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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
RNA modifications play a crucial role in diversifying the forms and functions of RNA within the cell. Natural modifications are widespread across all types of RNA molecules and vary in their complexity. Some of these modifications tune the RNA structure, while others participate in signaling pathways. An integral component of the ribosome is helix 69 (H69), which contains modifications that are highly conserved throughout all the domains of life. Synthetic modifications of nucleobases have been instrumental in investigation of the structure and function of both RNA and DNA, along with potential medicinal applications. Many non-naturally occurring modifications are used as structural probes and fluorophores to study nucleic acids while others are used for medicinal purposes. Despite the wealth of knowledge about RNA modifications, certain aspects regarding their physical properties are poorly understood. One of these important physical properties is the acid constant, or the pKa value. The pKa value for unmodified nucleobases is well understood, but modifications can impact the physical properties of RNA nucleotides, specifically the pKa values, resulting in downstream structural impacts. Additionally, the relationship between modification and pH level could impact the overall RNA structure, influencing the binding affinity of drugs to RNA targets.
Computational methods were employed to determine the impacts that modification have on the pKa values of nucleobases. A total of 87 nucleobases was optimized using ab initio quantum mechanical calculations with a hybrid implicit-explicit solvation system. The determined theoretical pKa values were compared to known experimental ones that were found in literature. The computational methodology employed in this work was found to provide accurate pKa values for the modified nucleobases. The determined pKa values of these modified nucleobases could provide insight into RNA structural behavior along with a greater understanding on how modifications impact the protonatable and deprotonable positions on the nucleobase.
Circular dichroism (CD) spectroscopy was utilized to investigate the structural significance of the modifications present in H69 from two different species, as well as the effects of varying solution pH environments and the presence of paromomycin. The modification landscape had a bigger impact on the CD spectra than did the pH environment or the presence of paromomycin. Complementary biophysical assays were used to provide support of the paromomycin binding to H69 These findings shed light on how modifications in H69 can influence its structural characteristics, providing insights into the functional implications of these modifications and their potential impact on ligand-binding interactions.
Recommended Citation
Mlotkowski, Alan Jacek, "The Impacts Of Modification On Acid Dissociation Constants Of Nucleobases And Structure Of Helix 69 Rna" (2023). Wayne State University Dissertations. 3930.
https://digitalcommons.wayne.edu/oa_dissertations/3930