Optimized Transcription of a Stem I Substitution in the Hammerhead Ribozyme and the Potential for Gene Therapy
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RNA is widely known to be an integral part of transcription and gene regulation. The RNA world hypothesis states that RNA is the precursor to life and biological processes as we know them today, but in order for this to be the case, RNA must be able to self-replicate. In 1987, Cech found RNA that is capable of catalysis and, therefore, self-replication. This kind of RNA came to be known as the ribozyme. A model system of ribozymes is the hammerhead ribozyme, due to its small size and its structure, three stems culminating in a catalytic core junction. Stems I and III are thought to have roles in conformational change for catalysis whereas stem II is known to help with catalytic core stability. This structure allows the hammerhead ribozyme to be capable of a diverse array of conformational changes and dynamic shifts. The present study was to continue the research of Olke Uhlenbeck and Brendan Nagler by substituting the stem I of the HH8 hammerhead ribozyme with the anticodon stem from an E. coli tRNA alanine and successfully finding large scale transcription conditions. Once large scale transcriptions were complete, rate of cleavage was to be analyzed. While it was again difficult to obtain the cleavage assays, optimal conditions were found for the transcription of the HH8 wild type and the HH8 stem I substituted mutant and large scale batches of RNA were produced for future use in cleavage assays. UV-Visible spectrometry was used to determine the concentrations of RNA made. The concentration of the synthesized short strand of HH8 wild type was found to be 0.176 M and the long strand was found to be 0.074 M. The concentration of the synthesized stem I substituted mutant was found to be 0.145 M for the short strand and 0.104 M for the long strand.