I

N, segment I

Fig. 5.2.10. Secondary structure of the branched ribozyme HP-RJBR and structure of the branching nucleoside. The arrow denotes the cleavage site. The single segments within the ribozyme strand are marked.

Fig. 5.2.11. Secondary structures of twin ribozymes. The arrows mark the cleavage sites. The substrates are fluorescein-labeled at both ends.
Fig. 5.2.11 (continued)

TW2. Fluorescein-labeled substrates were synthesized exclusively by solid-phase chemistry and the fluorescein residue was attached during this procedure [16].

HP-TW1 was assembled from the catalytic strand HP-TW1 and the substrate strand (Figure 5.2.11). The 34-mer substrate-RNA has a fluorescein-label at both the 3'- and the 5' -ends, to enable monitoring of the cleavage at both predicted sites by use of our previously developed quantitative assay of hairpin ribozyme activity [16]. Specific cleavage by the reverse-joined ribozyme unit within the twin ribozyme would produce a 9-mer and a 25-mer RNA; cleavage by the conventional hairpin ribozyme unit would produce a 5-mer and a 29-mer RNA. Both the 29-mer and the 25-mer fragments are still substrates for single cleavage at the second site. Formation of the 5-mer and 9-mer cleavage products is therefore expected to accelerate during reaction and these should be found as the main products (Figure 5.2.12).

Because the substrate is labeled at the 3 0-end and at the 5 0-end, short product RNAs and the longer product strands are both detectable. Analysis of the cleavage reaction shows that the substrate RNA is cleaved at two specific sites after incubation with the twin ribozyme, leading to the characteristic 5-mer and 9-mer fragments and to a 20-mer, which cannot be detected by laser-induced fluorescence, as main products. Whereas the fractions of the 5-mer and 9-mer produced increase with time, the fractions of the 25-mer and the 29-mer decrease after an initial increase, based on cleavage occurring at the second specific site (Figure 5.2.13). Similar behavior was observed for the cleavage reaction of HP-TW2 and HP-TW3.

Fig. 5.2.12. Schematic representation of cleavage of the 34-mer substrate RNA by HP-TW1 carrying a fluorescein label at both ends.
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