Verification of the Secondary Structure of RNA Diels - Alderases DA22/96

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Title: Verification of the Secondary Structure of RNA Diels - Alderases DA22/96
Author: Ju, Show-Yi Yang
Advisors: Dr. Stefan Franzen, Committee Chair
Dr. Steven A. Lommel, Committee Member
Dr. Stuart E Maxwell, Committee Member
Abstract: The Diels-Alder reaction which generates two carbon-carbon bonds during a [4 + 2] cycloaddition is biochemically and pharmacologically significant. In order to expand and explore the utility and the application of this reaction to the assembly of complex natural products, an in vitro SELEX approach was used to obtain RNA biocatalysts of this transformation. A DA22⁄96 was prepared by the sequence truncation from the parent DA22. An essential requirement for catalytic activity was the incorporation of 5-(4- pyridylmethyl) carboxamide-uridine in place of uridine during the transcription of the DA22⁄96. My research was interested in finding the secondary structure of the DA22⁄96. This particular ribozyme exhibited catalytic activity similar to an enzyme. Mutual information and covariation analysis of 50 different catalytic sequences were used to identify the potential secondary structural elements. The comparisons between the MFold program derived secondary structures and mutual information analysis plots suggests that there were several conserved structural elements in all active DA22⁄96. RNase mapping method was used to validate the predicted secondary structure of DA22⁄96. The mapping data is largely consistent with the predicted secondary structure of DA22⁄96. Diels-Alderase activity assay experiment proved that DA22⁄96 had true enzyme activity. In addition, results showed that the modified DA22⁄96 had the better turn over rate when copper was presented.
Date: 2007-12-15
Degree: MS
Discipline: Chemistry
URI: http://www.lib.ncsu.edu/resolver/1840.16/59


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