In vitro Selection of a hairpinribozyme for cleavage and ligation of substrates beyond the consensus sequence

Twin ribozymes are derived from hairpin ribozymes by tandem duplication. Their catalytic activity is based on the cleavage and ligation ability of the hairpin ribozyme. Twin ribozymes promote two site specific RNA-cleavages and two RNA-ligations. As a result, a specific fragment is cleaved out of the substrate-RNA, and subsequently becomes replaced by a separately added oligonucleotide that is ligated to the remaining parts of the former substrate. In the aggregate, these events mediate a specific exchange of RNA patches.

The twin ribozyme HP-TW anti ΔUCU^343-345 CTNNB1 catalyzes the fragment exchange reaction in the oncogenic CTNNB1 mRNA in vitro. The sequence of the substrate differs from the consensus sequence of the hairpin ribozyme, which was originally defined as Y^-2­N-1↓G⁺¹U⁺²Y⁺³B⁺⁴, with Y = C or U, and B = U, C or G. Not surprisingly, activity of a wild type hairpin ribozyme for the CTNNB1 substrate is very low. To improve the cleavage-and ligation activity, ribozyme variants with compensatory base substitutions can be rationally designed. However, in a more general approach, we decided to set-up an in vitro selection assay for the development of highly active hairpin ribozymes that are specific for the CTNNB1 substrate. Therein, cleavage and ligation should occur at 5’-A↓GCU-3’ sites.