Ribozymes
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RIBOZYMES Their Design and Use in Cancer
Philip C. Turner
School of Biological Sciences University of Liverpool Crown Street Liverpool L69 7ZB UK
1. INTRODUCTION Although ribozymes (catalytic RNA molecules) have not yet been used in human patients, their promising use in tissue culture cells, as well as transgenic and other animal models, suggests that their application to treat human disorders, including cancers, is imminent. The purpose of this chapter is to outline the structure of the most common and promising ribozymes and consider how they are designed and delivered before reviewing some of their most recent uses in the field of cancer. More detailed reviews of particular aspects of the various classes of ribozymes will be referred to below.
2. RIBOZYME TYPES The first ribozymes to be discovered were self-cleaving intons (Kruger et al., 1982) and the RNA component of the enzyme RNase P which cleaves tRNA precursor molecules to generate their mature end (Guerrier-Takada et al., 1983). Hammerhead ribozymes were discovered as parts of small pathogenic plant RNAs that undergo a selfcleaving reaction to generate monomeric genomes (Forster and Symons, 1987). Their small size, and the ease with which their reaction could be replicated in vitro (at neutral pH) enabled trans-cleaving hammerhead ribozymes to be rapidly developed (Uhlenbeck, 1987). Another self-cleaving ribozyme discovered in a plant virus pathogen was the somewhat larger hairpin ribozyme (Hampel and Triz, 1989) which also functions in viral replication to generate monomeric genomes. The human subviral pathogen hepatitis delta virus (HDV), which has around 1,700 nucleotides (nt) of single stranded, circular RNA Cancer Gene Therapy: Past Achievements and Future Challenges, edited by Habib Kluwer Academic/Plenum Publishers, New York, 2000.
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Figure 1. The hammerhead ribozyme showing the base pairing interaction with target RNA via helices I and III. The cleavage site is shown by an arrow.
in its genome is also replicated by producing linear genomic multimers that self cleave to monomers (Wu et al., 1989). Both of these ribozyme activities have also been studied and simplified for use in trans-cleaving applications (Branch and Robertson, 1991; Hampel et al., 1990). Other naturally occuring ribozymes are known, such as the Neurospora mitochondrial VS ribozymes (Saville and Collins, 1990), the newt ribozyme
(Marusic et al., 1997) and in vitro selection experiments have created novel ribozymes with unusual enzyme activities (Suga et al., 1998). At the present time these particular ribozymes are either not appropriate or have not yet been developed for use in transcleaving applications such as specific cleavage of target RNA molecules to down regulate gene expression and will not be considered further here.
2.1. Hammerhead Ribozymes Comparison of sequences of the hammerhead catalytic motif from many sources, together with extensive mutagenesis studies have enabled the conserved and important features of the hammerhead ribozyme to
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