Abstract

Ribozymes are RNA molecules with catalytic functions. These molecules can catalyze a self-cleaving reaction or bind to a target RNA molecule and cleave it. These properties make ribozymes perfect candidates for RNA therapeutics. Hammerhead ribozymes are a well-studied family of ribozymes and can be used to cleave specific mRNA molecules. These synthetically generated ribozymes can then regulate gene expression within cells. Normal PABPN1 protein has 10 alanine (GCG codon) repeats in its structure. Oculopharyngeal Muscular Dystrophy (OPMD), a hereditary disease with no cure, is caused by mutations that result in an increase in the number of GCG repeats in PABPN1 gene. The mutant proteins translated from genes that contain these repeat expansions are believed to be the disease-causing agents, hence targeting the expression of these proteins on the mRNA level is an attractive strategy using RNA therapeutics in OPMD treatment. To generate these RNA molecules, an evolutionary algorithm (Tri-Cleaver) was used to design ribozymes to be tested against two mutants of PABPN1 transcripts. Twenty-nine ribozymes that were designed using this algorithm were tested in human cells (HEK293) to investigate their effect on PABPN1 mutant transcripts with 13 and 17 alanine codon (GCG) repeats. These ribozymes were tested for 1) their efficiency to bind and cleave PABPN1 mRNA with 13 and 17 GCG repeats and 2) their selectivity for the mutant transcripts of PABPN1 gene. The results not only show the successful use of an evolutionary algorithm in designing trans-cleaving ribozymes that can selectively target repeat expansions, but also provide useful data to improve the algorithm’s later designs.