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Ribozymes
Kinds of Ribozymes Selected AbstractsSynthesis and Enzymic Hydrolysis of Oligoribonucleotides Incorporating 3-Deazaguanosine: The Importance of the Nitrogen-3 Atom of Single Conserved Guanosine Residues on the Catalytic Activity of the Hammerhead RibozymeHELVETICA CHIMICA ACTA, Issue 8 2003Frank Seela Four base-modified hammerhead ribozyme/substrate complexes were constructed in which single guanosine (1) residues were replaced by 3-deazaguanosine (2) in the positions G5, G8, GL2.1, and G12. The base-modified ribozyme complexes were prepared by solid-phase synthesis of oligoribonucleotides employing the novel phosphoramidite 3 derived from 2. Phosphoramidite 3 carried a phenoxyacetyl group at the amino function and a diphenylcarbamoyl residue at the oxo group of the nucleobase. The 2,-hydroxy group was blocked with a triisopropylsilyl residue. Kinetic analysis of the phosphodiester hydrolysis showed a moderate decrease of the ribozyme catalytic activity when the residues G5 or G8 were replaced by 3-deazaguanosine and a 200-fold decrease when G12 was substituted. A 6-fold catalytic increase occurred when 3-deazaguanosine was replacing GL2.1 in the loop region. The data indicate that the N(3) atom of compound 2, in particular at position G12 is critical for the ribozyme activity. [source] Structural Investigation of a High-Affinity MnII Binding Site in the Hammerhead Ribozyme by EPR Spectroscopy and DFT Calculations.CHEMBIOCHEM, Issue 10 2003Effects of Neomycin B on Metal-Ion Binding Abstract Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high-affinity MnIIbinding site in the hammerhead ribozyme. This binding site exhibits a dissociation constant Kdof 4.4 ,M in buffer solutions containing 1,M NaCl, as shown by titrations monitored by continuous wave (cw) EPR. A combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constant,of 0.7 MHz, an asymmetry parameter,of 0.4, and an isotropic hyperfine coupling constant of Aiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the MnIIligand sphere and can therefore be used to determine the structure of the binding site. A possible location for this binding site may be at the G10.1, A9 site found to be occupied by MnIIin crystals (MacKay et al., Nature 1994, 372, 68 and Scott et al., Science 1996, 274, 2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the MnIIsite in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese(II) center and the Iglo-II basis set for all other atoms yielded values of,(14N)=+0.80 MHz, ,=0.324, and Aiso(14N)=+2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that MnIIis released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 ,M) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry 1995, 34, 11,186). [source] Strategy to assess the efficiency of U1 RNA-hammerhead ribozyme constructs using GFP-tagged targetsEXPERIMENTAL DERMATOLOGY, Issue 5 2003Peter B. Cserhalmi-Friedman Abstract: The application of ribozymes for gene therapy of autosomal dominant diseases has become popularized in recent years. Further this technology has widespread utility in the treatment of any disease, acquired or inherited, by inhibition of gene expression. The design of ribozymes is usually accomplished using computer assisted design programs, however they are not very useful in predicting the behavior of the ribozyme in the in vivo setting. To overcome this technical challenge, we developed a simple in vivo strategy to accurately assess the efficiency of ribozyme cleavage that significantly enhances the computer based design programs. [source] Two conserved structural components, A-rich bulge and P4 XJ6/7 base-triples, in activating the group I ribozymesGENES TO CELLS, Issue 12 2002Yoshiya Ikawa Background: The A-rich bulge of the group I intron ribozyme, a highly conserved structural element in its P5 peripheral region, plays a significant role in activating the ribozyme. The bulge has been known to interact with the P4 stem forming P4 XJ6/7 base-triples in the conserved core. The base-triples by themselves have also been identified as a distinctive element responsible for enhancing the activity of the ribozyme. Results: A weakly active variant of the Tetrahymena ribozyme lacking the P5 extension was dramatically activated by the addition of an A-rich bulge at the peripheral region, or by replacement of the original P4 XJ6/7 base-triples in the core structure with more stabilized isosteric ones. Biochemical analyses showed that the two methods of activation affect the ribozyme differently. Conclusions: The long-range interaction between the A-rich bulge and P4 or additionally stabilized P4 XJ6/7 base-triples can contribute dramatically to activation of the Tetrahymena ribozyme. Both improve the kcat value, which represents the rate of the limiting step of the ribozyme reaction when its binding site is saturated with GTP. However, the bulge or the modified base-triples gave a moderate reduction or considerable increase, respectively, to the Km(GTP) value. [source] Significant activity of a modified ribozyme with N7-deazaguanine at G10.1: the double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymesGENES TO CELLS, Issue 8 2000Yuka Nakamatsu Background Several reports have appeared recently of experimental evidence for a double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme-mediated cleavage was analysed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to the N7 of nucleotide G10.1 is critical for efficient catalysis, despite the large distance (,20 Ĺ) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd2+ ion binds first to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site. Results In earlier discussions, it was difficult to completely exclude the possibility that La3+ ions might have replaced the P9 metal ion and, as a result, created conditions represented by the bell-shaped curve. In order to clarify this situation, we examined a chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1. We compared the kinetic properties of this ribozyme with those of the parental ribozyme (R34). Kinetic analysis revealed that, unlike the cases of added Cd2+ ions, the added La3+ ions did not replace the pre-existing P9 metal ion, and that the replacement of N7 by C7 at G10.1 reduced the catalytic activity to a limited extent. This result indicates that the binding of a Mg2+ ion to N7 at G10.1 is catalytically important but not indispensable. Most importantly, 7-deaza-R34 also yielded a bell-shaped curve upon addition of La3+ ions to the reaction mixture. Conclusions Since the data based on our experiments with 7-deaza-R34 are completely free from potential artefacts, due to the binding of a La3+ ion to N7 at G10.1, our results, that 7-deaza-R34 yielded a bell-shaped curve following the addition of La3+ ions to the Mg2+ -background reaction mixture, strongly supports the proposal that a double-metal-ion mechanism is operative in the cleavage reaction which is catalysed by hammerhead ribozymes. [source] RNase P RNA-mediated cleavageIUBMB LIFE, Issue 3 2009Leif A. Kirsebom Abstract Metal(II)-induced hydrolysis of RNA produce products with 5,-hydroxyls and 2,;3,-cyclic phosphates at the ends. Ribozymes are RNA molecules that act as catalysts. Some ribozymes that cleave RNA also generate 5,-hydroxyls and 2,;3,-cyclic phosphates whereas others produces 5,-phosphates and 3,-hydroxyls at the ends of the cleavage products. RNase P is an essential endoribonuclease involved in RNA processing. The catalytic RNA subunit of RNase P is a trans-acting ribozyme that cleaves various RNA substrates in vitro generating 5,-phosphates and 3,-hydroxyls as cleavage products. The activity depends on the presence of metal(II) ions such as Mg2+. RNase P RNA has therefore to facilitate a nucleophilic attack that generates the correct product ends and prevent metal(II)-induced hydrolysis of the RNA substrate. In this review, we will discuss our current understanding of the interactions between RNase P RNA and its substrate, role of specific residues with respect to catalysis and positioning of functionally important Mg2+ at and in the vicinity of the cleavage site that ensures that products with correct ends are generated. Moreover, we will discuss the composition of RNase P and its RNA subunit in an evolutionary perspective. © 2009 IUBMB IUBMB Life, 61(3):189,200, 2009 [source] Broadening the mission of an RNA enzymeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Michael C. Marvin Abstract The "RNA World" hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA-protein complex plays diverse roles in processing RNA, but its best-understood reaction is pre-tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment. J. Cell. Biochem. 108: 1244,1251, 2009. © 2009 Wiley-Liss, Inc. [source] Gene Knockdown: A Powerful Tool for Gene Function Study in FishJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 3 2008Surintorn Boonanuntanasarn So far, there are a number of fish genome projects, including experimental and economically important fish that provide available DNA sequence information. However, the function of a gene cannot be deduced only by its DNA sequence. Therefore, a technique with which to investigate the function of the fish gene is needed. Gene knockdown (GKD), or antisense technology, is now being used as a powerful technique to study gene functions in living organisms. GKD effects result from the introduction of an antisense molecule into living cells. The antisense agents bind to target messenger RNA, thus inactivating the target gene expression. The appropriately spatial inhibitory effects on protein production from corresponding gene resulted in the phenotypic change. Therefore, the function of the gene can be understood. To date, there are a number of antisense molecules that can affect efficient GKD in fish. These include antisense oligonucleotides, small interfering RNA, and ribozyme. These antisense molecules cause specific gene inhibitor effects with different mechanisms. The various antisense mechanism types facilitate a number of GKD applications with various approaches in animals. In this review, we demonstrate the characteristics of each antisense molecule, its mechanism, and its application, especially for gene functional analysis in fish. [source] Enhanced antiviral effect in cell culture of type 1 interferon and ribozymes targeting HCV RNAJOURNAL OF VIRAL HEPATITIS, Issue 6 2001D. G. Macejak We have recently shown that the replication of an HCV-poliovirus (PV) chimera that is dependent upon the hepatitis C virus (HCV) 5, untranslated region (UTR) can be inhibited by treatment with ribozymes targeting HCV RNA. To determine the antiviral effects of anti-HCV ribozyme treatment in combination with type 1 interferon (IFN), we analysed the replication of this HCV-PV chimera in HeLa cells treated with anti-HCV ribozyme and/or IFN-,2a, IFN-,2b, or consensus IFN. The anti-HCV ribozyme, or any of the IFNs alone have significant inhibitory effects on HCV-PV replication compared to control treatment (, 85%, P < 0.01). The maximal inhibition due to IFN treatment (94%, P < 0.01) was achieved with , 50 U/ml for either IFN-,2a or IFN-,2b compared to control treatment. A similar level of inhibition in viral replication could be achieved with a 5-fold lower dose of IFN if ribozyme targeting the HCV 5, UTR was given in combination. For consensus IFN, the dose could be reduced by > 12.5-fold if ribozyme targeting the HCV 5, UTR was given in combination. Conversely, the dose of ribozyme could be reduced 3-fold if given in combination with any of the IFN preparations. Moreover, treatment with low doses (1,25 U/mL) of IFN-,2a, IFN-,2b, or consensus IFN in combination with anti-HCV ribozyme resulted in > 98% inhibition of HCV-PV replication compared to control treatment (P < 0.01). These results demonstrate that IFN and ribozyme each have a beneficial antiviral effect that is augmented when given in combination. [source] LIM kinase-2 targeting as a possible anti-metastasis therapyTHE JOURNAL OF GENE MEDICINE, Issue 3 2004Eigo Suyama Abstract Background Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis. It is involved in the regulation of the actin cytoskeleton. Using the randomized ribozyme library, we initially found that metastatic human fibrosarcoma cells harboring ribozyme specific for ROCK lose their metastatic properties. In this study, we have determined the effect of ribozymes specific for LIM kinase-2 on metastatic and proliferative phenotypes of human fibrosarcoma cells. Methods We attempted to target LIM kinase-2 (LIMK-2) expression by hammerhead ribozymes (Rz) in human metastatic fibrosarcoma cells. An effective ribozyme was selected based on the expression analysis. Cells were stably transfected with Rz specifically effective for LIMK-2 and were examined for metastatic and proliferative properties. Results Analyses of cellular phenotypes such as cell proliferation, cell migration and colony-forming efficiency revealed that the suppression of LIMK-2 expression in human fibrosarcoma cells limits their migration and dense colony-forming efficiency without affecting cell proliferation rate or viability. Conclusions Specific targeting of metastatic and malignant properties of tumor cells by LIMK-2 ribozyme may serve as an effective therapy for invasive tumors with minimum effect on the surrounding normal cells. Copyright © 2004 John Wiley & Sons, Ltd. [source] Inclusion of weak high-resolution X-ray data for improvement of a group II intron structureACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010Jimin Wang It is common to report the resolution of a macromolecular structure with the highest resolution shell having an averaged I/,(I) , 2. Data beyond the resolution thus defined are weak and often poorly measured. The exclusion of these weak data may improve the apparent statistics and also leads to claims of lower resolutions that give some leniency in the acceptable quality of refined models. However, the inclusion of these data can provide additional strong constraints on atomic models during structure refinement and thus help to correct errors in the original models, as has recently been demonstrated for a protein structure. Here, an improved group II intron structure is reported arising from the inclusion of these data, which helped to define more accurate solvent models for density modification during experimental phasing steps. With the improved resolution and accuracy of the experimental phases, extensive revisions were made to the original models such that the correct tertiary interactions of the group II intron that are essential for understanding the chemistry of this ribozyme could be described. [source] Crystallization and X-ray diffraction analysis of an all-RNA U39C mutant of the minimal hairpin ribozymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2003Valerie Grum-Tokars The hairpin ribozyme is a naturally occurring catalytic RNA composed of two helix,loop,helix domains, A and B, that dock to form the biologically active enzyme. Previously, the crystal structure of the hairpin has been solved as a four-way helical junction that incorporated the U1A protein as an artificial crystal-packing motif [Rupert & Ferré-D'Amaré (2001), Nature (London), 410, 780,786]. Here, the crystallization of a minimal junctionless hairpin ribozyme 64-mer is reported in the absence of protein. Crystals grow in space group P6122, with unit-cell parameters a = 93.1, c = 123.2,Ĺ. Complete diffraction data have been collected to 3.35,Ĺ resolution. Structural analysis should provide details of intermolecular RNA docking, including the ground-state conformations of the U39C mutation relevant to hairpin catalysis. [source] Problem-solving test: Telomere replicationBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010József Szeberényi Terms to be familiar with before you start to solve the test: DNA replication, template, primer, linear DNA, antiparallel orientation, telomere, origin of replication, chromatid, replicon, short tandem repeats, Okazaki fragments, leading strand, lagging strand, ribozyme, promoter, enhancer, terminal transferase, DNA polymerases, reverse transcriptase, RNA polymerase, topoisomerase, retroviral vector, Southern blotting, restriction endonuclease. [source] The RNA-Cleaving Bipartite DNAzyme Is a Distinctive MetalloenzymeCHEMBIOCHEM, Issue 1 2006Anat R. Feldman Dr. Abstract 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. [source] Structural Investigation of a High-Affinity MnII Binding Site in the Hammerhead Ribozyme by EPR Spectroscopy and DFT Calculations.CHEMBIOCHEM, Issue 10 2003Effects of Neomycin B on Metal-Ion Binding Abstract Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high-affinity MnIIbinding site in the hammerhead ribozyme. This binding site exhibits a dissociation constant Kdof 4.4 ,M in buffer solutions containing 1,M NaCl, as shown by titrations monitored by continuous wave (cw) EPR. A combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constant,of 0.7 MHz, an asymmetry parameter,of 0.4, and an isotropic hyperfine coupling constant of Aiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the MnIIligand sphere and can therefore be used to determine the structure of the binding site. A possible location for this binding site may be at the G10.1, A9 site found to be occupied by MnIIin crystals (MacKay et al., Nature 1994, 372, 68 and Scott et al., Science 1996, 274, 2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the MnIIsite in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese(II) center and the Iglo-II basis set for all other atoms yielded values of,(14N)=+0.80 MHz, ,=0.324, and Aiso(14N)=+2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that MnIIis released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 ,M) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry 1995, 34, 11,186). [source] Electrochemically Induced Modulation of the Catalytic Activity of a Reversible Redoxsensitive RiboswitchELECTROANALYSIS, Issue 9 2008Denise Strohbach Abstract Over the past decade, RNA conformation has been shown to respond to external stimuli. Thus, dependent on the presence of a high affinity ligand, specifically designed ribozymes can be regulated in a classical allosteric way. In this scenario, a binding event in one part of the RNA structure induces conformational changes in a separated part, which constitutes the catalytic centre. As a result activity is switched on (positive regulation) or off (negative regulation). We have developed a hairpin aptazyme responding to flavine mononucleotide (FMN). Ribozyme activity is dependent on binding of FMN and thus is switched on in the presence of FMN in its oxidized form. Under reducing conditions, however, FMN changes its molecular geometry, which is associated with loss of binding and consequently down-regulation of ribozyme activity. While in previous experiments sodium dithionite was used for reduction of FMN, we now present an assay for electrochemically induced activity switching. We have developed an electrochemical microcell that allows for iterative cycles of reduction/oxidation of FMN in an oxygen free atmosphere and thus for reversible switching of ribozyme activity. The reaction proceeds in droplets of 3 to 10,,L at micro- to nanomolar concentrations of the reaction components. [source] Strategy to assess the efficiency of U1 RNA-hammerhead ribozyme constructs using GFP-tagged targetsEXPERIMENTAL DERMATOLOGY, Issue 5 2003Peter B. Cserhalmi-Friedman Abstract: The application of ribozymes for gene therapy of autosomal dominant diseases has become popularized in recent years. Further this technology has widespread utility in the treatment of any disease, acquired or inherited, by inhibition of gene expression. The design of ribozymes is usually accomplished using computer assisted design programs, however they are not very useful in predicting the behavior of the ribozyme in the in vivo setting. To overcome this technical challenge, we developed a simple in vivo strategy to accurately assess the efficiency of ribozyme cleavage that significantly enhances the computer based design programs. [source] Two conserved structural components, A-rich bulge and P4 XJ6/7 base-triples, in activating the group I ribozymesGENES TO CELLS, Issue 12 2002Yoshiya Ikawa Background: The A-rich bulge of the group I intron ribozyme, a highly conserved structural element in its P5 peripheral region, plays a significant role in activating the ribozyme. The bulge has been known to interact with the P4 stem forming P4 XJ6/7 base-triples in the conserved core. The base-triples by themselves have also been identified as a distinctive element responsible for enhancing the activity of the ribozyme. Results: A weakly active variant of the Tetrahymena ribozyme lacking the P5 extension was dramatically activated by the addition of an A-rich bulge at the peripheral region, or by replacement of the original P4 XJ6/7 base-triples in the core structure with more stabilized isosteric ones. Biochemical analyses showed that the two methods of activation affect the ribozyme differently. Conclusions: The long-range interaction between the A-rich bulge and P4 or additionally stabilized P4 XJ6/7 base-triples can contribute dramatically to activation of the Tetrahymena ribozyme. Both improve the kcat value, which represents the rate of the limiting step of the ribozyme reaction when its binding site is saturated with GTP. However, the bulge or the modified base-triples gave a moderate reduction or considerable increase, respectively, to the Km(GTP) value. [source] Significant activity of a modified ribozyme with N7-deazaguanine at G10.1: the double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymesGENES TO CELLS, Issue 8 2000Yuka Nakamatsu Background Several reports have appeared recently of experimental evidence for a double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme-mediated cleavage was analysed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to the N7 of nucleotide G10.1 is critical for efficient catalysis, despite the large distance (,20 Ĺ) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd2+ ion binds first to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site. Results In earlier discussions, it was difficult to completely exclude the possibility that La3+ ions might have replaced the P9 metal ion and, as a result, created conditions represented by the bell-shaped curve. In order to clarify this situation, we examined a chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1. We compared the kinetic properties of this ribozyme with those of the parental ribozyme (R34). Kinetic analysis revealed that, unlike the cases of added Cd2+ ions, the added La3+ ions did not replace the pre-existing P9 metal ion, and that the replacement of N7 by C7 at G10.1 reduced the catalytic activity to a limited extent. This result indicates that the binding of a Mg2+ ion to N7 at G10.1 is catalytically important but not indispensable. Most importantly, 7-deaza-R34 also yielded a bell-shaped curve upon addition of La3+ ions to the reaction mixture. Conclusions Since the data based on our experiments with 7-deaza-R34 are completely free from potential artefacts, due to the binding of a La3+ ion to N7 at G10.1, our results, that 7-deaza-R34 yielded a bell-shaped curve following the addition of La3+ ions to the Mg2+ -background reaction mixture, strongly supports the proposal that a double-metal-ion mechanism is operative in the cleavage reaction which is catalysed by hammerhead ribozymes. [source] RNase P RNA-mediated cleavageIUBMB LIFE, Issue 3 2009Leif A. Kirsebom Abstract Metal(II)-induced hydrolysis of RNA produce products with 5,-hydroxyls and 2,;3,-cyclic phosphates at the ends. Ribozymes are RNA molecules that act as catalysts. Some ribozymes that cleave RNA also generate 5,-hydroxyls and 2,;3,-cyclic phosphates whereas others produces 5,-phosphates and 3,-hydroxyls at the ends of the cleavage products. RNase P is an essential endoribonuclease involved in RNA processing. The catalytic RNA subunit of RNase P is a trans-acting ribozyme that cleaves various RNA substrates in vitro generating 5,-phosphates and 3,-hydroxyls as cleavage products. The activity depends on the presence of metal(II) ions such as Mg2+. RNase P RNA has therefore to facilitate a nucleophilic attack that generates the correct product ends and prevent metal(II)-induced hydrolysis of the RNA substrate. In this review, we will discuss our current understanding of the interactions between RNase P RNA and its substrate, role of specific residues with respect to catalysis and positioning of functionally important Mg2+ at and in the vicinity of the cleavage site that ensures that products with correct ends are generated. Moreover, we will discuss the composition of RNase P and its RNA subunit in an evolutionary perspective. © 2009 IUBMB IUBMB Life, 61(3):189,200, 2009 [source] Survivin as a target for new anticancer interventionsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2005Nadia Zaffaroni Abstract Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that has been implicated in both control of cell division and inhibition of apoptosis. Specifically, its anti-apoptosis function seems to be related to the ability to directly or indirectly inhibit caspases. Survivin is selectively expressed in the most common human neoplasms and appears to be involved in tumour cell resistance to some anticancer agents and ionizing radiation. On the basis of these findings survivin has been proposed and and attractive target for new anticancer interventions. Several preclinical studies have demonstrated that down-regulation of survivin expression/function, accomplished through the use of antisense oligonucleotides, dominant negative mutants, ribozymes, small interfering RNAs and cyclin-dependent kinase inhibitors, increased the apoptotic rate, reduced tumor-growth potential and sensitized tumor cells to chemotherapeutic drugs with different action mechanisms and ,-irradiation in in vitro and in vivo models of different human tumor types. [source] Broadening the mission of an RNA enzymeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Michael C. Marvin Abstract The "RNA World" hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA-protein complex plays diverse roles in processing RNA, but its best-understood reaction is pre-tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment. J. Cell. Biochem. 108: 1244,1251, 2009. © 2009 Wiley-Liss, Inc. [source] Antisense applications for biological controlJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Wei-Hua Pan Abstract Although Nature's antisense approaches are clearly impressive, this Perspectives article focuses on the experimental uses of antisense reagents (ASRs) for control of biological processes. ASRs comprise antisense oligonucleotides (ASOs), and their catalytically active counterparts ribozymes and DNAzymes, as well as small interfering RNAs (siRNAs). ASOs and ribozymes/DNAzymes target RNA molecules on the basis of Watson-Crick base pairing in sequence-specific manner. ASOs generally result in destruction of the target RNA by RNase-H mediated mechanisms, although they may also sterically block translation, also resulting in loss of protein production. Ribozymes and DNAzymes cleave target RNAs after base pairing via their antisense flanking arms. siRNAs, which contain both sense and antisense regions from a target RNA, can mediate target RNA destruction via RNAi and the RISC, although they can also function at the transcriptional level. A considerable number of ASRs (mostly ASOs) have progressed into clinical trials, although most have relatively long histories in Phase I/II settings. Clinical trial results are surprisingly difficult to find, although few ASRs appear to have yet established efficacy in Phase III levels. Evolution of ASRs has included: (a) Modifications to ASOs to render them nuclease resistant, with analogous modifications to siRNAs being developed; and (b) Development of strategies to select optimal sites for targeting. Perhaps the biggest barrier to effective therapies with ASRs is the "Delivery Problem." Various liposomal vehicles have been used for systemic delivery with some success, and recent modifications appear to enhance systemic delivery, at least to liver. Various nanoparticle formulations are now being developed which may also enhance delivery. Going forward, topical applications of ASRs would seem to have the best chances for success. In summary, modifications to ASRs to enhance stability, improve targeting, and incremental improvements in delivery vehicles continue to make ASRs attractive as molecular therapeutics, but their advance toward the bedside has been agonizingly slow. J. Cell. Biochem. 98: 14,35, 2006. © 2006 Wiley-Liss, Inc. [source] Inhibition of hepatitis B virus by lentiviral vector delivered antisense RNA and hammerhead ribozymesJOURNAL OF VIRAL HEPATITIS, Issue 4 2005K. L. Nash Summary., Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. Current treatments are limited and may be ineffective. Nucleic acid-mediated targeting of viral mRNA is an attractive and specific approach for viral infection and lentiviral vectors provide a means to express antisense sequences or ribozymes stably in target cells permitting continuous production within that cell and its progeny. To demonstrate long-term gene expression by lentiviral vectors in hepatocytes and to introduce lentiviral vectors expressing anti-HBV genes to assess their effect against HBV, lentiviral vectors expressing a reporter gene were assessed for longevity of gene expression in hepatocytes in vitro. Hammerhead ribozymes and antisense sequences targeting the HBV encapsidation signal (,), X or surface antigen on mRNAs were cloned into lentiviral vectors and used to transduce HBV expressing hepatocytes where the effect on HBV mRNA level was assessed using ribonuclease protection. Gene expression in hepatocytes from integrated vectors continued for over 4 months without selection. Antisense RNA targeting HBs mRNA reduced this transcript, whilst antisense RNA to HBX mRNA was ineffective. Sense RNAs corresponding to , and HBX mRNA also reduced HBV mRNA levels. Ribozymes targeting HBs and HBX mRNA effectively reduced HBV mRNA levels compared with inactive constructs indicating their effect to be enzymatic rather than antisense. Lentiviral vectors can produce long-term gene expression in hepatocytes and thus permit prolonged expression of antiviral genes targeting the HBV encapsidation signal, surface and X mRNAs as treatments for chronic HBV infection. [source] Conjugation mediates transfer of the Ll.LtrB group II intron between different bacterial speciesMOLECULAR MICROBIOLOGY, Issue 5 2004Kamila Belhocine Summary Some self-splicing group II introns (ribozymes) are mobile retroelements. These retroelements, which can insert themselves into cognate intronless alleles or ectopic sites by reverse splicing, are thought to be the evolutionary progenitors of the widely distributed eukaryotic spliceosomal introns. Lateral or horizontal transmission of introns (i.e. between species), although never experimentally demonstrated, is a well-accepted model for intron dispersal and evolution. Horizontal transfer of the ancestral bacterial group II introns may have contributed to the dispersal and wide distribution of spliceosomal introns present in modern eukaryotic genomes. Here, the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was used as a model system to address the dissemination of introns in the bacterial kingdom. We report the first experimental demonstration of horizontal transfer of a group II intron. We show that the Ll.LtrB group II intron, originally discovered on an L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, invades new sites using both retrohoming and retrotransposition pathways after its transfer by conjugation. Ll.LtrB lateral transfer is shown among different L. lactis strains (intraspecies) (retrohoming and retrotransposition) and between L. lactis and Enterococcus faecalis (interspecies) (retrohoming). These results shed light on long-standing questions about intron evolution and propagation, and demonstrate that conjugation is one of the mechanisms by which group II introns are, and probably were, broadly disseminated between widely diverged organisms. [source] Potato spindle tuber viroid: the simplicity paradox resolved?MOLECULAR PLANT PATHOLOGY, Issue 5 2007ROBERT A. OWENS SUMMARY Taxonomy: , Potato spindle tuber viroid (PSTVd) is the type species of the genus Posipiviroid, family Pospiviroidae. An absence of hammerhead ribozymes and the presence of a ,central conserved region' distinguish PSTVd and related viroids from members of a second viroid family, the Avsunviroidae. Physical properties: , Viroids are small, unencapsidated, circular, single-stranded RNA molecules which replicate autonomously when inoculated into host plants. Because viroids are non-protein-coding RNAs, designation of the more abundant, highly infectious polarity strand as the positive strand is arbitrary. PSTVd assumes a rod-like, highly structured conformation that is resistant to nuclease degradation in vitro. Naturally occurring sequence variants of PSTVd range in size from 356 to 361 nt. Hosts and symptoms: , The natural host range of PSTVd,cultivated potato, certain other Solanum spp., and avocado,appears to be quite limited. Foliar symptoms in potato are often obscure, and the severity of tuber symptoms (elongation with the appearance of prominent bud scales/eyebrows and growth cracks) depends on both temperature and length of infection. PSTVd has a broad experimental host range, especially among solanaceous species, and strains are classified as mild, intermediate or severe based upon the symptoms observed in sensitive tomato cultivars. These symptoms include shortening of internodes, petioles and mid-ribs, severe epinasty and wrinkling of the leaves, and necrosis of mid-ribs, petioles and stems. [source] Biologically Important Reactions Catalyzed by RNA MoleculesTHE CHEMICAL RECORD, Issue 5 2002Yutaka Ikeda Abstract The last few years have seen a considerable increase in our understanding of catalysis by naturally occurring RNA molecules called ribozymes. The biological functions of RNA molecules depend upon their adoption of appropriate three-dimensional structures. The structure of RNA has a very important electrostatic component, which results from the presence of charged phosphodiester bonds. Metal ions are usually required to stabilize the folded structures and/or catalysis. Some ribozymes utilize metal ions as catalysts, whereas others use the ions to maintain appropriate three-dimensional structures. In the latter case, the correct folding of the RNA structures can perturb the pKa values of the nucleotide(s) within a catalytic pocket such that they act as general acid/bases catalysts. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 307,318, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10031 [source] LIM kinase-2 targeting as a possible anti-metastasis therapyTHE JOURNAL OF GENE MEDICINE, Issue 3 2004Eigo Suyama Abstract Background Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis. It is involved in the regulation of the actin cytoskeleton. Using the randomized ribozyme library, we initially found that metastatic human fibrosarcoma cells harboring ribozyme specific for ROCK lose their metastatic properties. In this study, we have determined the effect of ribozymes specific for LIM kinase-2 on metastatic and proliferative phenotypes of human fibrosarcoma cells. Methods We attempted to target LIM kinase-2 (LIMK-2) expression by hammerhead ribozymes (Rz) in human metastatic fibrosarcoma cells. An effective ribozyme was selected based on the expression analysis. Cells were stably transfected with Rz specifically effective for LIMK-2 and were examined for metastatic and proliferative properties. Results Analyses of cellular phenotypes such as cell proliferation, cell migration and colony-forming efficiency revealed that the suppression of LIMK-2 expression in human fibrosarcoma cells limits their migration and dense colony-forming efficiency without affecting cell proliferation rate or viability. Conclusions Specific targeting of metastatic and malignant properties of tumor cells by LIMK-2 ribozyme may serve as an effective therapy for invasive tumors with minimum effect on the surrounding normal cells. Copyright © 2004 John Wiley & Sons, Ltd. [source] Principles of antidote pharmacology: an update on prophylaxis, post-exposure treatment recommendations and research initiatives for biological agentsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2010S Ramasamy The use of biological agents has generally been confined to military-led conflicts. However, there has been an increase in non-state-based terrorism, including the use of asymmetric warfare, such as biological agents in the past few decades. Thus, it is becoming increasingly important to consider strategies for preventing and preparing for attacks by insurgents, such as the development of pre- and post-exposure medical countermeasures. There are a wide range of prophylactics and treatments being investigated to combat the effects of biological agents. These include antibiotics (for both conventional and unconventional use), antibodies, anti-virals, immunomodulators, nucleic acids (analogues, antisense, ribozymes and DNAzymes), bacteriophage therapy and micro-encapsulation. While vaccines are commercially available for the prevention of anthrax, cholera, plague, Q fever and smallpox, there are no licensed vaccines available for use in the case of botulinum toxins, viral encephalitis, melioidosis or ricin. Antibiotics are still recommended as the mainstay treatment following exposure to anthrax, plague, Q fever and melioidosis. Anti-toxin therapy and anti-virals may be used in the case of botulinum toxins or smallpox respectively. However, supportive care is the only, or mainstay, post-exposure treatment for cholera, viral encephalitis and ricin , a recommendation that has not changed in decades. Indeed, with the difficulty that antibiotic resistance poses, the development and further evaluation of techniques and atypical pharmaceuticals are fundamental to the development of prophylaxis and post-exposure treatment options. The aim of this review is to present an update on prophylaxis and post-exposure treatment recommendations and research initiatives for biological agents in the open literature from 2007 to 2009. [source] Engineering Allosteric Regulation into Biological CatalystsCHEMBIOCHEM, Issue 18 2009Jacques Fastrez Prof. Abstract Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site. This implies that communication pathways must exist between these sites. While mechanisms of allosteric regulation were developed more than forty years ago, they continue to be revisited regularly. The improved understanding of these mechanisms has led in the past two decades to projects to transform several unregulated enzymes into allosterically regulated ones either by rational design or directed evolution techniques. More recently, ribozymes have also been the object of similar successful engineering efforts. In this review, after briefly summarising recent progress in the theories of allosteric regulation, several strategies to engineer allosteric regulations in enzymes and ribozymes are described and compared. These redesigned biological catalysts find applications in a variety of areas. [source] | |||||||||||||||||||