Comparison of Metal-Ion-Dependent Cleavages of RNA by a DNA Enzyme and a Hammerhead Ribozyme

He, Qiu-Chen; Zhou, Jinchuan; Zhou, Demin; Nakamatsu, Yuka; Baba, Tadashi; Taira, Kazunari

doi:10.1021/bm010095c

Cited by 24 publications

(23 citation statements)

References 122 publications

(287 reference statements)

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“…Whereas, solvent isotope effects usually arise from proton transfers between electronegative atoms (such as O or N) accompanying the bond-making and -breaking steps in the transition state, [25,26] an observed KSIE can also result from the difference in concentration of the chemically active species in the two solvents, [9] H 2 O and D 2 O. To differentiate between the two possibilities it is necessary to analyze the solvent isotope effect in the "plateau" region of the pL profile (in which pL refers to pH or pD).…”

Section: Kinetic Solvent Isotope Effect Experimentsmentioning

confidence: 99%

The RNA‐Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

et al. 2006

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Much interest has focused on the mechanisms of the five naturally occurring self-cleaving ribozymes, which, in spite of catalyzing the same reaction, adopt divergent strategies. These ribozymes, with the exception of the recently described glmS ribozyme, do not absolutely require divalent metal ions for their catalytic chemistries in vitro. A mechanistic investigation of an in vitro-selected, RNA-cleaving DNA enzyme, the bipartite, which catalyzes the same chemistry as the five natural self-cleaving ribozymes, found a mechanism of significant complexity. The DNAzyme showed a bell-shaped pH profile. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The DNAzyme was able to utilize manganese(II) as well as magnesium; however, with manganese it appeared to function complexed to either one or two of those cations. Titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base for activation of its nucleophile. Overall, the bipartite DNAzyme appeared to be kinetically distinct not only from the self-cleaving ribozymes but also from other in vitro-selected, RNA-cleaving deoxyribozymes, such as the 8-17, 10-23, and 614.

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Section: Kinetic Solvent Isotope Effect Experimentsmentioning

confidence: 99%

The RNA‐Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

et al. 2006

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“…The product of the transesterification is a 5′-OH terminus and a 2′,3′-cyclic phosphate. Additional information about this mechanism has been obtained via kinetic isotope studies [18][19][20] and chemical modifications such as thio substitution [21][22][23] at the scissile phosphate, although the mechanistic interpretation of these experimental studies remains a topic of discussion and some debate. Time-resolved x-ray crystallography 10,24,25 has become a powerful tool to elucidate structural information at different stages along the catalytic reaction coordinate that provides valuable insight into ribozyme activity.…”

Section: Introductionmentioning

confidence: 99%

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

et al. 2006

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Force field parameters specifically optimized for residues important in the study of RNA catalysis are derived from density-functional calculations in a fashion consistent with the CHARMM27 allatom empirical force field. Parameters are presented for residues that model reactive RNA intermediates and transition state analogs, thio-substituted phosphates and phosphoranes, and bound Mg 2+ and di-metal bridge complexes. Target data was generated via density-functional calculations at the B3LYP/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) level. Partial atomic charges were initially derived from the CHelpG electrostatic potential fitting and subsequently adjusted to be consistent with the CHARMM27 charges and Lennard-Jones parameters were determined to reproduce interaction energies with water molecules. Bond, angle and torsion parameters were derived from the density-functional calculations and renormalized to maintain compatibility with the existing CHARMM27 parameters for standard residues. The extension of the CHARMM27 force field parameters for the non-standard biological residues presented here will have considerable use in simulations of ribozymes, including the study of freeze-trapped catalytic intermediates, metal ion binding and occupation, and thio effects.

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“…Divalent cations play a crucial role in these mechanisms, as evidenced by a number of observations. For example, addition of La 3ϩ to the Mg 2ϩ -background reaction mixture inhibited the DNAzyme-catalyzed reactions, suggesting the replacement of catalytically and/or structurally important Mg 2ϩ by La 3ϩ (5). The function of divalent metal cations in DNAzyme activity is very complex and includes (i) stabilization of the transition state of reaction.…”

mentioning

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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Comparison of Metal-Ion-Dependent Cleavages of RNA by a DNA Enzyme and a Hammerhead Ribozyme

Cited by 24 publications

References 122 publications

The RNA‐Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

The RNA‐Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

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

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