Branched Reverse-joined Hairpin Ribozymes (HP-RJBR)

As mentioned above the specific structure of HP-RJ has a clear disadvantage for use as universal catalytic module. The substrate binding domain cannot be extended in the 5' direction, requiring HP-RJ to be placed exclusively at the 5' end of a multi-target ribozyme. To overcome this limitation we prepared the branched ribozyme HP-RJBR (Figure 5.2.10).

In this structure the catalytic domain is connected to the substrate binding domain via the 4-position of a thymidine analog, enabling extension of the substrate-binding domain in 5' direction by a natural phosphodiester bond at the 5 '-OH of the sugar unit belonging to the same nucleoside. Before RNA synthesis we prepared the modified monomer 2'-deoxy-N4-(n-6-hydroxyhexyl)-5-methylcytidine which, after suitable protection and on incorporation into the RNA, was used for assembly of the second dimension chain via the hexyl-OH group, resulting in the generation of a non-natural branch (Figure 5.2.10).

Activity tests showed that the chemically synthesized branched ribozyme catalyzed the cleavage of the substrate RNA shown in Figure 5.2.10.

Fig. 5.2.9. Secondary structure of the three-way junction hairpin ribozyme HP-TJ. The arrow denotes the cleavage site. The five helices (H-1 through H-5) are marked by bars. 5'-dangling nucleosides result from in-vitro transcription.
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