Computational Design and Experimental Validation of Switchable Ribozymes

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Objectives. In the next five years, I propose the following, specific, measurable and realizable objectives. A. Nucleotide-Repeat-Number Regulated Ribozymes (sRz). The computational design and repeated experimental testing of trans-acting hammerhead ribozymes (HHRz) that selectively cleave the transcripts of mutant genes with a greater number of repeats than the wild type- genes such as those causing repeat expansion disorders (REDs). B. Oligo Regulated Ribozymes (Ribosensors). The incremental design and testing of trans-acting hammerhead ribozymes, that efficiently and quickly cleave reporter gene transcripts, and that are switched-off by hybridizing DNA oligos, possibly amplified from distinct segments of pathogenic RNAs/DNAs. Methods. The goal is to design ribozymes that cleave the mutant transcript but leave the WT intact. However, these two transcripts are often identical, except for the (usually) trinucleotide repeat expansion of the mutant. Therefore, a `standard' ribozyme that binds to the mutant and cleaves it will bind to the WT and cleave it as well. This is not acceptable since the WT protein must be translated to avoid loss of function. However, it is possible to design selective ribozymes (sRz) that can differentiate between two transcripts of different lengths. This is achieved by augmenting a ribozyme with an extension region consisting of a sensor segment flanked by two linker segments. We have crafted TriCleaver, a customized evolutionary algorithm that discovers high-performing sRz automatically and efficiently. The same evolutionary algorithm can be used to design trans-acting ribozymes that target any transcript with available cut-sites for hammerhead ribozymes (preferably GUCs), but that are turned-off using a DNA (or indeed) RNA oligo, binding to a specially designed segment of stem II. Outcome. A. TriCleaver Algorithm, once optimized, will be incorporated into the latest version of our Ribosoft server, and will be available for all to generate a variety of repeat-expansion activated hammerhead ribozymes, selectively targeting mutant transcripts, responsible for repeat-expansion disorders (REDs). In the future, this will allow us to further build on our ongoing collaboration with MNIH, to generate in vivo results in cell cultures, showing the efficiency, specificity and generalizability of TriCleaver, which may lead to new genetic therapies for REDs. B. Oligo Biosensors. Once the principle of oligo-regulated hammerhead ribozymes is proven, I should be able to initiate a collaboration (and submit a Mitacs grant) with an interested biotech company (Bioboost Synbio Consulting inc. in Vancouver), for the development of taHHRzs, that regulate the activity of a reporter gene, upon binding of an rtPCR-amplified segment of a pathogenic viral RNA (e.g., hepatetis C), to the taHHRz. We will then be developing pathogenic RNA biosensors. C. Predictive model of cleavage efficiency of one type of hammerhead ribozyme.