Synthesis and Characterization of Peptide Nucleic Acids Incorporating Modified Nucleobases
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.