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Access Type
WSU Access
Date of Award
January 2023
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Christine S. Chow
Abstract
Modifications in RNA are known for their importance in facilitating cell function and are often found in functionally important regions. There are numerous nucleobase modifications, each having distinct biological impacts on RNA function. The modifications are often dynamically regulated in nature. Despite the high abundance of RNA modifications, many of their properties are poorly understood. Therefore, we employed a series of experimental and theoretical investigations to increase our understanding of modified nucleosides. By analyzing available sequencing data, information about modified nucleotide site preferences were uncovered. An examination of pseudouridine sequencing data by the region in which the modification takes place on the mRNA revealed certain trends in pseudouridylation. Pseudouridylated mRNAs had favored sequences surrounding the site of modification and displayed higher-than-expected enrichment of specific cellular functions. Furthermore, by analysis of pseudouridines in available three-dimensional structures, differences in RNA structural effects were observed that could be correlated with pseudouridylation.Although modified nucleosides are widely distributed in biology, one important physical property, the pKa value, has not been well studied. To determine the effects of modifications, ab initio quantum mechanical calculations in conjunction with an implicit-explicit solvation system were used to determine pKa values. This method yielded close approximations with known experimental values and provided insight into how modifications affect the pKa value at protonatable positions. Acrylamide is a toxin that humans are exposed to daily. Acrylamide has a currently unknown mechanism of action in neuronal damage. By comparing the reaction of acrylamide with unmodified and modified purines, significantly higher adduct formation was observed with inosine compared to guanosine or adenosine. Inosine is importance in neuronal homeostasis; therefore, the presence of this common modification has implications for acrylamide targeting in biological systems. The ribosome contains numerous RNA modifications located in important regions involved in ribosomal assembly and function. One hairpin in particular, helix 69, which is located on the large subunit, has three pseudouridine modifications that promote base flipping required for association with the small subunit. By examining peptides designed to target helix 69 in particular structural conformations, we gained some understanding of the binding preferences of these antimicrobial peptides. Overall, by performing experimental and theoretical studies of modifications gives us a better understanding of how they function in RNA structure and reactivity.
Recommended Citation
Jones, Evan Levi, "Experimental And Theoretical Investigations Of Modified Nucleotides" (2023). Wayne State University Dissertations. 3800.
https://digitalcommons.wayne.edu/oa_dissertations/3800