The Experts below are selected from a list of 1980 Experts worldwide ranked by ideXlab platform

Heisaburo Shindo - One of the best experts on this subject based on the ideXlab platform.

  • Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by Filter Binding Assay
    Biochemistry, 1993
    Co-Authors: Heisaburo Shindo, Hidetaka Torigoe, Akinori Sarai
    Abstract:

    The Filter Binding method was found to be a powerful method for studying the formation of triplexes composed of a single-stranded homopyrimidine and a duplex with a matched purine-pyrimidine tract. With this technique, we were able to determine thermodynamic and kinetic parameters for triplex formation between a homopyrimidine 19-mer (5'-TCCTCTTCTTTTCTTTCTT-3') and a duplex with sequence 5'-GCAGGAGAAGAAAAGAAAGAACG-3' for the purine strand. The experiments were performed over a wide pH range (3.8-7.4) and a temperature range of 0-35 degrees C. pH and temperature dependencies of the thermodynamic parameters were best explained in terms of a three-state model for triplex formation at low temperatures relative to the melting point. The main results were as follows: (1) pH dependence of the dissociation constants of the triplex is a result of the rapid acid-base equilibrium of pyrimidine single strands; (2) the association rate for triplex formation decreases with increasing pH in accordance with the dissociation constants; (3) the dissociation constant is virtually temperature-independent at low pH, while it becomes strongly temperature-dependent with increasing pH (these results can be explained in terms of a negative, non-zero delta Cp for triplex formation at low pH); (4) the association rate decreases with increasing temperature, and the resulting negative activation energy indicates that the triplex formation process involves a quasi-stable intermediate; (5) the triplex formation is a second-order reaction at low pH, whereas it can be interpreted as a third-order reaction at neutral pH, suggesting that different triplex formation pathways are observed depending on the pH.

  • Mechanism of DNA triplex formation and its specificity as studied by Filter Binding Assay.
    Nucleic acids symposium series, 1993
    Co-Authors: Heisaburo Shindo, Hidetaka Torigoe, Kamiya M, Sarai A
    Abstract:

    The Filter Binding method was found to be a very useful method for triple-helical formation of oligo DNA duplexes with homoprymidine single-strands. Using this method, we have obtained dissociation constants of triplexes and association rates of triple-helical formation of a variety of combinations of double-strands (23-mer) and pyrimidine single-strands as functions of pH and temperature. pH dependences of dissociation constants and association rates are theoretically discussed in terms of acid-base equilibrium of pyrimidine strands. Temperature-dependence of dissociation constants and association rates were considerably different between acidic and neutral pH range, suggesting that mechanism of triple-helical formation differs between two pH ranges. The results were best interpreted in terms of a three-state model for the triple-helical formation. Furthermore, the effect of mismatched sequences on the stability of the triplexes were also discussed.

  • Thermodynamic and Kinetic Analyses of DNA Triplex Formation: Application of Filter-Binding Assay
    Journal of biomolecular structure & dynamics, 1993
    Co-Authors: Akinori Sarai, Satoru Sugiura, Hidetaka Torigoe, Heisaburo Shindo
    Abstract:

    We have developed a simple and efficient method for studying equilibrium thermodynamics and kinetics of DNA triplex formation, which utilizes a Filter-Binding procedure. The application of this method to the triplex formation between a double-stranded homopurine-homopyrimidine and a single-stranded homopyrimidine oligonucleotides has demonstrated its ability in the quantitative estimation of equilibrium Binding constants and rate constants under various conditions. Thus, this simple method can serve as a powerful tool for the systematic analysis of sequence and environmental effects on the equilibrium and kinetic quantities in the triplex formation.

  • Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and Filter Binding Assay
    Archives of biochemistry and biophysics, 1992
    Co-Authors: Hitoshi Kurumizaka, Futoshi Kanke, Ushiho Matsumoto, Heisaburo Shindo
    Abstract:

    Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and Filter Binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by Binding of IHF when DNA had a consensus sequence for the Binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by Filter Binding Assay showed that IHF has indeed stronger affinity to DNA with the consensus Binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific Binding of IHF and that its Binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.

Akinori Sarai - One of the best experts on this subject based on the ideXlab platform.

  • construction of an artificial tandem protein of the c myb dna Binding domain and analysis of its dna Binding specificity
    Biochemical and Biophysical Research Communications, 1999
    Co-Authors: Koji Furukawa, Akinori Sarai, Haruki Nakamura
    Abstract:

    An artificial tandem protein was generated using the third repeat of the c-Myb DNA-Binding domain, and its DNA Binding affinity and specificity were analyzed by a Filter Binding Assay, isothermal titration calorimetry, and surface plasmon resonance. Although this artificial protein had the proper secondary structure, which is similar to the third repeat by itself, it could not bind to the expected base sequences specifically. Compared with the successful results of the zinc finger fusion proteins with novel sequence specificities, the cooperativity between the adjacent repeats, observed in the c-Myb-DNA complex, should also be required for the DNA recognition by the artificial tandem protein. Using the previous analyses of the DNA Binding specificities by Myb homologous proteins, the differences in the DNA recognition mechanisms between the animal and plant Myb domains are also discussed.

  • Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by Filter Binding Assay
    Biochemistry, 1993
    Co-Authors: Heisaburo Shindo, Hidetaka Torigoe, Akinori Sarai
    Abstract:

    The Filter Binding method was found to be a powerful method for studying the formation of triplexes composed of a single-stranded homopyrimidine and a duplex with a matched purine-pyrimidine tract. With this technique, we were able to determine thermodynamic and kinetic parameters for triplex formation between a homopyrimidine 19-mer (5'-TCCTCTTCTTTTCTTTCTT-3') and a duplex with sequence 5'-GCAGGAGAAGAAAAGAAAGAACG-3' for the purine strand. The experiments were performed over a wide pH range (3.8-7.4) and a temperature range of 0-35 degrees C. pH and temperature dependencies of the thermodynamic parameters were best explained in terms of a three-state model for triplex formation at low temperatures relative to the melting point. The main results were as follows: (1) pH dependence of the dissociation constants of the triplex is a result of the rapid acid-base equilibrium of pyrimidine single strands; (2) the association rate for triplex formation decreases with increasing pH in accordance with the dissociation constants; (3) the dissociation constant is virtually temperature-independent at low pH, while it becomes strongly temperature-dependent with increasing pH (these results can be explained in terms of a negative, non-zero delta Cp for triplex formation at low pH); (4) the association rate decreases with increasing temperature, and the resulting negative activation energy indicates that the triplex formation process involves a quasi-stable intermediate; (5) the triplex formation is a second-order reaction at low pH, whereas it can be interpreted as a third-order reaction at neutral pH, suggesting that different triplex formation pathways are observed depending on the pH.

  • Thermodynamic and Kinetic Analyses of DNA Triplex Formation: Application of Filter-Binding Assay
    Journal of biomolecular structure & dynamics, 1993
    Co-Authors: Akinori Sarai, Satoru Sugiura, Hidetaka Torigoe, Heisaburo Shindo
    Abstract:

    We have developed a simple and efficient method for studying equilibrium thermodynamics and kinetics of DNA triplex formation, which utilizes a Filter-Binding procedure. The application of this method to the triplex formation between a double-stranded homopurine-homopyrimidine and a single-stranded homopyrimidine oligonucleotides has demonstrated its ability in the quantitative estimation of equilibrium Binding constants and rate constants under various conditions. Thus, this simple method can serve as a powerful tool for the systematic analysis of sequence and environmental effects on the equilibrium and kinetic quantities in the triplex formation.

Hidetaka Torigoe - One of the best experts on this subject based on the ideXlab platform.

  • Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by Filter Binding Assay
    Biochemistry, 1993
    Co-Authors: Heisaburo Shindo, Hidetaka Torigoe, Akinori Sarai
    Abstract:

    The Filter Binding method was found to be a powerful method for studying the formation of triplexes composed of a single-stranded homopyrimidine and a duplex with a matched purine-pyrimidine tract. With this technique, we were able to determine thermodynamic and kinetic parameters for triplex formation between a homopyrimidine 19-mer (5'-TCCTCTTCTTTTCTTTCTT-3') and a duplex with sequence 5'-GCAGGAGAAGAAAAGAAAGAACG-3' for the purine strand. The experiments were performed over a wide pH range (3.8-7.4) and a temperature range of 0-35 degrees C. pH and temperature dependencies of the thermodynamic parameters were best explained in terms of a three-state model for triplex formation at low temperatures relative to the melting point. The main results were as follows: (1) pH dependence of the dissociation constants of the triplex is a result of the rapid acid-base equilibrium of pyrimidine single strands; (2) the association rate for triplex formation decreases with increasing pH in accordance with the dissociation constants; (3) the dissociation constant is virtually temperature-independent at low pH, while it becomes strongly temperature-dependent with increasing pH (these results can be explained in terms of a negative, non-zero delta Cp for triplex formation at low pH); (4) the association rate decreases with increasing temperature, and the resulting negative activation energy indicates that the triplex formation process involves a quasi-stable intermediate; (5) the triplex formation is a second-order reaction at low pH, whereas it can be interpreted as a third-order reaction at neutral pH, suggesting that different triplex formation pathways are observed depending on the pH.

  • Thermodynamic and Kinetic Analyses of DNA Triplex Formation: Application of Filter-Binding Assay
    Journal of biomolecular structure & dynamics, 1993
    Co-Authors: Akinori Sarai, Satoru Sugiura, Hidetaka Torigoe, Heisaburo Shindo
    Abstract:

    We have developed a simple and efficient method for studying equilibrium thermodynamics and kinetics of DNA triplex formation, which utilizes a Filter-Binding procedure. The application of this method to the triplex formation between a double-stranded homopurine-homopyrimidine and a single-stranded homopyrimidine oligonucleotides has demonstrated its ability in the quantitative estimation of equilibrium Binding constants and rate constants under various conditions. Thus, this simple method can serve as a powerful tool for the systematic analysis of sequence and environmental effects on the equilibrium and kinetic quantities in the triplex formation.

  • Mechanism of DNA triplex formation and its specificity as studied by Filter Binding Assay.
    Nucleic acids symposium series, 1993
    Co-Authors: Heisaburo Shindo, Hidetaka Torigoe, Kamiya M, Sarai A
    Abstract:

    The Filter Binding method was found to be a very useful method for triple-helical formation of oligo DNA duplexes with homoprymidine single-strands. Using this method, we have obtained dissociation constants of triplexes and association rates of triple-helical formation of a variety of combinations of double-strands (23-mer) and pyrimidine single-strands as functions of pH and temperature. pH dependences of dissociation constants and association rates are theoretically discussed in terms of acid-base equilibrium of pyrimidine strands. Temperature-dependence of dissociation constants and association rates were considerably different between acidic and neutral pH range, suggesting that mechanism of triple-helical formation differs between two pH ranges. The results were best interpreted in terms of a three-state model for the triple-helical formation. Furthermore, the effect of mismatched sequences on the stability of the triplexes were also discussed.

James J. Champoux - One of the best experts on this subject based on the ideXlab platform.

  • Assays for the preferential Binding of human topoisomerase I to supercoiled DNA.
    Methods in molecular biology (Clifton N.J.), 2009
    Co-Authors: Zheng Yang, James J. Champoux
    Abstract:

    To Assay the preferential Binding of eukaryotic type IB topoisomerases to supercoiled DNA, two methods are described that make use of a catalytically inactive mutant form of the enzyme. In the gel shift Assay, the preference for Binding to supercoiled plasmid DNA is detected in the presence of linear and nicked forms of the same DNA by a reduction in the mobility of the supercoiled plasmid during electrophoresis in agarose. The more quantitative Filter Binding Assay compares the ability of nicked and supercoiled forms of the circular DNA to compete for the Binding of a (3)H-labeled nicked DNA to the topoisomerase where the enzyme-DNA complexes are quantitated by the retention of the labeled DNA on a nitrocellulose membrane.

Andrew D. Ellington - One of the best experts on this subject based on the ideXlab platform.

  • UNIT 9.3 In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
    Current Protocols in Nucleic Acid Chemistry, 2000
    Co-Authors: Bradley Hall, Seyed Arshad, Kyunghyun Seo, Catherine Bowman, Meredith Corley, Sulay D. Jhaveri, Andrew D. Ellington
    Abstract:

    This unit describes the selection of aptamers from a pool of single-stranded RNA by Binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription. Curr. Protoc. Nucleic Acid Chem. 40:9.3.1-9.3.27. © 2010 by John Wiley & Sons, Inc. Keywords: aptamer; in vitro selection; affinity reagent; Filter Binding Assay; SELEX

  • Detecting immobilized protein kinase C isozymes with RNA aptamers.
    Analytical biochemistry, 1996
    Co-Authors: R. Conrad, Andrew D. Ellington
    Abstract:

    Nucleic acids that can bind tightly and specifically to protein targets can be selected from random sequence pools. Just as antibodies have been used to detect proteins in a variety of formats, it is possible the nucleic acid Binding species (aptamers) could be used to specifically detect proteins. Using a simple Filter Binding Assay, aptamers have been used to detect protein kinase C (PKC) introduced into a cell extract. Aptamers that were relatively nonspecific could be used to simultaneously detect different PKC isozymes, while aptamers with higher specificities could discriminate between related isozymes. These results demonstrate the feasibility of using aptamers as diagnostic tools to detect proteins.