Identification of base-pair preferences for optimal cleavage : compensatory mutagenesis in positions Cs4 and G11 of the sCYMV hairpin ribozyme
Radek, Katherine Amanda
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The hairpin ribozyme is an RNA enzyme capable of cleaving RNA substrates in a site-specific and phosphodiester cleavage reaction. This property has made the ribozyme useful for gene therapy applications where cleavage of a specific RNA would be optimal in inhibiting the further replication of a specific disease. This would be accomplished through the control of cellular RNA levels on specific genes. Three different hairpin ribozymes have been identified, including the tobacco ringspot virus satellite RNA (sTRSV), the arabis mosaic virus satellite RNA (sArMV), and the chicory yellow mottle virus satellite RNA (sCYMV). Of the 64 bases within the sTRSV ribozyme, six are required for catalytic activity. In addition, there is a base-pair preference in helix 2 between the substrate and the ribozyme (Cs4 and Gu). Few experiments have been performed on the sCYMV hairpin ribozyme, which is why I decided to perform base-pair mutations on this purine/purimidine pair. It has been suggested that in the sTRSV, Gu is the only required base for catalytic activity within any of the helices in the ribozyme. In order to further expand the understanding of the desired sequence for optimal cleavage of RNA substrates, positions Cs5 of the substrate and Gn of the ribozyme were evaluated for random base-pair mutations within the sCYMV hairpin ribozyme. Each of the mutations was analyzed for degrees of cleavage. As the sCYMV cassette was synthesized using Hindlll and BamHl primers, the Cs5 and Gil positions were inserted with random bases. The synthetic oligonucleotides were HPLC purified prior to their use in PCR primer dimer amplification. A restriction digest was conducted to cleave both the vector and PCR product at the desired restriction sites. After a gel extraction, the PCR insert was ligated into the vector and a bacterial transformation was performed. This was then followed by a complete plasmid preparation, after which dilutions were taken for DNA sequencing. After the sequencing had been completed, the base-pair mutations were now able to be determined. Although some mutations were successive among the nine desired compensatory mutations, six base-pair mutations had been achieved. These mutations include: Cs4G:GnC, Cs4A:GuU, Cs4A:Gn, Cs4:GnA, Cs4:GnC, and Cs4:GnU. These combinations were then linearized using Hindlll and transcribed with P-32 CTP. After transcription, each of the mutants was assessed according to the rate of cleavage using nonlinear regression analysis. The results of this experiment will show base-pair preferences for various degrees of cleavage among RNA substrates.