Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Dr. Robert H.E. Hudson

Abstract

The avid hybridization of peptide nucleic acid (PNA) to DNA and RNA along with the molecule’s biological stability has led it to be used in both antisense and antigene capacities. PNA acts against translation via a steric blockade mechanism. It is therefore reasonable to assume that increased heteroduplex stability could lead to increased potency. Two ways of doing this were explored. I) N-Terminal attachment of a platinous chloride chelating moiety to PNA complementary to Xenopus noggin was synthesized with the objective of selective, covalent platination of the target transcript in vivo. Phenotypes consistent with knockdown of the selected gene product were observed; however, it remains unclear whether this is solely due to site-specific platination. II) The use of the modified cytosine nucleobase [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (boPhpC) was previously shown to increase heteroduplex stability, putatively by interaction with guanine at both the Watson-Crick and Hoogsteen faces. PNAs containing this base were synthesized to target mutant huntingtin mRNA - of which the product is the causative agent of Huntington’s disease – and tested in patient derived fibroblasts where selective inhibition of mutant huntintin was observed with concomitant fluorescence imaging.

Modified nucleobases find use in fields ranging from materials science to cytogenetics and has been an area of much endeavour over the past years. Modifications at the 5-position of uracil abound but examples of similarly modified cytosine are lacking. Rapidly developing the inventory of cytosine analogues is a primary goal of ours. We aim to synthesize compounds that may be used as base discriminating fluorophores, created pre- or post-synthetically. Through the reaction of 5-ethynyldeoxycytidine with 1,3-dipoles such as nitrile oxides or azides a series of heterocycle-appended cytosine analogues have been prepared and their fluorescence properties studied. They exhibit moderate to good quantum yields and with high sensitivity to their environment and are considered good candidates for further use as base-discriminating fluorophores. Based on the known pyrrolocytosine scaffold, Indole-3-acetamide substituted deoxypyrrolocytidine (IAMpC) has shown the highest degree of solvatochromism for any pyrrolocytosine analogue known to date. The synthesis of oligodeoxynucleotides containing this base was carried out; however, final deprotection of the base does not proceed smoothly and the modification was found to be slightly destabilizing towards duplex formation.


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