Ribozyme parental

The fact that, in both selection experiments, new solutions regarding the structure of the functional molecules have been adopted demonstrates that the best sequence for binding is not necessarily the best sequence for performing catalysis. It seems likely that many of the sequence solutions could also have been selected from completely randomized pools. This notion is confirmed by the aforementioned study by Hager and Szostak [82], in which the mutated ATP-aptamer motif was also included in the starting library but where the resulting ribozyme had no relationship to the parent ATP-binding motif. 

The 5"-hydroxy group of neomycin B was converted to an amino group by treatment of the 5"-tosylated derivative with sodium azide, followed by reduction of the azide to the amino group by triphenylphosphine in the presence of an aqueous solution of sodium hydroxide. The resulting 5"-amino neomycin B was shown to be a better RNA binder, slowing down the ribozyme cleavage more effectively than the parent compound [73]. 

We tried to create variants of hammerhead ribozymes with deletions in the stem/loop II region and, fortunately, we found that some shortened forms of hammerhead ribozymes had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme (R32 Fig.lA). Moreover, the active species appeared to form dimeric structures with a common stem II (Fig. IB). In the active short ribozymes, the linker sequences that replaced the stem/loop II region were palindromic so that two short ribozymes were capable of forming a dimeric structure with a common stem II. In order to distinguish monomeric forms of conventional minizymes that have extremely low activity from our novel dimers with high-level activity, we... 

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). 

The studies described above demonstrated that our heterodimcric maxiz3mie was more active than the parental hammerhead ribozymes in vivo. 

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. 

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