RNA double cleavage by a hairpin-derived twin ribozyme.
The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.
Schmidt
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C
Humboldt-Universität zu Berlin, Institut für Chemie, Fachinstitut für Organische und Bioorganische Chemie, Hessische Strasse 1-2, 10115 Berlin, Germany.
Welz
R
R
Müller
S
S
D001483:Base Sequence; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*
D016337:RNA, Catalytic; D012313:RNA