Biomedical subjects

M Warashina

Publications and source records attributed to M Warashina.

At least 37 records · Page 2Linked to original sources

PubMed2000

Significant activity of a modified ribozyme with N7-deazaguanine at g10.1: the double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes.

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 ( approximately 20 A) 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.

Catalysis↗

PubMed2000

Construction of a ribozyme-expression system that effectively transports ribozymes to the cytoplasm.

In our previous studies, it was demonstrated that the activity of a ribozyme in vivo was governed by several parameters, which include a high level-expression of ribozyme, the intracellular stability of the ribozyme and colocalization of the ribozyme with its target RNA in the same cellular compartment. To generate ribozymes with significant activity in vivo, we have developed a ribozyme-expression system based on a human tRNA(Val) promoter. Our tRNA-embedded ribozymes produced by our ribozyme-expression system remain relatively stable in cultured cells with half-lives longer than 30 min. Moreover, tRNA-ribozymes with a cloverleaf structure were efficiently exported from the nucleus to the cytoplasm, where they would effectively cleave target RNAs. In the present study, we investigated the relationship between the secondary structure of the tRNA-ribozymes and the transport efficacy of them in mammalian cells by using a screening system in vivo. Furthermore, we also investigated the mechanism of the export of tRNA-embedded ribozymes both in mammalian cells and in Xenopus oocytes.

Animals↗

PubMed2000

Suppression of BCR-ABL mRNA by various ribozymes in HeLa cells.

Ribozymes are RNA molecules with enzymatic activity that can cleave target RNA molecules in a sequence specific manner. To date, various types of ribozyme have been constructed to cleave other RNAs and such trans-acting ribozymes include hammerhead, hairpin and HDV ribozymes. External guide sequence (EGS) can also induce the suppression of a gene-expression by taking advantage of cellular RNase P. Here we compared the activities of various functional RNA cleavers both in vitro and in vivo. The first purpose of this comparison was intended to determine the best ribozyme motif with the highest activity in cells. The second purpose is to know the correlation between the activities of ribozymes in vitro and in vivo. Our results indicated that the intrinsic cleavage activity of ribozymes is not the sole determinant that is responsible for the activity of a ribozyme in cultured cells.

Endoribonucleases↗

PubMed1999

Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression.

Chronic myelogenous leukemia (CML) is associated with the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation of chromosomes 9 and 22. In the case of L6 (b2a2) mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure. As an extension of our molecular engineering of maxizymes, as well as to improve their potential utility, we examined whether an analogous conformational change could be induced within a single molecule when two maxizymes were connected via a linker sequence. An active conformation was achieved by binding of the construct to the BCR-ABL junction in trans, with part of the linker sequence then acting as an antisense modulator in cis (within the complex) to adjust the overall structure. Results of studies in vitro in the presence of cetyltrimethylammonium bromide (CTAB) (but not in its absence) suggested that a certain kind of connected maxizyme (cMzB) might be able to undergo a desired conformational change and, indeed, studies in vivo confirmed this prediction. Therefore, we successfully created a fully functional, connected maxizyme and, moreover, we found that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of CTAB.

Allosteric Regulation↗

PubMed1999

CTAB-mediated enrichment for active forms of novel dimeric maxizymes.

We demonstrated previously that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). As a result of such a dimeric structure, heterodimeric maxizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In this case, active heterodimers are in equilibrium with inactive homodimers. Longer forms of common stem II can lead to enrichment of the active heterodimers in vitro. In this study, we investigated whether the cationic detergent CTAB, which is known to enhance strand displacement of nucleic acids, might inhibit the dimerization of maxizymes. Significantly, under all conditions examined, CTAB instead enhanced the activity of a variety of maxizymes, with the extent of enhancement depending on the conditions. The activity of our least stable, least active maxizyme was enhanced 100-fold by CTAB. The strand displacement activity of CTAB thus appears to enhance the conversion of alternative conformations of inactive maxizymes, with intra- and inter-molecular hydrogen bonds, to active forms. Thus, our smallest maxizyme can also be considered a potential candidate for a gene-inactivating agent in vivo, in view of the fact that various facilitators of strand displacement reactions are known to exist in vivo (indeed, a separate experiment in cell culture supported the conclusion that our smallest maxizyme is a good gene-inactivating agent). Although activities of ribozymes in vitro do not necessarily reflect their activities in vivo, our findings suggest that the activity of ribozymes in vivo can be better estimated by running ribozyme kinetics in the presence of CTAB in vitro.

Base Sequence↗

PubMed1999

tRNAVal-heterodimeric maxizymes with high potential as geneinactivating agents: simultaneous cleavage at two sites in HIV-1 Tat mRNA in cultured cells.

It has been demonstrated that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). Intracellular activities of heterodimeric maxizymes and conventional ribozymes, under the control of a human tRNAVal-promoter, were compared against the cleavage of HIV-1 tat mRNA. The pol III-driven maxizymes formed very active heterodimers, and they successfully cleaved HIV-1 tat mRNA in mammalian cells at two sites simultaneously. The cleaved fragments were identified directly by Northern blotting analysis. Despite the initial concerns that a complicated dimerization process and formation of inactive homodimers were involved in addition to the process of association with the target, the overall intracellular activities of tRNAVal-driven maxizymes were significantly higher in mammalian cells than those of two sets of independent, conventional hammerhead ribozymes that were targeted at the same two sites within HIV-1 tat mRNA. Because the tRNAVal-driven maxizymes tested to date have been more effective than tRNAVal-driven "standard" hammerhead ribozymes, the tRNAVal-driven heterodimeric maxizymes appear to have potential utility as gene-inactivating agents.

Animals↗

PubMed1999

Extremely high and specific activity of DNA enzymes in cells with a Philadelphia chromosome.

BACKGROUND: Chronic myelogenous leukemia (CML) results from chromosome 22 translocations (the Philadelphia chromosome) that creates BCR-ABL fusion genes, which encode two abnormal mRNAs (b3a2 and b2a2). Various attempts to design antisense oligonucleotides that specifically cleave abnormal L6 BCR-ABL fusion mRNA have not been successful. Because b2a2 mRNA cannot be effectively cleaved by hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to engineer specific cleavage of this chimeric mRNA. Nonspecific effects associated with using antisense molecules make the use of such antisense molecules questionable. RESULTS: The usefulness of DNA enzymes in specifically suppressing expression of L6 BCR-ABL mRNA in mammalian cells is demonstrated. Although the efficacy of DNA enzymes with natural linkages decreased 12 hours after transfection, partially modified DNA enzymes, with either phosphorothioate or 2'-O-methyl groups at both their 5' and 3' ends, remained active for much longer times in mammalian cells. Moreover, the DNA enzyme with only 2'-O-methyl modifications was also highly specific for abnormal mRNA. CONCLUSIONS: DNA enzymes with 2'-O-methyl modifications are potentially useful as gene-inactivating agents in the treatment of diseases such as CML. In contrast to conventional antisense DNAs, some of the DNA enzymes used in this study were highly specific and cleaved only abnormal BCR-ABL mRNA.

Cells, Cultured↗

PubMed1999

Probing of the secondary structure of maxizymes.

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. In order to probe the putative conformational changes, we used a weakly alkaline solution to hydrolyze differentially phosphodiester bonds that were located in different environments. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA.

Allosteric Regulation↗

PubMed1999

Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes.

Recently, hammerhead ribozyme-mediated cleavage was analyzed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role could be monitored of metal ions that are directly involved in the cleavage reaction. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies demonstrated that binding of a metal ion to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and N7 of nucleotide G10.1 is critical for efficient catalysis. In order to clarify the effect of this metal ion, we chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1.

Catalysis↗

PubMed1999

Selection of intracellularly active ribozymes in mammalian cells.

Ribozymes are expected to be useful as antiviral agents and powerful tools of functional analysis of unknown gene products in vivo. For use of ribozymes in vivo, they must be fully functional in the intracellular environment. Not all ribozymes selected in vitro would be expected to work in vivo, whereas ribozymes selected in the intracellular environment should retain their function in vivo. With the eventual aim of using ribozymes as antiviral agents or biological tools in mammalian cells, we then devised a novel selection system in mammalian cells of active ribozymes by targeting at a gene for the cyclin dependent kinase inhibitor (CDKI), p16INK4a. In this system, we found that p16INK4a-knockdown cells became malignant and they formed foci. In the mammalian system, we confirmed that the selected cells harbored the active ribozyme, indicating that our positive selection systems in vivo were operational.

3T3 Cells↗

PubMed1998

Effects of helical structures formed by the binding arms of DNAzymes and their substrates on catalytic activity.

As a part of our efforts to clarify structure-function relationships in reactions catalyzed by deoxyribozymes (DNAzymes), which were recently selected in vitro , we synthesized various chimeras and analyzed the kinetics of the corresponding cleavage reactions. We focused on the binding arms and generated helices composed of binding arms and substrates that consisted of RNA and RNA, of RNA and DNA or of DNA and DNA. As expected for the rate limiting chemical cleavage step in reactions catalyzed by DNAzymes, a linear relationship between log( k cat) and pH was observed. In all cases examined, introduction of DNA into the binding helix enhanced the rate of chemical cleavage. Comparison of CD spectra of DNAzyme. substrate complexes suggested that higher levels of B-form-like helix were associated with higher rates of cleavage of the substrate within the complex. To our surprise, the enhancement of catalytic activity that followed introduction of DNA into the binding helix (enhancement by the presence of more B-form-like helix) was very similar to that observed in the case of the hammerhead ribozymes that we had investigated previously. These data, together with other observations, strongly suggest that the reaction mechanism of metal-ion-dependent DNAzymes is almost identical to that of hammerhead ribozymes.

Base Sequence↗

PubMed1998

Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.

DNA↗

PubMed1998

A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo.

We have constructed an allosterically controllable novel enzyme (designated maxizyme) that can be transcribed in vivo under the control of a human tRNA(Val) promoter. The maxizyme has sensor arms that can recognize target sequences, and in the presence of such a target sequence only, it can form a cavity that can capture catalytically indispensable Mg2+ ions. As a target for a demonstration of the potential utility of the maxizyme, we chose BCR-ABL mRNA, the translated products of which cause chronic myelogenous leukemia. Only the maxizyme (but not conventional ribozymes) had extremely high specificity and high-level activity, not only in vitro but also in cultured cells including BV173 cells derived from a patient with a Philadelphia chromosome. The maxizyme induced apoptosis only in leukemic cells with this chromosome.

Allosteric Regulation↗

PubMed1998

Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

Base Sequence↗

PubMed1997

Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA.

With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo , especially for the treatment of CML, if we can develop exogenous delivery strategies.

Base Sequence↗

PubMed1997

Structural analysis of nucleic acids by using fluorescence resonance energy transfer (FRET).

We examined changes in the extent of fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In our system, FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. While, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. It should help us to estimate the suitability of designed functional molecules, such as antisense DNA and RNA and ribozymes, that target to specific sites.

DNA↗

PubMed1997

Comparison of activities between hammerhead ribozymes and DNA enzymes targeted to L6 BCR-ABL chimeric (b2a2) mRNA.

For the treatment of chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using DNA enzymes, that was newly selected by Santoro & Joyce and that can cleave RNA molecules with high activity, to cleave of L6 BCR-ABL fusion (b2a2) mRNA. By contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction, without any cleavage of the normal ABL or BCR mRNA.

Base Sequence↗

PubMed1997

Activities of tRNA-embedded dimeric minizymes.

Ribozymes have been shown to be potent inhibitors of gene expression and viral function. Effects of ribozyme-mediated repression to target gene in living cells are correlated with the amounts of expression and stabilities of ribozyme molecules. In our previous study, it was demonstrated that a minimized hammerhead ribozyme, minizyme, with high activity forms a dimeric structure with a common stem II. We constructed dimeric minizymes that could cleave the BCR-ABL chimeric (b2a2) mRNA which had been difficult target for conventional hammerhead ribozymes without damaging the normal ABL mRNA. In order to achieve high expression of these dimeric minizymes in vivo for the treatment of CML, we embedded the dimeric minizyme portion downstream of a tRNA(Val) promoter sequence which could be recognized by RNA polymerase III. We determined cleavage activities of tRNA-embedded dimeric minizymes and compared the activities between tRNA-embedded hammerhead ribozyme and tRNA-embedded dimeric minizymes. All tRNA-embedded dimeric minizymes tested were capable of cleavage the target substrate. The activity of the tRNA-embedded dimeric minizyme targeted at BCR-ABL mRNA was almost the same as that of the naked dimeric minizymes. Interestingly, the cleavage activity of tRNA-embedded dimeric minizymes was higher than that of tRNA-embedded conventional hammerhead ribozyme.

Base Sequence↗