Regulation and Activity of Twister Ribozymes Using Classical and High-throughput Approaches
- Author
- McKinley, Lauren
- Published
- [University Park, Pennsylvania] : Pennsylvania State University, 2024.
- Physical Description
- 1 electronic document
- Additional Creators
- Bevilacqua, Philip
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- etda.libraries.psu.edu , Connect to this object online.
- Graduate Program
- Restrictions on Access
- Restricted (PSU Only).
- Summary
- Catalytic RNA perform diverse cellular functions including the regulation of protein synthesis, splicing, and RNA degradation. Small nucleolytic ribozymes are one type of catalytic RNA that cleave their phosphodiester backbone in a site-specific manner without the assistance of proteins. To date, ten classes have been identified including the well characterized twister ribozyme, whose structural and catalytic properties have been elucidated. Advancements in bioinformatics has facilitated the discovery of over ten thousand putative twister ribozymes. However, fewer than one hundred of these candidates have been experimentally validated. Consequently, fundamental questions persist about the biological significance of twister ribozymes. This thesis investigates the regulation, activity, prevalence, and structure-function relationship of twister ribozymes using a combination of traditional and high-throughput experimental approaches. Chapter 2 examines how flanking sequence differentially regulates the self-cleavage activity of twister ribozymes from different genetic contexts in Oryza sativa (rice) both in vitro and in vivo. Chapter 3 explores the ability of twister ribozymes to cleave in trans under single- and multiple-turnover conditions, similar to proteinaceous enzymes. Chapter 4 is comprised of two objectives that employ high-throughput methodologies to (1) discover and (2) characterize putative twister ribozymes. It features the development of two predictive bioinformatic pipelines to identify putative ribozymes as well as an experimental Cleavage High-Throughput Assay (CHiTA) that quantifies ribozyme self-cleavage using massively parallel oligonucleotide synthesis and next-generation sequencing. Chapter 4 also provides a comprehensive analysis of how different structural imperfections influence twister self-cleavage as a means to better understand the structure-function relationship of twister ribozymes. Finally, Chapter 5 outlines potential future directions that employ CHiTA to discover, test, and engineer other types of RNAs.
- Other Subject(s)
- Genre(s)
- Dissertation Note
- Ph.D. Pennsylvania State University 2024.
- Technical Details
- The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
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