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.

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.

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.

RNA-protein hybrid ribozymes that efficiently cleave any mRNA independently of the structure of the target RNA.

Ribozyme activity in vivo depends on achieving high-level expression, intracellular stability, target colocalization, and cleavage site access. At present, target site selection is problematic because of unforeseeable secondary and tertiary RNA structures that prevent cleavage. To overcome this design obstacle, we wished to engineer a ribozyme that could access any chosen site. To create this ribozyme, the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, was attached to our ribozymes so that the helicase-bound, hybrid ribozymes would be produced in cells. This modification significantly enhanced ribozyme activity in vivo, permitting cleavage of sites previously found to be inaccessible. To confer cleavage enhancement, the CTE must retain helicase-binding activity. Binding experiments demonstrated the likely involvement of RNA helicase(s). We found that attachment of the RNA motif to our tRNA ribozymes leads to cleavage in vivo at the chosen target site regardless of the local RNA secondary or tertiary structure.

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.

Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells.

Nineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus. No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting step in vivo. For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules.

A novel approach to selection of functional proteins.

We present here a novel approach to connect phenotype and genotype in vitro by exploiting a strong interaction between RNA and protein. This strong interaction was used for the selection of functional proteins, such as dihydrofolate reductase (DHFR) and streptavidin, which were chosen as examples. The noncovalent but strong interaction should be useful as a novel tool for the future selection of functional proteins.

Allosterically controllable maxizyme-mediated suppression of progression of leukemia in mice.

Chronic myelogenous leukemia (CML) is a hematopoietic malignant disease associated with expression of a chimeric BCR-ABL gene. We recently succeeded in designing a novel allosterically controllable ribozyme, the maxizyme (Tanabe et al. Biomacromolecules 2000, 1, 108-117; Kuwabara et al. Biomacromolecules 2001, 2, 788-799), that not only specifically cleaves BCR-ABL mRNA and induces apoptosis in cultured CML cells but also shows significant inhibition against the growth of an established BV173 cell line in a mouse model (Tanabe et al. Nature 2000, 406, 473-474). As an extension of our studies, we tested the maxizyme against primary CML cells in the same mouse model. The maxizyme under the control of a tRNA(Val) promoter showed significant inhibition against the growth of the primary bone marrow cells from a Japanese patient with CML. Specifically, to examine the applicability of the maxizyme in the treatment of CML, we assessed the antitumor effect of the maxizyme in murine models of CML. Fourteen weeks after the injection of primary CML cells into a NOD-SCID mouse, the bone marrow of the mouse was filled with primary CML cells as a result of diffuse leukemia. In marked contrast, when maxizyme-expressing primary CML cells were injected, the mouse remained disease-free. These results further strengthen our earlier suggestion that the maxizyme technology might provide a useful approach to the treatment of CML.

Recognition of engineered tRNAs with an extended 3' end by Exportin-t (Xpo-t) and transport of tRNA-attached ribozymes to the cytoplasm in somatic cells.

Our recent analysis indicates that the cytoplasmic localization of tRNA-attached ribozymes (tRNA-Rz) is critical for its high-level intracellular activity, suggesting that mature mRNAs in the cytoplasm are more accessible to ribozymes than pre-mRNAs in the nucleus (Kato et al. J. Biol. Chem. 2001, 276, 15378-15385; Kuwabara et al. Nucleic Acids Res. 2001, 29, 2780-2788). Although studies in Xenopus oocytes led to the proposal that only correctly processed mature tRNAs are exported from nuclei in a RanGTP-dependent manner (Lund and Dahlberg Science 1998, 282, 2082-2085), our tRNA-Rz with an extended 3' end can also be exported to the cytoplasm in somatic cells. Xpo-t/RanGTP bound to tRNA-attached ribozymes in vitro and in somatic cells, with recognition basically resembling the recognition of mature tRNAs. In contrast, no binding to tRNA-attached ribozymes occurred in Xenopus oocytes. The injection of a nuclear extract of Xenopus oocytes together with tRNA-attached ribozymes inhibited the export of tRNA-attached ribozymes but not mature tRNAs in somatic cells, suggesting the existence of an inhibitor(s) of the Xpo-t-dependent export pathway. Moreover, the inhibitor(s) appears responsible for a proofreading mechanism that operates in oocytes.

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.

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.

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.

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.

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.

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.

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.

Maxizymes, novel allosterically controllable ribozymes, can be designed to cleave various substrates.

We demonstrated previously that an allosterically controllable novel ribozyme, designated the maxizyme, is a powerful tool for disruption of an abnormal chimeric RNA target [BCR-ABL (b2a2) mRNA], and we proposed that it might provide the basis for future gene therapy for the treatment of chronic myelogenous leukemia (Kuwabara et al. Mol. Cell 1998, 2, 617-627). The maxizyme has sensor arms that can recognize a specific sequence and, in the presence exclusively of such a specific sequence, it can form a cavity for capture of catalytically indispensable Mg2+ ions. Cleavage of the target RNA then occurs at a site distant from the specific sequence. Clearly, the specific sequences recognized by sensor arms should not be limited to those of the above mentioned abnormal chimeric target. Thus, to demonstrate the general applicability of maxizyme technology, we constructed maxizymes targeted to other mRNAs, such as PML-RAR alpha mRNA, sDLST mRNA, and BCR-ABL (b1a2) mRNA, that are not cleaved with high specificity by the wild-type hammerhead ribozyme. Specific and efficient cleavage in vitro of these mRNAs by the custom-designed maxizymes demonstrated clearly that maxizyme technology is not limited to a specific case but may have broad general applicability in molecular biology and, also, in a clinical setting.

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.