The Development of Hybrid Antigene Therapeutics Using Nucleic Acid Click Chemistry
Author | : Teresa Lauria |
Publisher | : |
Total Pages | : 0 |
Release | : 2020 |
Genre | : |
ISBN | : |
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Given their ability to break single- and double-strand DNA with high selectivity, the design of restriction endonuclease mimics has become an area of considerable research interest. One class of mimetics are copper artificial metallo-nucleases (AMNs) that prompt direct strand scission by oxidatively damaging duplex DNA. Therefore, the formation of metal-catalysed free radicals in the vicinity of nucleic acids provides a viable route to developing artificial gene editing tools. The aim of this research was to develop new copper-based AMNs for selective knockdown of the green fluorescent protein (GFP) gene. To achieve this, a novel pool of gene-targeted biomaterials was developed by hybridising intercalating azide-modified phenanthrene AMNs to triplex formation oligonucleotides (TFOs) using copper-catalysed alkyne-azide cycloaddition (CuAAC) 'click' chemistry. Upon isolating the family of AMN-TFO hybrids, the project focused on their triplex formation and stabilisation properties. In the presence of coordinated copper(II) ions and a reductant, AMN-TFOs selectively cleaved the GFP gene fragment and site-specific fragmentation patterns were identified using fluorophore-tagged sequences. Building on this first generation of hybrid AMNs, second generation hybrids were developed by clicking di-copper binding bis-phenanthroline ligands to alkyneTFOs. These di-copper(II) AMN-TFOs were prepared using both CuAAC and strainpromoted azide-alkyne cycloaddition (SPAAC) reactions and cleavage reactions with a GFP gene fragment were compared to first generation mononuclear AMN-TFO hybrids. The final aspect of this work focused on extending this technology to develop a novel class of luminescent probes. To achieve this, polypyridyl ruthenium(II) complexes bearing an azide-phenanthroline handle were prepared and conjugated to alkyne TFOs and their preliminary GFP-targeting and luminescence properties were identified.