Date of Award
2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Pharmaceutical Sciences
Department
Biomedical and Pharmaceutical Sciences
First Advisor
Deyu Li
Abstract
Chapter One will detail the synthesis, purification, and characterization of DNA oligonucleotides containing one of fifteen modified nucleic acid bases. For each modification, relevant background information is provided to highlight its biological significance. This chapter primarily focuses on the development of oligonucleotide stocks for use in future biochemical and cellular assays. Several of these modifications are recognized substrates of AlkB-family enzymes, while others remain uncharacterized as potential substrates.1-4 The discovery of novel substrates for AlkB-family enzymes can be facilitated through comprehensive in vitro biochemical assays, contingent upon the availability of well-characterized substrate stocks. The methods and findings presented here establish a foundation for future experiments aimed at identifying new AlkB-family enzyme substrates and elucidating their substrate promiscuity.5,6
Chapter Two explores the influence of pH conditions on AlkB-family enzyme-mediated oxidation of methylated DNA oligonucleotides. The experiments described in this chapter provide evidence that both substrate- and enzyme-related variables are significantly affected by pH.7,8 These findings underscore the importance of considering pH conditions when interpreting results from biochemical assays. Moreover, the data demonstrate that AlkB-family enzymes exhibit altered substrate preferences as pH conditions vary, indicating increased substrate promiscuity under certain conditions.
Since the discovery of the AlkB-family enzymes' catalytic function in 2002, several nucleic acid substrates have been identified and studied.9,10 Previous studies have shown that these enzymes preferentially oxidize protonated substrates over their neutral counterparts, although the full scope of this preference has not been thoroughly investigated.1 Preliminary data from our work suggest that mildly acidic pH conditions enhance the activity of AlkB-family homologs ABH2 and ABH3, potentially due to the pKa of the substrates.11-13 The stability of AlkB-family enzymes under extreme pH conditions is also a contributing factor, as some homologs appear more tolerant to pH extremes than others.
Chapter Three discusses the inhibition of AlkB-family enzymes and describes a series of in vitro biochemical experiments designed to screen various small-molecule compounds. The compounds included several anthraquinone-like molecules derived from a marine actinomycete species. They were specifically screened for their ability to inhibit ABH2-mediated oxidation of DNA oligonucleotides containing 3-methylcytosine (m3C). Rhein, a known anthraquinone-based inhibitor of AlkB-family enzymes, was used as a positive control due to its structural similarity to several of the tested actinomycete-derived compounds. Given that overexpression of AlkB-family enzymes has been implicated in multiple diseases, developing homolog-specific inhibitors could provide key insights into disease mechanisms.9,14
While a few of the evaluated compounds demonstrated inhibitory activity against ABH2-mediated oxidation of m3C, none exhibited stronger inhibition than Rhein. Although these compounds did not outperform Rhein, it is possible that they could inhibit other AlkB-family homologs, which warrants further investigation. Identification of selective inhibitors of AlkB-family enzymes remains a critical research goal, as it could enhance our understanding of the specific roles played by different homologs in cellular processes. Additionally, selective AlkB inhibitors could hold potential as a new class of therapeutic agents.
The experimental work required a wide range of specialized equipment. Oligonucleotide synthesis relied on a dedicated and reliable automated synthesis platform. Throughout the synthesis process, lyophilization was necessary for sample preparation. High-performance liquid chromatography (HPLC) was used for the purification and analysis of newly synthesized DNA oligonucleotides. Additionally, access to high-resolution liquid chromatography mass spectrometry (LC-MS) was critical for confirming the identity of the purified oligonucleotides, as HPLC alone was insufficient. The operation of these instruments required specific reagents and consumables for each platform.
Recommended Citation
Howarth, Samuel Dean, "AN INVESTIGATION OF ALKB-FAMILY DNA REPAIR ENZYMES AND THEIR INTERACTION WITH SYNTHETIC, MODIFIED OLIGONUCLEOTIDES" (2024). Open Access Dissertations. Paper 4427.
https://digitalcommons.uri.edu/oa_diss/4427
