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.

Recent advances in the elucidation of the mechanisms of action of ribozymes.

The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme-substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pK(a) of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Working at the cutting edge: the creation of allosteric ribozymes.

The appropriate folding of catalytic RNA is a prerequisite for effective catalysis. A novel ribozyme, the maxizyme, has been generated and its activity can be controlled allosterically. The maxizymes work both in vitro and in vivo indicating the potential utility of this novel class of ribozyme as a gene-inactivating agent with a biosensor function.

Allosterically controllable ribozymes with biosensor functions.

The concept of allosteric regulation has already been exploited in the creation of artificial ribozymes and the activities of certain ribozymes can be controlled allosterically by specific effectors. Ribozymes with such properties are in the spotlight as biosensors. Such artificial allosterically regulated ribozymes have potential utility as nucleic-acid-based biosensors.

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.

Differences among mechanisms of ribozyme-catalyzed reactions.

The catalytic properties of ribozymes depend on the sophisticated structures of the respective ribozyme-substrate complexes. Although it has been suggested that ribozyme-mediated cleavage of RNA occurs via a rather strictly defined mechanism, recent findings have clearly demonstrated the diversity of reaction mechanisms.

Allosterically controllable maxizymes cleave mRNA with high efficiency and specificity.

Ribozymes are small and versatile nucleic acids that can cleave RNA molecules at specific sites. However, because of the limited number of cleavable sequences on the target mRNA, in some cases conventional ribozymes do not have precise cleavage specificity. To overcome this problem, an allosteric version (a maxizyme) was developed that displayed activity and specificity in vivo. More than five custom-designed maxizymes have demonstrated sensor functions, which indicates that the technology might be broadly applicable in molecular biology and possibly in the clinic.

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.

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.

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme.

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G(8) and A(9), a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the (31)P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. (31)P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S-substrate complexes generated in the absence of Mg(2+) ions were still in an inactive form and that Mg(2+) ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.

Effects of cetyltrimethylammonium bromide on reactions catalyzed by maxizymes, a novel class of metalloenzymes.

We demonstrated previously that some shortened forms of hammerhead ribozymes had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme. Moreover, the active species appeared to form dimeric structures with a common stem II (in order to distinguish monomeric forms of conventional minizymes that have low activity from our novel dimers with high-level activity, the latter very active short ribozymes were designated 'maxizymes'). The dimers can be homodimeric (with two identical binding sequences) or heterodimeric (with two different binding sequences). In the case of heterodimers, they are in equilibrium with inactive homodimers. In this study, we investigated the effects of cationic detergent, cetyltrimethylammonium bromide (CTAB), on reactions catalyzed by a variety of maxizymes. The slope of close to unity in profiles of pH versus rate demonstrated that the deprotonation was important in catalysis and that the rate-limiting chemical step was followed in these reactions. Addition of appropriate amounts of CTAB enhanced the activity of a variety of maxizymes. The activity of our least stable, least active maxizyme was enhanced 100-fold by CTAB. Thus, CTAB effectively enhanced the conversion of kinetically trapped inactive conformations to active forms. Moreover, we suggest 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 appropriate amounts of CTAB.

Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme.

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. We designed the maxizyme in such a way that it was able to form an active core with which to capture the catalytically indispensable Mg2+ ions only in the presence of the BCR-ABL mRNA junction. In order to probe the putative conformational changes, we used a weakly alkaline solution (pH 9.2) in the presence of 25 mM Mg2+ ions to hydrolyze differentially phosphodiester bonds that were located in different environments. Phosphodiester bonds in single-stranded regions were clearly more susceptible to attack by alkali than those within a double-stranded helix. 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. Moreover, we demonstrated that the use of mild alkaline solutions to probe RNA structures is very informative.

[Effects of chronic ethanol feeding on lipid composition of rat liver plasma membrane--changes of membrane by acute ethanol loading or its withdrawal in vivo].

The effects of chronic ethanol feeding on lipid composition of rat liver plasma membrane were studied by following ethanol loading or its withdrawal in vivo. Male Wistar rats were pair-fed by a liquid diet containing ethanol as 36% of energy or an isocarolic amounts of glucose for 6 weeks. Chronic ethanol feeding resulted in an increase of cholesterol content and the cholesterol/phospholipid molar ratio in liver plasma membrane compared with pair-fed control rats. Acute ethanol (3 g/kg body weight) orally loading to rats fed ethanol chronically did not change any lipid composition of liver plasma membrane. However, withdrawal of ethanol for 2 days led cholesterol content and the cholesterol/phospholipid molar ratio of liver plasma membrane to be normal values. These data indicated that chronic ethanol feeding produced membrane alterlation that induced resistance to ethanol-induced membrane structural disordering (membrane tolerance), and that this alterlation resulted in a homeoviscous adaptation of liver plasma membrane.

Influence of sodium benzoate on the metabolism of o-xylene in the rat.

The main metabolites of o-xylene in urine are o-methylhippuric acid, o-toluic acid, o-toluic acid glucuronide, 3,4-dimethylphenol, 3,4-dimethylphenol conjugates and o-xylylmercapturic acid. The urinary excretion of o-toluic acid, o-toluic acid conjugates and o-xylene were increased by the prior administration of sodium benzoate. Conversely, the amounts of o-methylhippuric acid, 3,4-dimethylphenol conjugates and o-xylylmercapturic acid decreased by sodium benzoate pretreatment. In addition, the urinary excretion of o-methylhippuric acid was delayed by the pretreatment. The percentages of urinary excretion of the o-xylene metabolites were substantially changed by the pretreatment with sodium benzoate. These results therefore highlight a potential interaction of an air pollutant with a food additive, an interaction that remains to be established in man.

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.

Allosterically controlled single-chained maxizymes with extremely high and specific activity.

For the treatment of chronic myelogenous leukemia (CML), attempts have been made to design various ribozyme motifs that can specifically recognize and cleave BCR-ABL fusion mRNAs. In the case of L6 BCR-ABL 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 [Kuwabara, T.; et al. Mol. Cell 1998, 2, 617-627; Tanabe, T.; et al. Nature 2000, 406, 473-474]. Specifically, we tailored the maxizyme with molecular switching function: the maxizyme splices a cleavable GUC site, but only when it appears within a strand of mRNA that possesses the abnormal splice junction. We demonstrated that this approach is generalizable [Tanabe, T.; et al. Biomacromolecules 2000, 1, 108-117]. All the maxizymes designed in the past functioned as a result of the formation of a dimeric structure. Questions have been asked whether a similar molecular switching might be possible within a single molecule when two monomer units of the maxizyme were connected via a linker sequence. We found that an analogous conformational change could not be induced within a single molecule when two maxizyme units were simply connected via a nonregulatable linker sequence. However, an active conformation was achieved by the introduction of an antisense modulator within the linker sequence that adjusted the overall structure to the correct form. Results of studies in cultured cells suggested that the desired conformational change could indeed be induced within the modified single-chained maxizyme and such a construct caused apoptosis only in leukemic cells with the Philadelphia chromosome.

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.

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.

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.