With PQDT Open, you can read the full text of open access dissertations and theses free of charge.
About PQDT Open
Search
8-Oxo-7,8-dihydroguanosine (8-oxoG) is a result of oxidative damage on the nucleobase Guanosine. 8-OxoG has been well characterized in DNA, but little is known of its function in RNA. The purpose of this study was to understand how 8-oxoG influences enzyme reactivity, RNA structure, and RNA function. First, endoribonucleases Ribonuclease A (RNase A) and Ribonuclease T1 (RNase T1) were studied to understand 8-oxoG recognition. It was found that 8-oxoG becomes a substrate for RNase A, while it was not recognized by RNase T1. The functional group on C8 causes an anti to syn flip which exposes new hydrogen bonding patterns similar to that of Uridine, which allowed RNase A reactivity. The conformational change, though, causes 8-oxoG to lose its ability to be a substrate for RNase T1 due to sterics and adverse hydrogen bonding induced by the exocyclic functional group at C8. The information gathered from the endoribonucleases was then applied to study how 8-oxoG begets structural and functional changes to a well-studied aptamer, the theophylline binding aptamer. 8-OxoG insertion caused a 100-fold increase in dissociation constant (Kd) for the theophylline binding aptamers modified at positions G25 and G26, while a modification at G11 prevented the aptamer from binding to theophylline. RNase A and T1 degradation data also yielded different degradation sites in the modified aptamers compared to the canonical, indicating a change in structure. Following, the reactivity of exoribonuclease XRN1 was studied in the presence of oligomers containing one to three 8-oxoG modifications due to XRN1’s role in oxidized mRNA surveillance. XRN1 was found to stall at 8-oxoG sites as a function of number of 8-oxoG present when more than one modification was introduced. The data combined helps provide insights into how 8-oxoG may affect RNA decay and surveillance mechanisms.
Advisor: | Resendiz, Marino J.E. |
Commitee: | Fisk, John D., Ren, Xiaojun, Wang, Haobin |
School: | University of Colorado at Denver |
Department: | Chemistry |
School Location: | United States -- Colorado |
Source: | MAI 58/06M(E), Masters Abstracts International |
Source Type: | DISSERTATION |
Subjects: | Chemistry, Biochemistry |
Keywords: | 8-OxoG, Microscale thermophoresis, RNase A, RNase T1, Theophylline binding aptamer, XRN1 |
Publication Number: | 13865629 |
ISBN: | 978-1-392-27529-0 |