Thesis Format

Integrated Article

Synthesis and Evaluation of Nucleobase Modifications for Peptide Nucleic Acid Studies and Quantitative Analysis of Fluorescent Imidazolocytosines as Potential G-Quadruplex Disruptors

Author

Zi Wang, university of western ontarioFollow

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Hudson, Robert H. E.

Abstract

The overall theme of this thesis is the design of modified nucleic acid compounds to cultivate desired properties to further potential biological and medical uses. This work is divided into two topics: the synthesis of modified PNA oligomers, and the spectroscopic and molecular binding ability of modified cytosine compounds.

The iconic double helix nucleic acid structure can become a triple helix structure with the addition of a third strand via Hoogsteen bonding, in either the parallel or antiparallel motif. However, both motifs consisting of naturally occurring nucleobases suffer from deficiencies that limit their versatility or stability. We designed N⁹-inosine (hypoxanthine) nucleic acid monomers in order to build parallel triple helices, as well as N⁷-Inosine and N⁷-isoguanine nucleic acid monomers in order to build antiparallel triple helices. Our goal was to incorporate these synthetic nucleic acids into peptide nucleic acid (PNA) oligomer strands, an artificial nucleic acid structure isomorphous to, and with important advantages over, the natural nucleic acids DNA and RNA. Oligomer synthesis was confirmed via HPLC and mass spectroscopy, though technical issues have so far prevented N⁷-isoguanine PNA monomer formation.

G-quadruplex (G4) is a secondary structure formed in nucleic acid sequences that are guanine rich, and their presence in the human body in levels that are too high or too low can lead to serious medical conditions. Therefore, it is of great interest to design easily accessible small molecules that can unwind or destabilize G4s as conditions require. We have synthesized a series of imidazolocytosine compounds that are designed to destabilize G4s, and quantified their binding strength to guanine via isothermal calorimetry (ITC) and NMR titration. We found that relative to ordinary cytosine-guanine binding, most of our imidazolocytosine analogues have a stronger bond to guanine, with an association constant K_a approximately one order of magnitude greater, for a bond strength that is 6-8 kJ/mol stronger under the conditions measured. Further quantitative studies in physiological conditions are warranted.

The imidazolocytosine analogues synthesized are also fluorescent, a property that is of great interest for the purpose of a variety of biomedical applications. We measured their photophysical properties, including extinction coefficient and quantum yield, and attempted to find a pattern between their structural features and fluorescent properties.

Lastly, in collaboration with Dr. David Monchaud of the University of Burgundy, the G4-unwinding properties of an array of molecules, including pyrrolocytosines and imidazolocytosines, were accessed using a variety of techniques in search of an appropriate G4-unwinder.

Summary for Lay Audience

Nucleic acids are ubiquitous in life, and their rich chemical properties allow for huge possibilities for chemical modifications in order to suit diagnostic or medicinal uses. Modifications to nucleic acids can enhance their desired properties and suppress unwanted features, and incorporating these modified nucleic acids into oligomers has the potential for customizing medical treatments, among other possibilities.

Peptide nucleic acid (PNA) is a modified nucleic acid with enhanced properties, and our goal is to design durable and robust PNA oligomers in order to bind to double helices, creating robust triple helix structures. We synthesized a variety of modified nucleic acid monomers such as N⁷-inosine and N⁹-inosine PNA monomers, and successfully incorporated them into oligomers, while also synthesizing N⁷-­isoguanine monomers.

We designed and synthesized imidazolocytosines by modifying cytosine for two purposes: for their fluorescent properties, as well as to study their binding interactions with guanines in order to identify candidates for G4 disruptors, a less studied but underrated and highly important medical research topic, as unchecked g-quadruplex formation in cells can lead to serious health conditions. We catalogued their fluorescent properties, and obtained potential fluorescent markers in different colours, a highly useful trait. We also quantitatively measured their binding strength to guanine via both NMR titration and isothermal calorimetry, and found that under the given conditions imidazolocytosines have a binding strength to guanosine 6-8kJ/mol stronger than that of unmodified cytosines, making them potential candidates as G4 disruptors, while our collaborator Dr. Monchaud and his team analyzes G4 disruption in vitro.

Recommended Citation

Wang, Zi, "Synthesis and Evaluation of Nucleobase Modifications for Peptide Nucleic Acid Studies and Quantitative Analysis of Fluorescent Imidazolocytosines as Potential G-Quadruplex Disruptors" (2024). Electronic Thesis and Dissertation Repository. 10076.
https://ir.lib.uwo.ca/etd/10076