No abstract

Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y−2N−1*G+1U+2Y+3B+4, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions −2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U−2G−1*G+1U+2A+3A+4 sites. Substrates with G−1*G+1U+2A+3 sites were previously shown to be processed by the wild‐type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight‐fold) and ligation (100‐fold). In particular, we show that A+3 and A+4 are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G6→U, owing to restoration of a Watson–Crick base pair in helix 1. In this ribozyme–substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A7 with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A7, C7 and G7. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.

RNA repair is an emerging strategy for gene therapy. Conventional gene therapy typically relies on the addition of the corrected DNA sequence of a defective gene to restore gene function. As an additional option, RNA repair allows alteration of the sequence of endogenous messenger RNAs (mRNAs). mRNA sequence alteration is either facilitated by intracellular spliceosome machinery or by the intrinsic catalytic activity of trans-acting ribozymes. Previously we developed twin ribozymes, derived from the hairpin ribozyme, by tandem duplication and demonstrated their potential for patchwise RNA repair. Herein we describe the development of such a twin ribozyme for potential repair of a deletion mutation in the oncogenic CTNNB1-ΔS45 mRNA. We demonstrate that hairpin ribozyme units within the twin ribozyme can be adapted to efficiently cleave/ligate non-consensus substrates by introduction of compensatory mutations in the ribozyme. Thus, we show the twin ribozyme mediated repair of truncated CTNNB1 transcripts (up to 1000 nt length). Repair of the entire CTNNB1-ΔS45 mRNA, although apparently possible in general, is hampered in vitro by the secondary structure of the transcript.

Kinetic analysis of ribozyme reactions is a common method to evaluate and compare activities of catalytic RNAs. The hairpin ribozyme catalyzes the reversible cleavage of a suitable RNA substrate at a specific site. Hairpin ribozyme variants as an allosteric ribozyme responsive to flavine mononucleotide and a hairpin-derived twin ribozyme that catalyzes two cleavage reactions and two ligation events with the result of a fragment exchange have been developed by rational design and were kinetically characterized. Herein, protocols for preparation of ribozymes and dye-labeled substrates as well as for analysis of cleavage, ligation, and fragment exchange reactions are provided.