Maxizymes

Kuwabara, T., Warashina, M., Tanabe, T., Tani, K., Asano, S. and Taira, K. (1998) A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo. Mol. Cell, 2, 617-627. 

Ribozymes cleave RNA molecules at specifrc sites. In order to modify and extend the number of cleavable target sequences on the mRNA, an allosteric version of a ribozyme, a maxizyme, in vivo activity and specificity was developed several custom-designed maxizymes showed sensor functions, thus indicating the applicability of the technology for the design of maxizymes with use in analytics and therapy. 

Tlie term maxizyme is chosen for the designed ribozymes because they ai c highly active, minimized dimeric ribozymes [minimized, active, X-shaped (functioning as a dimer), intelligent (allosterically controllable) ribozymes... 

Tomoko Kuwabara, Masaki Warashina and Kazunari Raira, Allosterically controllable maxizymes cleave mRNA with high efficiency and specificity, TIBTECH, 18 (2000), 462 68. 

RIBOZYME TECHNOLOGY 2 NOVEL ALLOSTERICALLY CONTROLLABLE RIBOZYMES, THE MAXIZYMES, WITH CONSIDERABLE POTENTIAL AS GENEINACTIVATING AGENTS... 

Figure I. Secondary strucnires of (A) a wild-lype hammerhead ribozyme (R32), (B) a maxizyme that is capable of forming a homodimer and (C) the heterodimeric maxizyme. The effects of the concentration of the uncleavable pseudosubstrate on the cleavage activity (Vq) of the heterodimeric maxizyme is also shown in Figure IC.

DISCOVERY OF MAXIZYMES THAT ACT AS A DIMERIC FORM OF SHORT RIBOZYMES... 

Since the signals due to imino protons of the maxizyme were broader than those of R32 and since other signals were also detected, it appeared that the homodimeric maxizyme existed in the presence of other contaminating conformers. The NMR spectra of imino protons changed with changes in temperature (Fig.2B and 2C). 

Figure 2. The NMR spectra of a hammerhead ribozyme (R32) and of the maxizyme. (A) Apparent secondary structures of R32 and the homodimeric maxizyme are shown on the leit, and the NMR spectra of imino proton of R32 and a homodimeric maxizyme [in 0.1 M NaCl, 10 mM sodium phosphate buffer (pH 7.0) at 5 C] are shown on the right. Effects of temperature on the NMR spectra of the imino protons of R32 (B) and the homodimeric maxizyme (C) are also shown.

CONSTRUCTION OF tRNAVAL.EMBEDDED MAXIZYMES AND DETECTION OF THE FORMATION OF DIMERIC STRUCTURES BY tRNAVAL EMBEDDED MAXIZYMES... 

More direct evidence for the formation of dimers by the tRNA al. embedded homodimeric maxizyme (tRNAVal Mz Fig.3B)was provided by gel-shift analysis in the absence of the substrate. As controls, we also analyzed tRNAVal transcripts that contained a nonsense sequence between the tRNAVal portion and the terminator sequence, as well as transcripts of the gene for the tRNAVal-embedded parental hammerhead ribozyme (tRNAVal.R32). Shifted bands (dimers) were observed only in the case of the tRNAVal-Mz (Fig.3B). 

Figure 3. The dimeric form of a tRNAV hembedded maxi2yine. (A) A model of the tRNA-embedded dimeric maxizyme (tRNAVafMz). In this model, the internal loop or linker is ignored and is assumed to be an A-form duplex. (B) Detection of the dimeric form of tRNAVal-Mz. Gel-shift analysis revealed that the dimerization occurred only in the case of tRNAV l-Mz, S - P-labeled tRNAV hribozyme (2 nM) was incubated with increasing amounts of the respective non-radiolabeled RNA in 25 mM MgCl2 and 50 mM Tris-HCl (pH 8.0) at 37 C for 20 minutes. The reaction products were separated on a non-denaturing gel.

The luciferase activity recorded when we used only the Tat-expression vector (pCD-SRa tat) was taken as 100%. Ribozyme- and Tat-expression vectors were used at a molar ratio of 2 1 for co-transfection of LTR-Luc HeLa cells. The results shown in Figure 4C are the averages of results from five sets of experiments. As shown in Figure 4C, the tRNAVal-dimeric maxizyme was extremely effective (D-L/R >90% inhibition), despite the fact that the SRa promoter, which controlled the transcription of the target... 

Figure 4. The heterodimcric maxizyme that can cleave an mRNA at two sites simultaneously. (A) Schematic representation of the heteradimeric maxizyme that cleave an mRNA at two sites simultaneously. (B) Assay system for measurements of activities of tRNA -ribozymes in LTR-Luc HeLa cells and (C) the effects of tRNA hribozymes on the Tat-mediated transcription of the chimeric LTR-Luc gene. Luciferasc activity was normalized by reference to the efficiency of transfection, which was determined by monitoring activity of a co-transfected gene for p-galactosidase.

The novel tRNA -driven heterodimeric maxizymes, which can cleave one substrate at two independent sites simultaneously, can be designed very easily and, thus, they should be very useful tools in vivo. Nevertheless, it remains true that two independent iRNA l-driven parental riboz5mies can also cleave such a substrate at the two different sites, albeit with lower efficiency In the following section, we shall describe tRNA Ldnven heterodimeric maxizymes that specifically cleave a chimeric mRNA with high selectivity while conventional ribozymes failed to do so. 

Figure 5. Types of translocation and the possible sites of cleavage within notmal ABL mRNA and abnormal BCR-ABL fusion mRNAs by conventional ribozymes (A) and the design of the novel maxizyme (B).

Figure 6. Secondary structures of the active (A) and inactive maxizymes (B).

In order to prove in vitro that ehanges in the conformation of our heterodimeric maxizyme depended on the presence or absence of the abnormal b2a2 mRNA, we prepared a short 16-nucleotide (nt) BCR-ABL substrate (SI6) that corresponded to the target (cleavage) site indicated by capital letters in the upper panel of Figure 6B (within exon 2 of ABL). 

Figure 7. Allosteric control of the activity of the maxii rme in vitro. The speciflcily of maxizyme-medialied cleavage was examined by incubating iRNA h nven component(s) with the 5 - P-labeled 16 in the presence and in the absence of an allosteric effector molecule, namely, either a normal ABL effector or a BCR-ABL effector. MzL and/or MzR were incubated at 0.1 pM with 2 nM 5 - P-tabeled SI6. When applicable, the concentration of the effector, 20-mer ABL or 28-mcr BCR-ABL, was 1 pM. The catalytic action of ions is indicated by Mg scissors .

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