growing interest in hydrogel materials that are formed via peptide self-assembly for tissue engineering applications. Peptide based materials are excellent candidates for diverse applications in biomedical field due to their responsive behavior and complex self-assembled structures. However, there is very limited information on the self-assembly and resultant network and mechanical properties of these types of hydrogels. The main goal of this dissertation is to investigate the self-assembly mechanism and viscoelastic properties of hydrogels that can be altered by changing solution conditions as well as the primary structure of the peptide. These hydrogels are formed via intramolecular folding and consequent self-assembly of 20 amino acid long beta-hairpin peptide molecules (Max1). The peptide molecules are locally amphiphilic with two linear strands of alternating hydrophobic valine and hydrophilic lysine amino acids connected with a Dproline-LProline turn sequence. Circular dichroism and FTIR spectroscopy show that at physiological conditions peptides are unfolded in the absence of salt. By raising the ionic strength of the solution electrostatic interactions between charged lysines are screened and the peptide arms are forced into a beta-sheet secondary structure stabilized by the turn sequence. These folded molecules intermolecularly assemble via hydrophobic collapse and hydrogen bonding into a three dimensional network. Folding and self-assembly of these molecules can also be triggered by increasing temperature and/or pH of the peptide solution. In addition, the random-coil to beta-sheet transition of the beta-hairpin peptides is pH and, with proper changes in the peptide sequence, thermally reversible. Rheological measurements demonstrate that the resultant supramolecular structure forms an elastic material, whose structure, and thus modulus, can be tuned by magnitude of the stimulus. Hydrogels recover their initial viscoelastic

  9. Molecular dynamics of β-hairpin models of epigenetic recognition motifs.

    Science.gov (United States)

    Zheng, Xiange; Wu, Chuanjie; Ponder, Jay W; Marshall, Garland R

    2012-09-26

    The conformations and stabilities of the β-hairpin model peptides of Waters (Riemen, A. J.; Waters, M. L. Biochemistry 2009, 48, 1525; Hughes, R. M.; Benshoff, M. L.; Waters, M. L. Chemistry 2007, 13, 5753) have been experimentally characterized as a function of lysine ε-methylation. These models were developed to explore molecular recognition of known epigenetic recognition motifs. This system offered an opportunity to computationally examine the role of cation-π interactions, desolvation of the ε-methylated ammonium groups, and aromatic/aromatic interactions on the observed differences in NMR spectra. AMOEBA, a second-generation force field (Ponder, J. W.; Wu, C.; Ren, P.; Pande, V. S.; Chodera, J. D.; Schnieders, M. J.; Haque, I.; Mobley, D. L.; Lambrecht, D. S.; DiStasio, R. A., Jr.; Head-Gordon, M.; Clark, G. N.; Johnson, M. E.; Head-Gordon, T. J. Phys. Chem. B 2010, 114, 2549), was chosen as it includes both multipole electrostatics and polarizability thought to be essential to accurately characterize such interactions. Independent parametrization of ε-methylated amines was required from which aqueous solvation free energies were estimated and shown to agree with literature values. Molecular dynamics simulations (100 ns) using the derived parameters with model peptides, such as Ac-R-W-V-W-V-N-G-Orn-K(Me)(n)-I-L-Q-NH(2), where n = 0, 1, 2, or 3, were conducted in explicit solvent. Distances between the centers of the indole rings of the two-tryptophan residues, 2 and 4, and the ε-methylated ammonium group on Lys-9 as well as the distance between the N- and C-termini were monitored to estimate the strength and orientation of the cation-π and aromatic/aromatic interactions. In agreement with the experimental data, the stability of the β-hairpin increased significantly with lysine ε-methylation. The ability of MD simulations to reproduce the observed NOEs for the four peptides was further estimated for the monopole-based force fields, AMBER, CHARMM, and

  10. Dedicated Beamline Facilities for Catalytic Research. Synchrotron Catalysis Consortium (SCC)

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jingguang [Columbia Univ., New York, NY; Frenkel, Anatoly [Yeshiva Univ., New York, NY (United States); Rodriguez, Jose [Brookhaven National Lab. (BNL), Upton, NY (United States); Adzic, Radoslav [Brookhaven National Lab. (BNL), Upton, NY (United States); Bare, Simon R. [UOP LLC, Des Plaines, IL (United States); Hulbert, Steve L. [Brookhaven National Lab. (BNL), Upton, NY (United States); Karim, Ayman [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Mullins, David R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Overbury, Steve [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2015-03-04

    Synchrotron spectroscopies offer unique advantages over conventional techniques, including higher detection sensitivity and molecular specificity, faster detection rate, and more in-depth information regarding the structural, electronic and catalytic properties under in-situ reaction conditions. Despite these advantages, synchrotron techniques are often underutilized or unexplored by the catalysis community due to various perceived and real barriers, which will be addressed in the current proposal. Since its establishment in 2005, the Synchrotron Catalysis Consortium (SCC) has coordinated significant efforts to promote the utilization of cutting-edge catalytic research under in-situ conditions. The purpose of the current renewal proposal is aimed to provide assistance, and to develop new sciences/techniques, for the catalysis community through the following concerted efforts: Coordinating the implementation of a suite of beamlines for catalysis studies at the new NSLS-II synchrotron source; Providing assistance and coordination for catalysis users at an SSRL catalysis beamline during the initial period of NSLS to NSLS II transition; Designing in-situ reactors for a variety of catalytic and electrocatalytic studies; Assisting experimental set-up and data analysis by a dedicated research scientist; Offering training courses and help sessions by the PIs and co-PIs.

  11. Determination of low-energy structures of a small RNA hairpin using Monte Carlo–based techniques

    Indian Academy of Sciences (India)

    Sudhanshu Shanker; Pradipta Bandyopadhyay

    2012-07-01

    The energy landscape of RNA is known to be extremely rugged, and hence finding low-energy structures starting from a random structure is a challenging task for any optimization algorithm. In the current work, we have investigated the ability of one Monte Carlo–based optimization algorithm, Temperature Basin Paving, to explore the energy landscape of a small RNA T-loop hairpin. In this method, the history of the simulation is used to increase the probability of states less visited in the simulation. It has been found that using both energy and end-to-end distance as the biasing parameters in the simulation, the partially folded structure of the hairpin starting from random structures could be obtained.

  12. β-Sheet 13C Structuring Shifts Appear only at the H-bonded Sites of Hairpins

    Science.gov (United States)

    Shu, Irene; Stewart, James M.; Scian, Michele; Kier, Brandon L.

    2011-01-01

    The 13C chemical shifts measured for designed β hairpins indicate that the structuring shifts (upfield for Cα and C′, downfield for Cβ) previously reported as diagnostic for β structuring in protein appear only at the H-bonded strand residues. The resulting periodicity of structuring shift magnitudes is not, however, a consequence of H-bonding status; rather, it reflects a previously unrecognized alternation in the backbone torsion angles of β strands. This feature of hairpins is also likely to be present in proteins. The study provides reference values for the expectation shifts for 13C sites in β structures that should prove useful in the characterization of the folding equilibria of β sheet models. PMID:21214243

  13. Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots.

    Science.gov (United States)

    Liu, Siming; Yoder, John I

    2016-11-29

    Understanding the functions encoded by plant genes can be facilitated by reducing transcript levels by hairpin RNA (hpRNA) mediated silencing. A bottleneck to this technology occurs when a gene encodes a phenotype that is necessary for cell viability and silencing the gene inhibits transformation. Here we compared the use of two chemically inducible plant promoter systems to drive hpRNA mediated gene silencing in transgenic, hairy roots. We cloned the gene encoding the Yellow Fluorescence Protein (YFP) into the dexamethasone inducible vector pOpOff2 and into the estradiol induced vector pER8. We then cloned a hpRNA targeting YFP under the regulation of the inducible promoters, transformed Medicago truncatula roots, and quantified YFP fluorescence and mRNA levels. YFP fluorescence was normal in pOpOff2 transformed roots without dexamethasone but was reduced with dexamethasone treatment. Interestingly, dexamethasone removal did not reverse YFP inhibition. YFP expression in roots transformed with pER8 was low even in the absence of inducer. We used the dexamethasone system to silence acetyl-CoA carboxylase gene and observed prolific root growth when this construct was transformed into Medicago until dexamethasone was applied. Our study shows that dexamethasone inducibility can be useful to silence vital genes in transgenic roots.

  14. Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression.

    Science.gov (United States)

    Yu, Han; Jiang, Xiaoou; Tan, Kar Tong; Hang, Liting; Patzel, Volker

    2015-10-15

    Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system.

  15. Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides.

    Science.gov (United States)

    Oparin, Peter B; Mineev, Konstantin S; Dunaevsky, Yakov E; Arseniev, Alexander S; Belozersky, Mikhail A; Grishin, Eugene V; Egorov, Tsezi A; Vassilevski, Alexander A

    2012-08-15

    A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

  16. Differential effects of alpha-helical and beta-hairpin antimicrobial peptides against Acanthamoeba castellanii.

    Science.gov (United States)

    Sacramento, R S; Martins, R M; Miranda, A; Dobroff, A S S; Daffre, S; Foronda, A S; De Freitas, D; Schenkman, S

    2009-07-01

    In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and P6, corresponding to the N-terminus of the pore-forming protein from Triatoma infestans, a blood-sucking insect, and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells. P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.

  17. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  18. The inverse autotransporter intimin exports its passenger domain via a hairpin intermediate.

    Science.gov (United States)

    Oberhettinger, Philipp; Leo, Jack C; Linke, Dirk; Autenrieth, Ingo B; Schütz, Monika S

    2015-01-16

    Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation.

  19. Comparison of Cytotoxic Activity in Leukemic Lineages Reveals Important Features of β-Hairpin Antimicrobial Peptides.

    Science.gov (United States)

    Buri, Marcus V; Torquato, Heron F Vieira; Barros, Carlos Castilho; Ide, Jaime S; Miranda, Antonio; Paredes-Gamero, Edgar J

    2017-07-01

    Several reports described different modes of cell death triggered by antimicrobial peptides (AMPs) due to direct effects on membrane disruption, and more recently by apoptosis and necrosis-like patterns. Cytotoxic curves of four β-hairpin AMPs (gomesin, protegrin, tachyplesin, and polyphemusin) were obtained from several human leukemic lineages and normal monocytes and Two cell lines were then selected based on their cytotoxic sensitivity. One was sensitive to AMPs (K562) and the other resistant (KG-1) and their effect compared between these lineages. Thus, these lineages were chosen to further investigate biological features related with their cytotoxicities to AMPs. Stimulation with AMPs produced cell death, with activation of caspase-3, in K562 lineage. Increase on the fluidity of plasmatic membrane by reducing cholesterol potentiated cytotoxicity of AMPs in both lineages. Quantification of internal and external gomesin binding to the cellular membrane of both K562 and KG-1 cells showed that more peptide is accumulated inside of K562 cells. Additionally, evaluation of multi-drug resistant pumps activity showed that KG-1 has more activity than K562 lineage. A comparison of intrinsic gene patterns showed great differences between K562 and KG-1, but stimulation with gomesin promoted few changes in gene expression patterns. Differences in internalization process through the plasma membrane, multidrug resistance pumps activity, and gene expression pattern are important features to AMPs regulated cell death. J. Cell. Biochem. 118: 1764-1773, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Stability and immunogenicity properties of the gene-silencing polypurine reverse Hoogsteen hairpins.

    Science.gov (United States)

    Villalobos, Xenia; Rodríguez, Laura; Prévot, Jeanne; Oleaga, Carlota; Ciudad, Carlos J; Noé, Véronique

    2014-01-06

    Gene silencing by either small-interference RNAs (siRNA) or antisense oligodeoxynucleotides (aODN) is widely used in biomedical research. However, their use as therapeutic agents is hindered by two important limitations: their low stability and the activation of the innate immune response. Recently, we developed a new type of molecule to decrease gene expression named polypurine reverse Hoogsteen hairpins (PPRHs) that bind to polypyrimidine targets in the DNA. Herein, stability experiments performed in mouse, human, and fetal calf serum and in PC3 cells revealed that the half-life of PPRHs is much longer than that of siRNAs in all cases. Usage of PPRHs with a nicked-circular structure increased the binding affinity to their target sequence and their half-life in FCS when bound to the target. Regarding the innate immune response, we determined that the levels of the transcription factors IRF3 and its phosphorylated form, as well as NF-κB were increased by siRNAs and not by PPRHs; that the expression levels of several proinflammatory cytokines including IL-6, TNF-α, IFN-α, IFN-ß, IL-1ß, and IL-18 were not significantly increased by PPRHs; and that the cleavage and activation of the proteolytic enzyme caspase-1 was not triggered by PPRHs. These determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response.

  1. Stereopositional Outcome in the Packing of Dissimilar Aromatics in Designed β-Hairpins.

    Science.gov (United States)

    Makwana, Kamlesh Madhusudan; Mahalakshmi, Radhakrishnan

    2016-03-14

    A popular strategy in the de novo design of stable β-sheet structures for various biomedical applications is the incorporation of aromatic pairs at the non-hydrogen-bonding (NHB) position. However, it is important to explicitly understand how aryl pair packing at the NHB region is coordinated with backbone structural rearrangements, and to delineate the benefits and drawbacks associated with stereopositional choice of dissimilar aromatic pairs. Here, we probe the consequences of flipped Trp/Tyr pairs by using engineered permutants at the NHB position of dodecapeptide β-hairpins, proximal and distal to the turn. Extensive conformational analysis of these peptides using NMR and CD spectroscopy reveal that a classic Edge-to-Face and Face-to-Edge geometry at the proximal and distal aromatic pairs, respectively, in YW-WY, is the most stabilizing. Such a preferred packing geometry in YW-WY results in a highly twisted β-sheet backbone, with Trp always providing a 'Face' orientation to its dissimilar aromatic partner Tyr. Flipping the proximal and/or distal aromatic pair distorts the ideal T-shaped geometry, and results in alternate aryl arrangements that can adversely affect strand twist and β-sheet stability. Our study reveals the existence of a strong stereopositional influence on the packing of dissimilar aromatic pairs. Our findings highlight the importance of modeling physical interaction forces while designing protein and peptide structures for functional applications.

  2. High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants.

    Directory of Open Access Journals (Sweden)

    Pu Yan

    Full Text Available With the wide use of double-stranded RNA interference (RNAi for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.

  3. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core.

    Science.gov (United States)

    Hoop, Cody L; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M; Boatz, Jennifer C; Mandal, Abhishek; Lewandowski, Józef R; Wetzel, Ronald; van der Wel, Patrick C A

    2016-02-09

    Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

  4. The effect of active fluctuations on the dynamics of particles, motors and hairpins

    CERN Document Server

    Vandebroek, Hans

    2016-01-01

    Inspired by recent experiments on the dynamics of particles and polymers in artificial cytoskeletons and in cells, we introduce a modified Langevin equation for a particle in an environment that is a viscoelastic medium and that is brought out of equilibrium by the action of active fluctuations caused by molecular motors. We show that within such a model, the motion of a free particle crosses over from superdiffusive to subdiffusive as observed for tracer particles in an {\\it in vitro} cytoskeleton or in a cell. We investigate the dynamics of a particle confined by a harmonic potential as a simple model for the motion of the tethered head of kinesin-1. We find that the probability that the head is close to its binding site on the microtubule can be enhanced by a factor of two due to active forces. Finally, we study the dynamics of a particle in a double well potential as a model for the dynamics of DNA-hairpins. We show that the active forces effectively lower the potential barrier between the two minima and ...

  5. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  6. Coexpression of Escherichia coli RNase Ⅲ in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

    Institute of Scientific and Technical Information of China (English)

    Jae Man Lee; Yoshito Kojin; Tsuneyuki Tatsuke; Hiroaki Mon; Yoshitaka Miyagawa; Takahiro Kusakabe

    2013-01-01

    Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells,although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs(dsRNAs).To solve this problem,we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA(siRNA).Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm,but were not effectively diced into siRNAs.Interestingly,RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase Ⅲ,although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.

  7. Oligodeoxynucleotide binding to (CTG) · (CAG) microsatellite repeats inhibits replication fork stalling, hairpin formation, and genome instability.

    Science.gov (United States)

    Liu, Guoqi; Chen, Xiaomi; Leffak, Michael

    2013-02-01

    (CTG)(n) · (CAG)(n) trinucleotide repeat (TNR) expansion in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)(n) · (CAG)(n) structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)(45) · (CAG)(45) causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)(45) · (CAG)(45) lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)(45) · (CAG)(45) expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.

  8. Probing the Gaseous Structure of a β-Hairpin Peptide with H/D Exchange and Electron Capture Dissociation

    Science.gov (United States)

    Straus, Rita N.; Jockusch, Rebecca A.

    2016-12-01

    An improved understanding of the extent to which native protein structure is retained upon transfer to the gas phase promises to enhance biological mass spectrometry, potentially streamlining workflows and providing fundamental insights into hydration effects. Here, we investigate the gaseous conformation of a model β-hairpin peptide using gas-phase hydrogen-deuterium (H/D) exchange with subsequent electron capture dissociation (ECD). Global gas-phase H/D exchange levels, and residue-specific exchange levels derived from ECD data, are compared among the wild type 16-residue peptide GB1p and several variants. High protection from H/D exchange observed for GB1p, but not for a truncated version, is consistent with the retention of secondary structure of GB1p in the gas phase or its refolding into some other compact structure. Four alanine mutants that destabilize the hairpin in solution show levels of protection similar to that of GB1p, suggesting collapse or (re)folding of these peptides upon transfer to the gas phase. These results offer a starting point from which to understand how a key secondary structural element, the β-hairpin, is affected by transfer to the gas phase. This work also demonstrates the utility of a much-needed addition to the tool set that is currently available for the investigation of the gaseous conformation of biomolecules, which can be employed in the future to better characterize gaseous proteins and protein complexes.

  9. Streak instability and generation of hairpin-vortices by a slotted jet in channel crossflow: Experiments and linear stability analysis

    Science.gov (United States)

    Philip, Jimmy; Karp, Michael; Cohen, Jacob

    2016-01-01

    Streaks and hairpin-vortices are experimentally generated in a laminar plane Poiseuille crossflow by injecting a continuous jet through a streamwise slot normal to the crossflow, with air as the working media. Small disturbances form stable streaks, however, higher disturbances cause the formation of streaks which undergo instability leading to the generation of hairpin vortices. Particular emphasis is placed on the flow conditions close to the generation of hairpin-vortices. Measurements are carried out in the cases of natural and phase-locked disturbance employing smoke visualisation, particle image velocimetry, and hot-wire anemometry, which include, the dominant frequency, wavelength, and the disturbance shape (or eigenfunctions) associated with the coherent part of the velocity field. A linear stability analysis for both one- and two-dimensional base-flows is carried out to understand the mechanism of instability and good agreement of wavelength and eigenfunctions are obtained when compared to the experimental data, and a slight under-prediction of the growth-rates by the linear stability analysis consistent with the final nonlinear stages in transitional flows. Furthermore, an energy analysis for both the temporal and spatial stability analysis revels the dominance of the symmetric varicose mode, again, in agreement with the experiments, which is found to be governed by the balance of the wallnormal shear and dissipative effects rather than the spanwise shear. In all cases the anti-symmetric sinuous modes governed by the spanwise shear are found to be damped both in analysis and in our experiments.

  10. Probing the Gaseous Structure of a β-Hairpin Peptide with H/D Exchange and Electron Capture Dissociation

    Science.gov (United States)

    Straus, Rita N.; Jockusch, Rebecca A.

    2017-02-01

    An improved understanding of the extent to which native protein structure is retained upon transfer to the gas phase promises to enhance biological mass spectrometry, potentially streamlining workflows and providing fundamental insights into hydration effects. Here, we investigate the gaseous conformation of a model β-hairpin peptide using gas-phase hydrogen-deuterium (H/D) exchange with subsequent electron capture dissociation (ECD). Global gas-phase H/D exchange levels, and residue-specific exchange levels derived from ECD data, are compared among the wild type 16-residue peptide GB1p and several variants. High protection from H/D exchange observed for GB1p, but not for a truncated version, is consistent with the retention of secondary structure of GB1p in the gas phase or its refolding into some other compact structure. Four alanine mutants that destabilize the hairpin in solution show levels of protection similar to that of GB1p, suggesting collapse or (re)folding of these peptides upon transfer to the gas phase. These results offer a starting point from which to understand how a key secondary structural element, the β-hairpin, is affected by transfer to the gas phase. This work also demonstrates the utility of a much-needed addition to the tool set that is currently available for the investigation of the gaseous conformation of biomolecules, which can be employed in the future to better characterize gaseous proteins and protein complexes.

  11. Substrate catalysis enhances single-enzyme diffusion.

    Science.gov (United States)

    Muddana, Hari S; Sengupta, Samudra; Mallouk, Thomas E; Sen, Ayusman; Butler, Peter J

    2010-02-24

    We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.

  12. Constant domain-regulated antibody catalysis.

    Science.gov (United States)

    Sapparapu, Gopal; Planque, Stephanie; Mitsuda, Yukie; McLean, Gary; Nishiyama, Yasuhiro; Paul, Sudhir

    2012-10-19

    Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

  13. Catalysis of Forster Resonances in Rubidium

    Science.gov (United States)

    Win, A. L.; Williams, W. D.; Sukenik, C. I.

    2016-05-01

    When two ultracold Rydberg atoms collide they may change their quantum state if the total electronic energy of the two atoms before and after the collision is about the same. This process can be made resonant by tuning the energy levels of the atoms with an electric field, via the Stark shift, so that the energy difference between incoming and outgoing channels vanishes. This condition is known as a ``Forster resonance.'' We have studied a particular Forster resonance in rubidium: 34p + 34p --> 34s + 35s, by investigating the time dependence of the state change in an ultracold environment. Furthermore, we have added 34d state atoms to the mix and observed an enhancement of 34s atom production. We attribute this enhancement to a catalysis effect whereby the 34d atoms alter the spatial distribution of 34p atoms that participate in the energy transfer interaction. We will present results from the experiment and compare them to model calculations. Present address: Department of Physics, Smith College, Northampton, MA.

  14. Ferroelectric based catalysis: Switchable surface chemistry

    Science.gov (United States)

    Kakekhani, Arvin; Ismail-Beigi, Sohrab

    2015-03-01

    We describe a new class of catalysts that uses an epitaxial monolayer of a transition metal oxide on a ferroelectric substrate. The ferroelectric polarization switches the surface chemistry between strongly adsorptive and strongly desorptive regimes, circumventing difficulties encountered on non-switchable catalytic surfaces where the Sabatier principle dictates a moderate surface-molecule interaction strength. This method is general and can, in principle, be applied to many reactions, and for each case the choice of the transition oxide monolayer can be optimized. Here, as a specific example, we show how simultaneous NOx direct decomposition (into N2 and O2) and CO oxidation can be achieved efficiently on CrO2 terminated PbTiO3, while circumventing oxygen (and sulfur) poisoning issues. One should note that NOx direct decomposition has been an open challenge in automotive emission control industry. Our method can expand the range of catalytically active elements to those which are not conventionally considered for catalysis and which are more economical, e.g., Cr (for NOx direct decomposition and CO oxidation) instead of canonical precious metal catalysts. Primary support from Toyota Motor Engineering and Manufacturing, North America, Inc.

  15. Inverse Magnetic Catalysis in Bottom-Up Holographic QCD

    CERN Document Server

    Evans, Nick; Scott, Marc

    2016-01-01

    We explore the effect of magnetic field on chiral condensation in QCD via a simple bottom up holographic model which inputs QCD dynamics through the running of the anomalous dimension of the quark bilinear. Bottom up holography is a form of effective field theory and we use it to explore the dependence on the coefficients of the two lowest order terms linking the magnetic field and the quark condensate. In the massless theory, we identify a region of parameter space where magnetic catalysis occurs at zero temperature but inverse magnetic catalysis at temperatures of order the thermal phase transition. The model shows similar non-monotonic behaviour in the condensate with B at intermediate T as the lattice data. This behaviour is due to the separation of the meson melting and chiral transitions in the holographic framework. The introduction of quark mass raises the scale of B where inverse catalysis takes over from catalysis until the inverse catalysis lies outside the regime of validity of the effective descr...

  16. Kinetic evolutionary behavior of catalysis-select migration

    Institute of Scientific and Technical Information of China (English)

    Wu Yuan-Gang; Lin Zhen-Quan; Ke Jian-Hong

    2012-01-01

    We propose a catalysis-select migration driven evolution model of two-species (A- and B-species) aggregates,where one unit of species A migrates to species B under the catalysts of species C,while under the catalysts of species D the reaction will become one unit of species B migrating to species A.Meanwhile the catalyst aggregates of species C perform self-coagulation,as do the species D aggregates.We study this catalysis-select migration driven kinetic aggregation phenomena using the generalized Smoluchowski rate equation approach with C species catalysis-select migration rate kernel K(k;i,j) =Kkij and D species catalysis-select migration rate kernel J(k;i,j) =Jkij.The kinetic evolution behaviour is found to be dominated by the competition between the catalysis-select immigration and emigration,in which the competition is between JD0 and KC0 (D0 and C0 are the initial numbers of the monomers of species D and C,respectively).When JD0 - KC0 > 0,the aggregate size distribution of species A satisfies the conventional scaling form and that of species B satisfies a modified scaling form.And in the case of JDo - KCo < 0,species A and B exchange their aggregate size distributions as in the above JD0 - KC0 > 0 case.

  17. Center for Catalysis at Iowa State University

    Energy Technology Data Exchange (ETDEWEB)

    Kraus, George A.

    2006-10-17

    The overall objective of this proposal is to enable Iowa State University to establish a Center that enjoys world-class stature and eventually enhances the economy through the transfer of innovation from the laboratory to the marketplace. The funds have been used to support experimental proposals from interdisciplinary research teams in areas related to catalysis and green chemistry. Specific focus areas included: • Catalytic conversion of renewable natural resources to industrial materials • Development of new catalysts for the oxidation or reduction of commodity chemicals • Use of enzymes and microorganisms in biocatalysis • Development of new, environmentally friendly reactions of industrial importance These focus areas intersect with barriers from the MYTP draft document. Specifically, section 2.4.3.1 Processing and Conversion has a list of bulleted items under Improved Chemical Conversions that includes new hydrogenation catalysts, milder oxidation catalysts, new catalysts for dehydration and selective bond cleavage catalysts. Specifically, the four sections are: 1. Catalyst development (7.4.12.A) 2. Conversion of glycerol (7.4.12.B) 3. Conversion of biodiesel (7.4.12.C) 4. Glucose from starch (7.4.12.D) All funded projects are part of a soybean or corn biorefinery. Two funded projects that have made significant progress toward goals of the MYTP draft document are: Catalysts to convert feedstocks with high fatty acid content to biodiesel (Kraus, Lin, Verkade) and Conversion of Glycerol into 1,3-Propanediol (Lin, Kraus). Currently, biodiesel is prepared using homogeneous base catalysis. However, as producers look for feedstocks other than soybean oil, such as waste restaurant oils and rendered animal fats, they have observed a large amount of free fatty acids contained in the feedstocks. Free fatty acids cannot be converted into biodiesel using homogeneous base-mediated processes. The CCAT catalyst system offers an integrated and cooperative catalytic

  18. SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    ZHAI Xiao-hui; LIU Min; GUO Xiao-juan; WANG Si-cen; ZHANG Hong-xia; GUO You-min

    2011-01-01

    Background The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines,SKOV-3.Methods hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid gadolinium (Ⅲ) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).Results The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r=0.990, P <0.01) and concentration-dependent manner (r=0.964, P <0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.Conclusions hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

  19. Function and anatomy of plant siRNA pools derived from hairpin transgenes

    Directory of Open Access Journals (Sweden)

    Lee Kevin AW

    2007-11-01

    Full Text Available Abstract Background RNA interference results in specific gene silencing by small-interfering RNAs (siRNAs. Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs will retain cross-species function and that transgenic plants expressing heterologous siRNAs might serve as useful siRNA bioreactors. Here we report a detailed evaluation of the above proposition and present evidence regarding structural features of siRNAs extracted from plants. Results Testing the gene silencing capacity of plant-derived siRNAs in mammalian cells proved to be very challenging and required partial siRNA purification and design of a highly sensitive assay. Using the above assay we found that plant-derived siRNAs are ineffective for gene silencing in mammalian cells. Plant-derived siRNAs are almost exclusively double-stranded and most likely comprise a mixture of bona fide siRNAs and aberrant partially complementary duplexes. We also provide indirect evidence that plant-derived siRNAs may contain a hitherto undetected physiological modification, distinct from 3' terminal 2-O-methylation. Conclusion siRNAs produced from plant hairpin transgenes and extracted from plants are ineffective for gene silencing in mammalian cells. Thus our findings establish that a previous claim that transgenic plants offer a cost-effective, scalable and sustainable source of siRNAs is unwarranted. Our results also indicate that the presence of aberrant siRNA duplexes and possibly a plant-specific siRNA modification, compromises the gene silencing capacity of plant-derived siRNAs in mammalian cells.

  20. Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

    Science.gov (United States)

    Yu, Jiankun; Zhang, Jinmin; Xing, Haonan; Sun, Yanping; Yang, Zhen; Yang, Tianzhi; Cai, Cuifang; Zhao, Xiaoyun; Yang, Li; Ding, Pingtian

    2016-01-01

    Two different disulfide (SS)-containing poly(amidoamine) (PAA) polymers were constructed using guanidino (Gua)-containing monomers (ie, arginine [Arg] and agmatine [Agm]) and N,N′-cystamine bisacrylamide (CBA) by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA)-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the observed enhancement of transfection efficiency and low cytotoxicity. Overall, two newly synthesized Gua-SS-PAAs polymers demonstrated great potential to be used as shRNA carriers for gene-therapy applications. PMID:27994462

  1. Effect of DNA Hairpin Loops on the Twist of Planar DNA Origami Tiles

    Science.gov (United States)

    Li, Zhe; Wang, Lei; Yan, Hao; Liu, Yan

    2012-01-01

    The development of scaffolded DNA origami, a technique in which a long single-stranded viral genome is folded into arbitrary shapes by hundreds of short synthetic oligonucleotides, represents an important milestone in DNA nanotechnology. Recent findings have revealed that two-dimensional (2D)DNA origami structures based on the original design parameters adopt a global twist with respect to the tile plane, which may be because the conformation of the constituent DNA (10.67 bp/turn) deviates from the natural B-type helical twist (10.4 bp/turn). Here we aim to characterize the effects of DNA hairpin loops on the overall curvature of the tile and explore their ability to control, and ultimately eliminate any unwanted curvature. A series of dumbbell-shaped DNA loops were selectively displayed on the surface of DNA origami tiles with the expectation that repulsive interactions among the neighboring dumbbell loops and between the loops and the DNA origami tile would influence the structural features of the underlying tiles. A systematic, atomic force microscopy (AFM) study of how the number and position of the DNA loops influenced the global twist of the structure was performed, and several structural models to explain the results were proposed. The observations unambiguously revealed that the first generation of rectangular shaped origami tiles adopt a conformation in which the upper right (corner 2) and bottom left (corner 4) corners bend upward out of the plane, causing linear superstructures attached by these corners to form twisted ribbons. Our experimental observations are consistent with the twist model predicted by the DNA mechanical property simulation software CanDo. Through the systematic design and organization of various numbers of dumbbell loops on both surfaces of the tile, a nearly planar rectangular origami tile was achieved. PMID:22126326

  2. High-Spin Cobalt Hydrides for Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Holland, Patrick L. [Univ. of Rochester, NY (United States)

    2013-08-29

    Organometallic chemists have traditionally used catalysts with strong-field ligands that give low-spin complexes. However, complexes with a weak ligand field have weaker bonds and lower barriers to geometric changes, suggesting that they may lead to more rapid catalytic reactions. Developing our understanding of high-spin complexes requires the use of a broader range of spectroscopic techniques, but has the promise of changing the mechanism and/or selectivity of known catalytic reactions. These changes may enable the more efficient utilization of chemical resources. A special advantage of cobalt and iron catalysts is that the metals are more abundant and cheaper than those currently used for major industrial processes that convert unsaturated organic molecules and biofeedstocks into useful chemicals. This project specifically evaluated the potential of high-spin cobalt complexes for small-molecule reactions for bond rearrangement and cleavage reactions relevant to hydrocarbon transformations. We have learned that many of these reactions proceed through crossing to different spin states: for example, high-spin complexes can flip one electron spin to access a lower-energy reaction pathway for beta-hydride elimination. This reaction enables new, selective olefin isomerization catalysis. The high-spin cobalt complexes also cleave the C-O bond of CO2 and the C-F bonds of fluoroarenes. In each case, the detailed mechanism of the reaction has been determined. Importantly, we have discovered that the cobalt catalysts described here give distinctive selectivities that are better than known catalysts. These selectivities come from a synergy between supporting ligand design and electronic control of the spin-state crossing in the reactions.

  3. Catalysis-by-design impacts assessment

    Energy Technology Data Exchange (ETDEWEB)

    Fassbender, L L; Young, J K [Pacific Northwest Lab., Richland, WA (USA); Sen, R K [Sen (R.K.) and Associates, Washington, DC (USA)

    1991-05-01

    Catalyst researchers have always recognized the need to develop a detailed understanding of the mechanisms of catalytic processes, and have hoped that it would lead to developing a theoretical predictive base to guide the search for new catalysts. This understanding allows one to develop a set of hierarchical models, from fundamental atomic-level ab-initio models to detailed engineering simulations of reactor systems, to direct the search for optimized, efficient catalyst systems. During the last two decades, the explosions of advanced surface analysis techniques have helped considerably to develop the building blocks for understanding various catalytic reactions. An effort to couple these theoretical and experimental advances to develop a set of hierarchical models to predict the nature of catalytic materials is a program entitled Catalysis-by-Design (CRD).'' In assessing the potential impacts of CBD on US industry, the key point to remember is that the value of the program lies in developing a novel methodology to search for new catalyst systems. Industrial researchers can then use this methodology to develop proprietary catalysts. Most companies involved in catalyst R D have two types of ongoing projects. The first type, what we call market-driven R D,'' are projects that support and improve upon a company's existing product lines. Project of the second type, technology-driven R D,'' are longer term, involve the development of totally new catalysts, and are initiated through scientists' research ideas. The CBD approach will impact both types of projects. However, this analysis indicates that the near-term impacts will be on market-driven'' projects. The conclusions and recommendations presented in this report were obtained by the authors through personal interviews with individuals involved in a variety of industrial catalyst development programs and through the three CBD workshops held in the summer of 1989. 34 refs., 7 figs., 7 tabs.

  4. Biodiesel forming reactions using heterogeneous catalysis

    Science.gov (United States)

    Liu, Yijun

    Biodiesel synthesis from biomass provides a means for utilizing effectively renewable resources, a way to convert waste vegetable oils and animal fats to a useful product, a way to recycle carbon dioxide for a combustion fuel, and production of a fuel that is biodegradable, non-toxic, and has a lower emission profile than petroleum-diesel. Free fatty acid (FFA) esterification and triglyceride (TG) transesterification with low molecular weight alcohols constitute the synthetic routes to prepare biodiesel from lipid feedstocks. This project was aimed at developing a better understanding of important fundamental issues involved in heterogeneous catalyzed biodiesel forming reactions using mainly model compounds, representing part of on-going efforts to build up a rational base for assay, design, and performance optimization of solid acids/bases in biodiesel synthesis. As FFA esterification proceeds, water is continuously formed as a byproduct and affects reaction rates in a negative manner. Using sulfuric acid (as a catalyst) and acetic acid (as a model compound for FFA), the impact of increasing concentrations of water on acid catalysis was investigated. The order of the water effect on reaction rate was determined to be -0.83. Sulfuric acid lost up to 90% activity as the amount of water present increased. The nature of the negative effect of water on esterification was found to go beyond the scope of reverse hydrolysis and was associated with the diminished acid strength of sulfuric acid as a result of the preferential solvation by water molecules of its catalytic protons. The results indicate that as esterification progresses and byproduct water is produced, deactivation of a Bronsted acid catalyst like H2SO4 occurs. Using a solid composite acid (SAC-13) as an example of heterogeneous catalysts and sulfuric acid as a homogeneous reference, similar reaction inhibition by water was demonstrated for homogeneous and heterogeneous catalysis. This similarity together with

  5. Gold Nanoparticle-Biological Molecule Interactions and Catalysis

    Directory of Open Access Journals (Sweden)

    Jonathan G. Heddle

    2013-09-01

    Full Text Available This review gives a brief summary of the field of gold nanoparticle interactions with biological molecules, particularly those with possible catalytic relevance. Gold nanoparticles are well known as catalysts in organic chemistry but much is unknown regarding their potential as catalysts of reactions involving biological molecules such as protein and nucleic acids. Biological molecules may be the substrate for catalysis or, if they are the ligand coating the gold particle, may be the catalyst itself. In other cases biological molecules may form a template upon which gold nanoparticles can be precisely arrayed. As relatively little is currently known about the catalytic capabilities of gold nanoparticles in this area, this review will consider templating in general (including, but not restricted to, those which result in structures having potential as catalysts before going on to consider firstly catalysis by the gold nanoparticle itself followed by catalysis by ligands attached to gold nanoparticles, all considered with a focus on biological molecules.

  6. Phosphorus Lewis acids: emerging reactivity and applications in catalysis.

    Science.gov (United States)

    Bayne, J M; Stephan, D W

    2016-02-21

    Part of the renaissance in main group chemistry has been a result of the focus on reactivity. This has led to the development of applications in stoichiometric reactivity and catalysis. In this tutorial review, we focus attention on the role of phosphorus-based Lewis acids in such advances. While early literature recognizes the role of P(iii) and P(v) electrophiles in coordination chemistry, it has generally been more recent studies that have focused on applications of this Lewis acidity. Applications of these novel P-based Lewis acids in stoichiometric reactivity, Lewis acid catalysis and frustrated Lewis pair (FLP) reactivity are reviewed. These advances demonstrate that P-based Lewis acids are a powerful tool for further developments in metal-free catalysis.

  7. Catalysis induced by radiations; Catalisis inducida por radiaciones

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez B, J.; Gonzalez J, J. C., E-mail: jaime.jimenez@inin.gob.m [ININ, Departamento de Quimica, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2010-07-01

    In Mexico is generated a great quantity of residuals considered as dangerous, for its capacity of corrosion, reactivity, toxicity to the environment, inflammability and biological-infectious potential. It is important to mention that the toxic compounds cannot be discharged to the sewerage systems and much less to the receiving bodies of water. The usual treatment that receives the dangerous residuals is the incineration and the bordering. The incineration is an efficient form of treating the residuals, but it can be dioxins source and benzofurans, being the phenol and chloro phenol the precursors of these compounds. At the present time the radiolytic degradation of organic compounds has been broadly studied, especially the 4-chloro phenol and of same form the photo catalysis of organic compounds. However the combination of both processes, called radio catalysis is barely reported. In this work the results of the experiments realized for to degrade the 4-chloro phenol by means of radio catalysis are reported. (Author)

  8. Hydrogen Production by Homogeneous Catalysis: Alcohol Acceptorless Dehydrogenation

    DEFF Research Database (Denmark)

    Nielsen, Martin

    2015-01-01

    in hydrogen production from biomass using homogeneous catalysis. Homogeneous catalysis has the advance of generally performing transformations at much milder conditions than traditional heterogeneous catalysis, and hence it constitutes a promising tool for future applications for a sustainable energy sector......, and are fundamental for the thrive of almost all business fields. The latter include the industries of agriculture, food additives, pharmaceuticals, electronics, plastic, fragrances, and more. Today, the major source of both energy and bulk chemicals is fossil fuels, being responsible for more than 80 % of the energy...... dehydrogenation. The third chapter, Biorelevant Substrates, concentrates on the use of alcohols such as ethanol, which are biomass related. The topic is alcohol acceptorless dehydrogenation reactions for both H2 production and the concurrent synthetic application. Finally, Chap. 4, Substrates for H2 Storage...

  9. Ceramics in Environmental Catalysis:Applications and Possibilities%Ceramics in Environmental Catalysis: Applications and Possibilities

    Institute of Scientific and Technical Information of China (English)

    Nitin LABHSETWAR; P.DOGGALI; S.RAYALU; R.YADAV; T.MISTUHASHI; H.HANEDA

    2012-01-01

    Environmental catalysis has been steadily growing because of the advances in its scientific and engineering aspects,as well as due to the new environmental challenges in the industrial era.The development of new catalysts and materials is essential for new technologies for various environmental applications.Ceramics play important roles in various environmental applications including the identification,monitoring,and quantification of pollutants and their control.Ceramics have important applications as sensors and photocatalysts,and they are extensively used as catalyst carriers and supports.Many ceramics are being explored as catalysts for pollution control applications.Their low cost,thermal and chemical stability,and capability of being tailored make them especially attractive for pollution control applications.Although a wide variety of materials have been developed as catalyst supports,this area is still of interest with new or modified catalyst supports being frequently reported.It is of equal importance to develop new or modified processes for the loading of catalysts on specific supports.Applications like chemical looping combustion (CLC) and other catalytic combustion processes are raising the demands to a new scale.We have been working on the development of both new and modified support materials,including mesoporous materials without structural order for possible applications in CLC and other catalytic reactions.Successful attempts have been made in the modification of conventional γ-Al2O3 and improved synthesis processes for supporting perovskite type catalysts.Our research on environmental catalysis applications of ceramic materials and processes are also briefly discussed.

  10. Role of catalysis in sustainable production of synthetic elastomers

    Indian Academy of Sciences (India)

    Vivek K Srivastava; Madhuchhanda Maiti; Ganesh C Basak; Raksh V Jasra

    2014-03-01

    Elastomer business plays a significant role in the transportation industry. In fact, elastomers make the world move. Due to limited availability of natural rubber, synthetic elastomers bridge the gap between demand and supply in today’s growing tyre and automobile industry.With more than ∼10000 KTA total world productions, the impact of synthetic elastomer business cannot be overlooked. The need of synthetic elastomers for tyre and automobile industries is stringently specific. Catalysis plays an inevitable role in achieving the growing demand of specific synthetic elastomers. The present study will describe how catalysis plays a significant role in the sustainable development of elastomers with special reference to polybutadiene rubber.

  11. New and future developments in catalysis activation of carbon dioxide

    CERN Document Server

    Suib, Steven L

    2013-01-01

    New and Future Developments in Catalysis is a package of books that compile the latest ideas concerning alternate and renewable energy sources and the role that catalysis plays in converting new renewable feedstock into biofuels and biochemicals. Both homogeneous and heterogeneous catalysts and catalytic processes will be discussed in a unified and comprehensive approach. There will be extensive cross-referencing within all volumes. This volume presents a complete picture of all carbon dioxide (CO2) sources, outlines the environmental concerns regarding CO2, and critica

  12. KCC1: First Nanoparticle developed by KAUST Catalysis Center

    KAUST Repository

    Basset, Jean-Marie

    2010-08-01

    KCC1 is the first Nanoparticle developed by KAUST Catalysis Center. Director of KAUST Catalysis Center, Dr. Jean-Marie Basset, Senior Research Scientist at KCC, Dr. Vivek Polshettiwar, and Dr. Dongkyu Cha of the Advanced Nanofabrication Imaging & Characterization Core Laboratory discuss the details of this recent discovery. This video was produced by KAUST Visualization Laboratory and KAUST Technology Transfer and Innovation - Terence McElwee, Director, Technology Transfer and Innovation - IP@kaust.edu.sa This technology is part of KAUST\\'s technology commercialization program that seeks to stimulate development and commercial use of KAUST-developed technologies. For more information email us at ip@kaust.edu.sa.

  13. A Possible Macroscopic-Photo-Catalysis Mechanism in Solar Furnace

    Science.gov (United States)

    Ho, Tsohsiu; Qing, Cheng-Rui; Chen, Ying-Tian

    2011-05-01

    Based on the experimental results of Chen et al. to use the solar furnace and medium frequency induction furnace to extract boron impurity from metallurgical silicon, we propose a strong radiation catalysis mechanism to explain the difference of reaction rates in these two furnaces. The postulate assuming the photons striking on the material not only increase the thermal energy of the molecules of reactants but also lower down the energy barrier of the reaction to speed up the chemical reaction. It is believed the photon catalysis mechanism is universal in most of high temperature chemical reactions and looking forward to more evidences for the postulate proposed in this article.

  14. A Possible Macroscopic-Photo-Catalysis Mechanism in Solar Furnace

    Institute of Scientific and Technical Information of China (English)

    HO Tsohsiu; QING Cheng-Rui; CHEN Ying-Tian

    2011-01-01

    Based on the experimental results of Chen et al.to use the solar furnace and medium frequency induction furnace to extract boron impurity from metallurgical silicon, we propose a strong radiation catalysis mechanism to explain the difference of reaction rates in these two furnaces.The postulate assuming the photons striking on the material not only increase the thermal energy of the molecules of reactants but also lower down the energy barrier of the reaction to speed up the chemical reaction.It is believed the photon catalysis mechanism is universall in most of high temperature chemical reactions and looking forward to more evidences for the postulate proposed in this article.

  15. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

    Science.gov (United States)

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer.

  16. Enantioselective conjugate additions of α-amino radicals via cooperative photoredox and Lewis acid catalysis.

    Science.gov (United States)

    Ruiz Espelt, Laura; McPherson, Iain S; Wiensch, Eric M; Yoon, Tehshik P

    2015-02-25

    We report the highly enantioselective addition of photogenerated α-amino radicals to Michael acceptors. This method features a dual-catalyst protocol that combines transition metal photoredox catalysis with chiral Lewis acid catalysis. The combination of these two powerful modes of catalysis provides an effective, general strategy to generate and control the reactivity of photogenerated reactive intermediates.

  17. Seventh BES (Basic Energy Sciences) catalysis and surface chemistry research conference

    Energy Technology Data Exchange (ETDEWEB)

    1990-03-01

    Research programs on catalysis and surface chemistry are presented. A total of fifty-seven topics are included. Areas of research include heterogeneous catalysis; catalysis in hydrogenation, desulfurization, gasification, and redox reactions; studies of surface properties and surface active sites; catalyst supports; chemical activation, deactivation; selectivity, chemical preparation; molecular structure studies; sorption and dissociation. Individual projects are processed separately for the data bases. (CBS)

  18. Isoporphyrin Intermediate in Heme Oxygenase Catalysis

    Science.gov (United States)

    Evans, John P.; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

    2008-01-01

    Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the α-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin π-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of α-meso-phenylheme-IX, α-meso-(p-methylphenyl)-mesoheme-III, and α-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the α-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced α-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation. PMID:18487208

  19. A conceptual translation of homogeneous catalysis into heterogeneous catalysis: homogeneous-like heterogeneous gold nanoparticle catalyst induced by ceria supporter.

    Science.gov (United States)

    Li, Zhen-Xing; Xue, Wei; Guan, Bing-Tao; Shi, Fu-Bo; Shi, Zhang-Jie; Jiang, Hong; Yan, Chun-Hua

    2013-02-07

    Translation of homogeneous catalysis into heterogeneous catalysis is a promising solution to green and sustainable development in chemical industry. For this purpose, noble metal nanoparticles represent a new frontier in catalytic transformations. Many challenges remain for researchers to transform noble metal nanoparticles of heterogeneous catalytic active sites into ionic species of homogeneous catalytic active sites. We report here a successful design on translating homogeneous gold catalysis into a heterogeneous system with a clear understanding of the catalytic pathway. This study initiates a novel concept to immobilize a homogeneous catalyst based on electron transfer between supporting base and supported nanoparticles. Meanwhile, on the basis of theoretical calculation, it has deepened the understanding of the interactions between noble metal nanoparticles and the catalyst support.

  20. The long-range P3 helix of the Tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations.

    Science.gov (United States)

    Mitchell, David; Jarmoskaite, Inga; Seval, Nikhil; Seifert, Soenke; Russell, Rick

    2013-08-09

    RNAs are prone to misfolding, but how misfolded structures are formed and resolved remains incompletely understood. The Tetrahymena group I intron ribozyme folds in vitro to a long-lived misfolded conformation (M) that includes extensive native structure but is proposed to differ in topology from the native state (N). A leading model predicts that exchange of the topologies requires unwinding of the long-range, core helix P3, despite the presence of P3 in both conformations. To test this model, we constructed 16 mutations to strengthen or weaken P3. Catalytic activity and in-line probing showed that nearly all of the mutants form the M state before folding to N. The P3-weakening mutations accelerated refolding from M (3- to 30-fold) and the P3-strengthening mutations slowed refolding (6- to 1400-fold), suggesting that P3 indeed unwinds transiently. Upon depletion of Mg(2+), the mutations had analogous effects on unfolding from N to intermediates that subsequently fold to M. The magnitudes for the P3-weakening mutations were larger than in refolding from M, and small-angle X-ray scattering showed that the ribozyme expands rapidly to intermediates from which P3 is disrupted subsequently. These results are consistent with previous results indicating unfolding of native peripheral structure during refolding from M, which probably permits rearrangement of the core. Together, our results demonstrate that exchange of the native and misfolded conformations requires loss of a core helix in addition to peripheral structure. Further, the results strongly suggest that misfolding arises from a topological error within the ribozyme core, and a specific topology is proposed.