Twin Ribozyme Research Articles

In vitro repair of a defective EGFP transcript and translation into a functional protein.

Twin ribozymes mediate the exchange of a short patch of RNA against an exogenous oligonucleotide within a suitable RNA substrate. Thus, twin ribozymes are promising tools for RNA repair, i.e. for the treatment of genetic disorders at the mRNA level. A number of twin ribozyme-mediated RNA fragment exchange reactions have been successfully demonstrated using short model substrates. Herein we show for the first time a twin ribozyme-mediated in vitro repair of a full-length transcript and translation into a functional protein. The system is based on the repair of a designed mutant EGFP mRNA containing the four-base deletion ΔACTC (190-193). Upon twin ribozyme-mediated replacement of a patch of 15 nucleotides with an externally added repair oligonucleotide (19 mer) the wild type sequence of the EGFP transcript could be restored with 32% yield. This is the first time that such a high twin ribozyme-mediated repair yield, so far observed only for short model substrates, has been obtained for a full-length mRNA. Translation of the repaired EGFP-ΔACTC mRNA produces functional EGFP, as detected by the restored fluorescence.

Design and characterization of a twin ribozyme for potential repair of a deletion mutation within the oncogenic CTNNB1-ΔS45 mRNA.

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.

The many faces of the hairpin ribozyme: Structural and functional variants of a small catalytic rna

The hairpin ribozyme is a small catalytic RNA that has been reengineered resulting in a number of variants with extended or even new functions. Thus, manipulation of the hairpin ribozyme structure has allowed for activity control by external effectors, namely oligonucleotides, flavine mononucleotide, and adenine. Hairpin ribozyme-derived twin ribozymes that mediate RNA fragment exchange reactions as well as self-processing hairpin ribozymes were designed. Furthermore, several hairpin ribozyme variants have been engineered for knock down of specific RNA substrates by adapting the substrate-binding domain to the specific target sequence. This review will focus on hairpin ribozymes possessing structural extensions/variations and thus functionally differing from the parent hairpin ribozyme.

Twin ribozyme mediated removal of nucleotides from an internal RNA site

Over the past two decades, the structure and mechanism of catalytic RNA have been extensively studied; now ribozymes are understood well enough to turn them into useful tools. After we have demonstrated the twin ribozyme mediated insertion of additional nucleotides into a predefined position of a suitable substrate RNA, we here show that a similar type of twin ribozyme is also capable of mediating the opposite reaction: the site-specific removal of nucleotides. In particular, we have designed a twin ribozyme that supports the deletion of four uridine residues from a given RNA substrate. This reaction is a kind of RNA recombination that in the specific context of gene therapy mimics, at the level of RNA, the correction of insertion mutations. As a result of the twin ribozyme driven reaction, 17% of substrate are converted into the four nucleotides shorter product RNA.

Site‐Specific Fluorescent and Affinity Labelling of RNA by Using a Small Engineered Twin Ribozyme

Site‐Specific Fluorescent and Affinity Labelling of RNA by Using a Small Engineered Twin Ribozyme

Efficient RNA ligation by reverse‐joined hairpin ribozymes and engineering of twin ribozymes consisting of conventional and reverse‐joined hairpin ribozyme units

In recent years major progress has been made in elucidating the mechanism and structure of catalytic RNA molecules, and we are now beginning to understand ribozymes well enough to turn them into useful tools. Work in our laboratory has focused on the development of twin ribozymes for site-specific RNA sequence alteration. To this end, we followed a strategy that relies on the combination of two ribozyme units into one molecule (hence dubbed twin ribozyme). Here, we present reverse-joined hairpin ribozymes that are structurally optimized and which, in addition to cleavage, catalyse efficient RNA ligation. The most efficient variant ligated its appropriate RNA substrate with a single turnover rate constant of 1.1 min(-1) and a final yield of 70%. We combined a reverse-joined hairpin ribozyme with a conventional hairpin ribozyme to create a twin ribozyme that mediates the insertion of four additional nucleotides into a predetermined position of a substrate RNA, and thus mimics, at the RNA level, the repair of a short deletion mutation; 17% of the initial substrate was converted to the insertion product.

RNA Synthesis by T7 RNA Polymerase Supported Primer Extension

Transcription of RNA molecules from synthetic DNA templates with T7 RNA polymerase is a common procedure for the preparation of long RNA molecules. However, enzymatic synthesis does not allow for site-specific incorporation of modified nucleotides. RNA synthesis by chemical methods on the other side can satisfy this purpose, but it is limited to RNA fragments of about 80 nucleotides at maximum. We aimed to combine both chemical and enzymatic procedures to synthesise RNA molecules by RNA primed transcription with T7 RNA polymerase. To this end we have chemically synthesised a fluorescein labelled RNA primer and studied elongation of this primer by T7 RNA polymerase on a single-stranded DNA template. We show that the enzyme is capable of extending the primer to the full-length product. The 34-mer RNA that has been synthesised by RNA primed transcription served as substrate for a twin ribozyme and was successfully cleaved in the expected manner.

Site-directed alteration of RNA sequence mediated by an engineered twin ribozyme.

Repair of deletion mutations at the RNA level: A small synthetic twin ribozyme supports the insertion of four additional nucleotides into a predefined site of an arbitrarily chosen substrate RNA (see scheme). This method of RNA repair constitutes a novel approach to the therapy of medically relevant mutations.

Flanking sequences with an essential role in hydrolysis of a self-cleaving group I-like ribozyme.

DiGIR1 is a group I-like ribozyme derived from the mobile twin ribozyme group I intron DiSSU1 in the nuclear ribosomal DNA of the myxomycete Didymium iridis. This ribozyme is responsible for intron RNA processing in vitro and in vivo at two internal sites close to the 5'-end of the intron endo-nuclease open reading frame and is a unique example of a group I ribozyme with an evolved biological function. DiGIR1 is the smallest functional group I ribozyme known from nature and has an unusual core organization including the 6 bp P15 pseudoknot. Here we report results of functional and structural analyses that identify RNA elements critical for hydrolysis outside the DiGIR1 ribozyme core moiety. Results from deletion analysis, disruption/compensation mutagenesis and RNA structure probing analysis all support the existence of two new segments, named P2 and P2.1, involved in the hydrolysis of DiGIR1. Significant decreases in the hydrolysis rate, k (obs), were observed in disruption mutants involving both segments. These effects were restored by compensatory base pairing mutants. The possible role of P2 is to tether the ribozyme core, whereas P2.1 appears to be more directly involved in catalysis.

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.

Rational Design and Synthesis of Ribozymes

We review our procedure for the rational design and the synthesis of oligoribonucleotides with particular reference to our recent studies of the development of a hairpin-derived twin ribozyme. Our work focuses on the design of ribozymes that are derived from naturally occurring structures but posses extended catalytic activities. Currently we are studying ribozymes that are capable of site-specific RNA double cleavage. We have designed these “twin ribozymes” by combining of two catalytic motifs in one molecule. In addition to kinetic analysis of the cleavage reaction, we are interested in the general phenomena of RNA folding and the characterization of RNA structure–function relationships. The developed twin ribozymes are particularly well suited for this purpose. They have to fold into a complex structure to achieve catalytic activity. Since there is a direct correlation between conformation and activity, the process of folding can be followed by monitoring the cleavage reaction. The twin ribozymes were synthesized by a combination of phosphoramidite chemistry and in vitro transcription techniques. We report the synthesis of a powerful reagent to replace tetrazole for activation of RNA phosphoramidites in automated solid-phase synthesis, allowing for coupling yields of >99%. Furthermore, we introduce a fast quantitative assay of ribozyme activity which is based on fluorescently labeled oligoribonucleotide substrates and DNA sequencer technology.