Maxizymes dimeric

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... 

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

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... 

There have been many prior attempts using various approaches to construct artificial allosteric enzymes, but there have been no examples reported that can be used in vivo. The experimental results described alxtve represent the first successful construction of an allosteric enzyme that functions at the tissue culture level. The maxizyme is new in that it imparts a sensor function to the short ribozymes that function as a dimer. By using this sensor function, it has become possible to specifically cleave abnormal chimeric mRNA only, without affecting the normal mRNA which should not be cleaved. This could not be achieved with the previously used ribozymes. There is more potent inhibition of expression of abnormal mRNA by the maxizyme than the previously used ribozymes, as has been demonstrated in cells derived from patients. Currently, the only known the effective... 

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