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Application ofTwin Ribozymes

After having shown that combination of two catalytic modules in one molecule results in twin ribozymes with double cleavage activity we wondered whether twin ribozymes can also catalyze RNA ligation and thus be used for catalysis of RNA fragment-exchange reactions. If all fragments (substrates and cleavage products)

Fig. 5.2.13. Time course of the HP-TW1 cleavage reaction.

remain bound to the ribozyme, the hairpin ribozyme mentioned above is a better ligase than an endonuclease, it ligates twice as fast as it cleaves its substrate [11]. Because of rapid dissociation of cleavage products in the minimal construct as shown in Figure 5.2.5, however, the fragments are not available for re-ligation and substrate cleavage is the predominant reaction. This in turn, opens up control of the activity of the ribozyme by duplex stability. Substrates that are weakly bound to the ribozymes should be preferentially cleaved, whereas strongly bound substrates should be ligated.

On the basis of this assumption we designed the twin ribozyme HP-TW2 (Figure 5.2.11) for catalysis of an RNA exchange reaction. HP-TW2 consists of two catalytic modules, a reverse-joined (left unit) and a three-way junction hairpin ribozyme (right unit). On binding of the substrate a GGGAGA loop is formed in the central region of the ribozyme, because no complementary bases are provided in the substrate sequence. Thus after cleavage at both predicted sites a 14-mer RNA fragment is produced which, because of helix destabilization by the loop, should easily dissociate from the ribozyme. The incoming fragment to be ligated contains the six additional complementary bases and thus forms a more stable duplex with the ribozyme than the removed 14-mer (Figure 5.2.14). Preliminary results suggest that HP-TW2 catalyzes the exchange of those two fragments, although still with rather low yield.

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