Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Professor Robert H. E. Hudson

Abstract

Peptide Nucleic Acid (PNA) has shown great potential in molecular diagnostics, antisense/antigene therapy and nanotechnology. Like other synthetic nucleic acids and artificial analogues, PNA has been extensively modified to achieve better performance in these applications. To efficiently develop PNA probes for molecular diagnosis, this thesis is focused on versatile functionalization of PNA via post-synthetic click chemistry.

Chapter 2 presents the synthesis of quencher-free PNA molecular beacons (MBs) targeting a cystic fibrosis transmembrane conductance regulator (CFTR) sequence mutation. To avoid the tedious synthesis of functionalized PNA monomers for probe development, a simple approach to modify PNA oligomers by post-synthetic on-resin click chemistry was developed. Precursor PNA MBs were prepared by incorporation of azide-containing monomers into the oligomer by automatic solid-phase peptide synthesis and subsequent derivatization with pyrene moieties by copper-catalyzed azide-alkyne cycloaddition (CuAAC) produced the functional MBs. Two pyrene-based quencher-free PNA molecular beacons, a stemless MB and one possessing a stem-loop structure, both targeting a portion of the cystic fibrosis gene, were successfully synthesized by this method. Fluorescence studies showed that the stem-loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct and showed promise as MB for detection of this CFTR mutation.

Chapter 3 reports the synthesis of PNA MBs targeting CFTR mutation via a “click-couple-click” method. Unlike the well-known synthetic procedure of conventional PNA MBs that requires different protection strategies on the sites where fluorophores and quenchers are attached, an on-resin “click-couple-click” approach that includes microwave-assisted on-resin CuAAC of a quencher to PNA, coupling of an azide-containing PNA monomer and another on-resin CuAAC of a fluorophore without need of protecting reactive sites. By using this method, various PNA MBs have been successfully synthesized and characterized.

Chapter 4 discloses the simultaneous, multiple conjugation of alkynyl-(Gd(III)-DOTA) to a PNA oligomer possessing azide residues in a single post-synthetic CuAAC reaction. The resulting probe, (Gd(III)-DOTA)4-PNA, has potential as a MRI contrast agent. Conceptually by binding to the target, poly(rA) tail of mRNA, Gd ions would be significantly loaded to a localized microenvironment, which may improve the enhancement of contrast in MR images. The in vitro MR characteristics of this probe is presented.

With the interest in the conjugation of gold nanoparticles (AuNPs) to PNA for the development of targeted contrast agents for CT imaging, preliminary studies have been carried out by conjugating an oligopeptide to AuNPs. Chapter 5 introduces a new method combining an interfacial strain-promoted azide-alkyne cycloaddition and post assembly deprotection (SPAAC-PAD) for the well-defined functionalization of small, water-soluble gold nanoparticles with oligopeptides. This approach will enable the fabrication of gold nanoparticles with a high degree of complexity with biomolecules for a variety of applications in targeted cancer diagnosis and therapies.

Available for download on Thursday, March 15, 2018


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