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Please use this identifier to cite or link to this item: http://hdl.handle.net/10155/316

Issue Date:  4
Title: The synthesis and in vitro evaluation of siRNAs modified at the backbone with a novel triazole-based internucleotide linkage and abasic alkyl-chain linkages of varying length
Authors: Efthymiou, Tim
Publisher : University of Ontario Institute of Technology
Degree : Doctor of Philosophy (PhD)
Department : Applied Bioscience
Supervisor : Desaulniers, Jean-Paul
Keywords: Nucleic acids
SiRNA
Click chemistry
Triazole
Backbone
Abstract: For over a decade, the use of double-stranded short interfering RNAs (siRNAs) to silence the expression of genes associated with disease at the translational level has gained much attention. Using a highly conserved endogenous pathway within cells, siRNA technology displays a high degree of target specificity and potency. However, traits required for the successful design of siRNA-based therapeutics such as resistance to nuclease-mediated degradation, improved cell membrane permeability and reduced off-target toxicity, are compromised by the native structure of duplex RNA′s charged backbone. This study therefore focused on synthesizing novel and neutrally-charged triazole-linked nucleoside dimer analogs which were incorporated throughout siRNA duplexes using DMTphosphoramidite chemistry, in order to attenuate the negative contributions of RNA′s native backbone. In order to further elucidate the mechanism of the RNA interference (RNAi) pathway, additional siRNAs were synthesized containing commercially available abasic alkyl spacers. Through the robust copper(I)-catalyzed Huisgen [3+2] cycloaddition, azide and alkyne nucleic acid monomers were joined through the heterocyclic linkage in nearly quantitative yields, producing the novel triazole-linked uracil-uracil (UtU) and cytosine-uracil (CtU) dimers. Results from cell-based assays indicate that triazole-modified siRNAs are capable of silencing the transiently-expressed reporter gene firefly luciferase and the endogenous gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a dose-dependent manner. In addition, modifying the 3′- overhangs of siRNAs with the triazole backbone linkage, gave rise to increased nuclease resistance well beyond that of wild-type siRNA. Duplex RNAs containing abasic spacer linkages in the sense strand also maintained activity while targeting the luciferase reporter gene, indicating the capability to tune the efficacy of siRNA constructs by altering their thermodynamic profiles, in addition to providing evidence for an alternative RNAi mechanism. The studies describe herein, emphasize the compatibility of these novel backbone modifications with Watson-Crick interactions and with the RNA pathway.
Appears in Collections:Electronic Theses and Dissertations (Public)
Faculty of Science - Doctoral Dissertations

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