Lactose Repressor-Operator DNA Interactions: Kinetic Analysis by a Surface Plasmon Resonance Biosensor

Frostellkarlsson, Aa; Fägerstam, Lars G.; Magnusson, Göran

doi:10.1006/abio.1993.1484

Cited by 135 publications

(79 citation statements)

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“…15 l of 5Ј end-biotinylated RNA at 100 nM in 50 mM Tris, pH 8.0, was injected at the flow rate of 5 l/min and consequently immobilized on the bound avidin to give a response of ϳ500 resonance units, an arbitrary unit specific for the BIAcore instrument. The levels of immobilized RNA were within the low levels that have to be used to ensure that the observed binding rate will be limited by the reaction kinetics rather than by the mass transport effects of the injected DNAzyme (19). In typical experiments, DNAzyme flowed in two channels of the sensor; the first one contained the RNA substrate attached to avidin, and the second was without the RNA substrate.…”

Section: Methodsmentioning

confidence: 99%

Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity

Cieślak

Szymański

Adamiak

et al. 2003

Journal of Biological Chemistry

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The intracellular ability of the "10 -23" DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mM Mg 2؉ , a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10 -23 DNAzyme induced by Mg 2؉ . For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg 2؉ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg 2؉ may occur in several distinct stages. The first stage, observed at 0.5 mM Mg 2؉ , corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mM Mg 2؉ , flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mM Mg 2؉ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.The typical DNAzyme, 1 known as the "10 -23" model, has tremendous potential in gene suppression for both target validation and therapeutic applications (1). It is capable of cleaving single-stranded RNA at specific sites under simulated physiological conditions and can be used to control even complex biological processes such as tumor angiogenesis. For example, DNAzymes to ␤1 and ␤3 mRNA reduced expression of targeted integrin subunits in endothelial cells and blocked proliferation, migration, and network formation in a fibrin and Matrigel™ matrix (2). In a cell culture system, a 10 -23 deoxyribozyme designed against 12-lipoxygenase mRNA specifically down-regulated expression of this protein and its metabolites, which are known to play a crucial role in tumor angiogenesis (3). Similarly, the DNAzyme to VEGFR2 mRNA cleaved its substrate efficiently and inhibited the proliferation of endothelial cells with a concomitant reduction of VEGFR2 mRNA and blocked tumor growth in vivo (4).The origins of the DNAzyme catalytic activity are not yet fully understood, but the observed rate enhancements probably are generated by a number of factors, including metal ion and nucleobase catalysis and local stereochemical effects. The 10 -23 DNAzyme has been developed using an in vitro selection strategy on the basis of it...

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Section: Methodsmentioning

confidence: 99%

Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity

Cieślak

Szymański

Adamiak

et al. 2003

Journal of Biological Chemistry

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“…2 281 association and dissociation rate constants (k~ and kd~) can be extrapolated from the data in sensorgrams [22]. The summary of the calculated data is given in Table 1.…”

Section: Association and Dissociation Rates For Non-specific Binding mentioning

confidence: 99%

“…The BIAcore (biospecific interaction analysis) system, developed recently by Pharmacia Biosensor to measure surface plasmon resonance (SPR), allows interactions between two macromolecules to be monitored in real time [21,22]. This system has been used for analysis of interactions between *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

“…Abbreviations: ds, double stranded; Km dissociation constant; BIA, biospecific interaction analysis; SPR, surface plasmon resonance; RU, resonance unit(s); k~, association rate constant; kalss, dissociation rate constant; KA, association constant the repressor of the lac operon and the relevant DNA sequence as well as for analysis of the action of the co-repressor in the methionine repressor-operator complex in E. coli [22,23]. In the present study, we analyzed the kinetic parameters of interactions between the Antp homeodomain and fragments of ds DNA under various different conditions with respect to salt concentration and temperature.…”

Section: Introductionmentioning

confidence: 99%

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Kinetics of binding of Antp homeodomain to DNA analyzed by measurements of surface plasmon resonance

Seimiya¹,

Kurosawa²

1996

FEBS Letters

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The kinetics of binding of the Antp homeodomain to three kinds of DNA fragment were analyzed by measurements of surface plasmon resonance at various temperatures. Non-specific and specific binding of the homeodomain to DNA was examined. In the case of non-specific binding, the association rate constant (kass) was estimated to be 1.41-2.62X 105 M -1 s -1 and the dissociation rate constant (kdiss) was 1.36-3.10 X 10 -2 s -1, thus, the dissociation constant (KI)) was 0.847-1.72 × 10 7 M. The association seemed to be driven by entropy. In the case of specific binding, by contrast, the enthalpy term seemed to contribute more to the binding than did the entropy term. The kass was 2.04-2.59X105 M -1 s -1 and the kdiss was 0.759-1.16X10 -3 s -1, thus, the /to was 2.93-5.69X 10 -9 M. These values were measured under the condition of 150 mM NaCl. Both interactions were strongly dependent on the concentration of NaC1. The /to at 50 mM NaCI became several tens of times smaller than those at 150 mM. Possible reasons for the differences between non-specific and specific interactions are discussed.

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“…The binding of an antibody to an immobilized antigen, as visualized in real time with BlAcore, was used to determine the kinetics of such interactions (Karlsson et al, 1991;O'Shannessy et al, 1993). BIAcore technology has been used to study receptor-ligand interactions (Cunningham and Wells, 1993;Zhou et a!., 1993) and to characterize signal transduction pathways (Panayotou et al, 1993;Schuster et al, 1993), protein to DNA interactions (Bondesson et a!., 1993), and antibodies (Holliger et a!., 1993;Ma!mqvist, 1993). BlAcore sensogram showed that there is no competition between the binding of G-90 and B-1531 to the immobilized GDNF on the sensor chip, again indicating that G-90 recognizes a distinct antigenie epitope from B-1531.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Two Distinct Monoclonal Antibodies Specific for Glial Cell Line‐Derived Neurotrophic Factor

Xu¹,

Pong²,

et al. 1998

Journal of Neurochemistry

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Abstract:Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-i 531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BlAcore data suggested that G-90 and B-1531 recognize different epitopes: POE confirmed that B-1531 binds to NH 2-terminal peptides between amino acids 18 and 37, whereas 0-90 does not; BlAcore data showed that B-i 531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-i 531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by 0-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to downregulate GDNF-induced biological activity. Key Words: Glial cell line-derived neurotrophic factor-Monoclonal antibodies-Motoneurons-Dopaminergic neuronsWestern blot-ELISA.

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Lactose Repressor-Operator DNA Interactions: Kinetic Analysis by a Surface Plasmon Resonance Biosensor

Cited by 135 publications

References 0 publications

Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity

Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity

Kinetics of binding of Antp homeodomain to DNA analyzed by measurements of surface plasmon resonance

Characterization of Two Distinct Monoclonal Antibodies Specific for Glial Cell Line‐Derived Neurotrophic Factor

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