RNA double cleavage by a hairpin-derived twin ribozyme

Abstract

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.

Figure 1

Secondary structures of the studied ribozymes. The four helices (H-1–H-4) in HP-WT, HP-RJ, HP-RJTL and HP-WTSV1 are indicated by bars. The arrows denote the sites of cleavage. Vector nucleotides resulting from in vitro transcription of HP-RJTL, HP-WTSV1 and HP-DS1 with T7 RNA polymerase are indicated in lower case. (a) Conventional hairpin ribozyme HP-WT with wild-type sequence. The fluorescently 5′- and 3′-labelled substrate S-WT is shown. (b) Reverse-joined hairpin ribozyme HP-RJ derived from HP-WT by dissecting the loop A and loop B domains at the hinge and rejoining them via an A₆ linker in reverse order. (c) Reverse-joined ribozyme HP-RJTL derived from HP-RJ with helix 3 capped by the UUCG tetraloop. (d) Conventional hairpin ribozyme with sequence variation HP-WTSV1. Base pairs which are reversed in comparison to HP-WT are highlighted in blue. (e) Twin ribozyme HP-DS1 derived from combination of the monoribozymes HP-RJTL and HP-WTSV1. The sequence introduced as spacer between both ribozyme units is highlighted in red.

Figure 2

Primary data on the cleavage of fluorescein 5′-labelled S-WT by the wild-type hairpin ribozyme HP-WT (lane 1) and of fluorescein 5′-labelled S-WTSV1 by the hairpin ribozyme with varied sequence HP-WTSV1 (lane 2). Cleavage conditions: 20 nM ribozyme, 200 nM substrate, 10 mM Tris–HCl pH 7.5, 12 mM MgCl₂, 37°C. Lane 3 shows the migration of a separately synthesised fluorescein 5′-labelled RNA (5′-AGACA-3′) corresponding to the sequence of the 5mer fragment resulting from cleavage of S-WTSV1 by HP-WTSV1. cp, 2′,3′-cyclic phosphate. All lanes show autoscaled data (ordinate, fluorescence intensity in arbitrary units).

Figure 3

Fluorescein building blocks used for RNA labelling by solid phase synthesis. (a) 5,6-Carboxy isomer of the fluorescein phosphoramidite building block used for 5′-labelling. (b) Polymer-bound fluorescein building block used for 3′-labelling.

Figure 4

RNA double cleavage by the twin ribozyme HP-DS1. The coloured squares refer to the sequences of the cleavage products. (a) Primary data on the cleavage of S-DS1 by HP-DS1 in the presence of Mg²⁺ after gel electrophoresis on the ALF DNA sequencer. Cleavage conditions: 20 nM HP-DS1, 200 nM S-DS1, 10 mM Tris–HCl pH 7.5, 12 mM MgCl₂, 37°C. (b) Primary data on the cleavage of S-DS1 by HP-DS1 in the presence of Mg²⁺ and spermine after gel electrophoresis on the ALF DNA sequencer. Cleavage conditions: 20 nM HP-DS1, 200 nM S-DS1, 10 mM Tris–HCl pH 7.5, 7 mM MgCl₂, 5 mM spermine, 37°C. All lanes in (a) and (b) show autoscaled data (ordinate, fluorescence intensity in arbitrary units). (c) Formation of 5mer (blue), 9mer (black), 25mer (red) and 29mer (green) fragments as a function of time. In a separate experiment, which involved cleavage of a double labelled substrate RNA at one specific position, the contribution of the fluorescein label at the 5′-end and the fluorescein label at the 3′-end to the fluorescence intensity of the substrate RNA was determined to be 3:2. This factor was used for standardising peak areas in quantification of the twin ribozyme cleavage reaction.