Thesis Format
Integrated Article
Degree
Master of Science
Program
Chemistry
Supervisor
Hudson, Robert H. E.
Abstract
Peptide Nucleic Acid (PNA), is a mimic of natural nucleic acids with exceptional binding properties. As such, numerous applications in biochemistry, medicine and biotechnology for PNA exist or have been proposed so far. Pseudo-complementary PNAs containing diaminopurine and thiouracil base pairs have been prepared and are shown to bind with high specificity and efficiency to complementary targets in double-stranded DNA by a mechanism termed ‘‘double duplex invasion’’ in which the duplex is unwound and both DNA strands are targeted simultaneously, each by one of the two pseudo-complementary PNAs. Chapter 2 describes a method for the preparation of modified nucleic acids and PNA oligomerization which can be used to study different pseudo-complementarity and base pairing and open up the possibility for the preparation of PNA oligomers with interesting new properties. Within the realm of PNA synthesis, we have examined different benzyl-type protections for the thiouracil nucleobase in PNA synthesis and oligomerization. We have also demonstrated for the first time that PNA oligomer synthesis can proceed without thiouracil protecting group on sulfur. Finally, the thermal stability of PNA bindings and pseudo-complementary PNA were studied.
As a part of the study of pseudo-complementary nucleobases, an optimized method of assaying DNA strand invasion by PNA was investigated. Traditional methods require time-consuming gel assays and lack an easily quantifiable marker. Instead, our method incorporates the fluorescent cytidine base analogue phenylpyrrolocytidine (PhpC) into a DNA strand such that upon invasion by PNA fluorescence increases when excited. We also synthesized pseudo-complementary PNA sequences that can undergo DNA invasion; no gels are required as all measurements are performed in solution. Nucleic acid detection takes place by changing either the reporter’s fluorescence intensity or the colour of its fluorescence. The use of fluorescent probes for nucleic acid detection has attracted much attention due to its efficiency, the ease of synthesis and availability of commercial reporters that facilitate the probe synthesis.
Chapter 3 describes the synthesis of a novel quinazolinone-based uracil PNA monomers. Their photophysical properties were evaluated in different solvents and at varying temperatures. These luminescent PNA monomers show solvatochromism and solvent viscosity-dependent emission which indicate that they have the potential to act fluorescent reporters of hybridization events once incorporated into PNA oligomers which will be done as future work for this study.
Summary for Lay Audience
Peptide Nucleic Acid (PNA), is a mimic of natural nucleic acids, also known as the building blocks of DNA, with exceptional binding properties. As such, numerous applications in biochemistry, medicine and biotechnology for PNA exist or have been proposed so far. Pseudo-complementary PNAs containing diaminopurine and thiouracil base pairs have been prepared and are shown to bind with high specificity and efficiency to complementary targets in double-stranded DNA by a mechanism termed ‘‘double duplex invasion’’ in which the duplex is unwound and both DNA strands are targeted simultaneously, each by one of the two pseudo-complementary PNAs. The present work describes a method for the preparation of modified nucleic acids, and PNA oligomerization which can be used to study different pseudo-complementarity and base pairing and open up the possibility for the preparation of PNA oligomers with interesting new properties. Oligomerization is the process of making oligomers which are molecular complexes of chemicals that consist of repeating units. Within the realm of PNA synthesis, we have examined different acid-labile protections for the thiouracil nucleobase in PNA synthesis and oligomerization. We have also demonstrated for the first time that PNA oligomer synthesis can proceed without thiouracil protecting group on sulfur. Finally, the thermal stability of PNA bindings and pseudo-complementary PNA were studied. As a part of the study of pseudo-complementary nucleobases, an optimized method of assaying, or making, DNA strand invasion by PNA was investigated. Traditional methods require time consuming gel assays and lack an easily quantifiable marker. Instead, our method incorporates the fluorescent cytidine base analogue phenylpyrrolocytidine (PhpC) into a DNA strand such that upon invasion by PNA fluorescence increases when excited. We also synthesized pseudo-complementary PNA sequences that can undergo DNA invasion; no gels are required for this process, as all measurements are performed in solution.
Nucleic acid detection takes place by changing either the reporter’s fluorescence intensity or the colour of its fluorescence. The use of fluorescent probes for nucleic acid detection has attracted much attention due to its efficiency, the ease of synthesis and availability of commercial reporters that facilitate the probe synthesis. Chapter 3 describes the synthesis of a novel quinazoline-based uracil PNA monomers. The study of photophysical properties in different solvents and temperatures show that they can be good fluorescent reporters upon hybridization with the complementary DNA sequence by incorporation of these PNA monomers into PNA oligomers as the future work for this study.
Recommended Citation
Heidari, Ali, "Synthesis and Characterization of Peptide Nucleic Acids Incorporating Modified Nucleobases" (2019). Electronic Thesis and Dissertation Repository. 6415.
https://ir.lib.uwo.ca/etd/6415
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