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http://hdl.handle.net/11375/29827
Title: | Elucidating Evolutionary Mechanisms and Variants of the Hammerhead Ribozyme Using In Vitro Selection |
Authors: | Brill, Jake |
Advisor: | Li, Yingfu |
Department: | Biochemistry and Biomedical Sciences |
Keywords: | Hammerhead Ribozyme;In Vitro Selection;RNA World;Functional Nucleic Acids;RNA Evolution |
Publication Date: | 2024 |
Abstract: | The RNA World Hypothesis posits that RNA enzymes (ribozymes) catalyzed biochemical reactions in primitive cells prior to the emergence of proteins. However, the evolutionary mechanisms that gave rise to functional RNA sequences on early Earth remains largely unclear. Using a bottom-up approach that combines in vitro selection and high-throughput sequencing, we demonstrate how a self-cleaving RNA enzyme, the Hammerhead Ribozyme (HHR), may have evolved from non-catalytic sequences in the RNA World. Multiple starting libraries were generated by progressively increasing the number of randomized positions in the ribozyme’s catalytic core. The HHR was selected from each of these libraries following several rounds of amplification and enrichment. Deep sequencing analysis was then used to track evolutionary trends that gave rise to the wild-type sequence during each selection. This novel approach revealed a wide range of functional HHR variants. Notably, we discovered active hammerhead variants with mutations to previously identified essential nucleotides, shedding new light on the sequence requirements of the full-length, cis-acting ribozyme. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids. Altogether, the in vitro evolution of ribozymes shows how complex molecules might have emerged from non-catalytic polymers in the RNA world, contributing to our understanding of the origin of life on Earth. |
URI: | http://hdl.handle.net/11375/29827 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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Brill_Jake_S_finalsubmission202405_MSc.pdf | MSc Thesis | 2.36 MB | Adobe PDF | View/Open |
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