Electronic Thesis and Dissertation Repository

Thesis Format



Doctor of Philosophy




Hudson, Robert H.E.


Nucleic acids have been extensively studied not only for their importance in biological systems as the medium of genetic information but also for their potential uses in therapeutic, diagnostic and other biological applications. As such, modified oligonucleotides and oligonucleotide analogues have drawn the attention of researchers from various disciplines. Modification of oligonucleotides can enhance their desired characteristics and engender unique properties, such as fluorescence, giving rise to a variety of applications. Peptide nucleic acid (PNA) is an oligonucleotide mimic with a pseudo-peptide backbone based on N-(2-aminoethyl)glycine that is renowned for high target binding affinity and resistance to enzyme degradation. These properties of PNA are useful in their application as sequence-selective probes and other bioanalytical applications.

Thus, the overall theme of this thesis was the synthesis and evaluation of nucleobase-modified PNA and DNA monomers and the investigation of their effects on the physicochemical and photophysical characteristics of oligonucleotides and analogues.

The nucleobase modification 5-nitrouracil has only been investigated in-silico and these studies indicate that the 5-nitrouracil-adenine base pair is more energetically favourable than the uracil-adenine base-pair. We have constructed an experimental system to investigate the base-pairing ability of 5-nitrouracil in the context of a PNA oligomer. PNA oligomers possessing 5-nitrouracil substitution for uracil were studied in both duplex and triplex binding modes. 5-nitrouracil destabilized in the duplex forming sequence. However, in the triplex study, 5-nitrouracil formed stronger hydrogen bonds with adenine when it was incorporated into the Hoogsteen strand of a bis-PNA triplex. The discrimination of matched binding versus mismatched binding was also investigated. This study showed that 5-nitroU maintained the specificity for the matching complementary base pair.

We have also investigated the synthesis of modified nucleobases which possess dark fluorophore properties so that they act as a quencher when paired with an appropriate fluorophore. Two fluorophore systems, that change conformation and switch between quenched and fluorescent states are known as molecular beacons. This work contributes to the design of a new model for a molecular beacon in which a base-pairing competent fluorescent nucleobase and a nucleobase quencher are incorporated in the stem region of a stem-loop sequence. An improved synthesis of the 5-(4-(N,N-dimethylamino)phenylazo-yl)uracil (DMPAU) PNA analog was achieved. Subsequently, its ability to hydrogen bond to adenine was determined by NMR methods, and it was found to associate with adenine as strongly as thymine, with a Ka ~ 120 in chloroform. The ability of DMPAU to quench the fluorescence of the intrinsically fluorescent 6-phenylpyrrolocytosine (PhpC) and a selection of other common fluorophores was examined. These results allow us to predict that the DMPAU-PhpC make a suitable fluorophore-quencher pair for molecular beacon development.

Finally, an extension of the DMPAU base modification was developed for the 2′-deoxynucleoside, which resulted in 5-(4-(N,N-dimethylamino)phenylazo-yl)-2′-deoxyuridine (DMPAdU). A new synthetic route for starting with 2′-deoxyuridine was developed to avoid the need for a stereochemically-controlled glycoside bond formation which has been problematic in past syntheses from our lab. During the synthesis, the method for selective acylation of 2′-deoxyuridine was studied. The optimized condition for the diazotization of 5-amino-2′-deoxyuridine without glycosidic bond breakage was described. With the photophysical characterization of DMPAdU, the quenching ability was tested against 6-phenylpyrrolo-2′-deoxycytidine (PhpdC) fluorophore.

Summary for Lay Audience

Nucleic acids have been a useful tool in biological applications; thus, many modifications have been made to nucleic acids to alter and give additional characteristics. Among the modified DNA, peptide nucleic acid (PNA) has been heavily investigated due to its excellent binding target DNA and ability to find the exact target. Such characteristics allow for PNAs to be used in therapeutic and diagnostic applications. Also, researchers have studied different modifications on a nucleobase and their backbone and their effects in adding or eliminating characteristics. This thesis focuses on the development of novel design and synthesis of PNA/DNA building blocks with structural nucleobase modifications that add novel properties. Photophysical characteristics of modified nucleobases and the binding ability of nucleic acids were investigated.

According to previous studies, the introduction of a nitro (-NO2) group to uracil should result in stronger pairing with its base pair partner, adenine, thereby improving binding; thus, we hypothesized 5-nitrouracil containing PNA to have better binding when it replaced natural nucleobase. Correspondingly, in Chapter 2, 5-nitrouracil containing PNA monomer was made, and then the PNA chains to test their improved binding.

Molecular beacon is a useful biological tool that can detect certain sequences of nucleic acids. To create a molecular beacon, we need a light source (fluorophore) and a switch (quencher). In Chapter 3, two modified nucleobases DMPAU and nitrophenylpC nucleobases could act as the switch. After making these nucleobase switch, their ability to turn ON/OFF and to pair with their nucleobase partners were tested