Maxizymes tRNAVAL

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). 

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

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... 

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