Small interfering RNAs (siRNAs) and antisense oligonucleotides (AONs) use natural cellular machinery to enact sequence-specific mRNA inhibition. The development of synthetic oligonucleotides (ONs) as potent and specific agents for silencing the expression of target genes has provided incredibly useful tools for molecular biology, functional genomics, and biochemistry research. Gene silencing nucleic acids are under active investigation in the clinic for the treatment of a wide variety of diseases. While promising, ON therapeutics face several challenges. siRNAs and AONs are rapidly degraded in the body, may cause undesired side effects, and the delivery of active ONs to target cells remains a significant challenge. Chemical modification of the natural nucleic acid structure can address several of these shortcomings. This thesis describes a series of studies directed towards the development of fluorinated nucleic acid analogues as gene silencing agents with improved biological and chemical properties. A screen of chemically modified siRNA constructs revealed that 2′F-ANA, a fluorinated DNA analogue, can be combined with 2′F-RNA or LNA, rigid RNA analogues, to produce potent gene silencing agents devoid of natural RNA. Several rules were developed to optimize the potency of these chemically modified siRNAs, including a method for creating duplex binding asymmetry within 21mer duplexes through alternating 2′F-ANA and 2′F-RNA inserts along the siRNA passenger strand. These active siRNAs have enhanced nuclease stability, and reduced immunostimulatory properties.Thermal stability studies revealed that 1-1 alternations of 2′F-ANA and 2′F-RNA are destabilizing in siRNA duplexes, whereas 3-3 alternations are neutral. siRNAs incorporating 3-3 and 1-1 2′F-ANA/2′F-RNA motifs produce potent gene silencing. Experiments suggest that the gene silencing cellular machinery, the RISC complex, readily binds the 5′ terminus of siRNA guide strands modified with 2′F-ANA and 2′F-RNA. Gene silencing siRNAs chemically modified with 2′F-ANA, 2′F-RNA, and LNA were applied towards silencing the expression of several endogenous and therapeutically relevant genes, demonstrating the versatility and therapeutic potential of these constructs. Genes linked to antiviral (4E-BP1 and 4E-BP2) and anticancer (Bcl-2 and DRR) applications were readily silenced with chemically modified siRNAs and AONs in a variety of disease models.Phosphorothioated (PS) 2′F-ANA oligonucleotides were investigated as AONs targeting the cancer-linked Bcl-2 and AR genes. Results demonstrate that PS-2′F-ANA oligonucleotides are fully compatible with gymnotic cellular delivery, a carrier-free method for achieving cellular uptake in tissue culture. In a second delivery approach, chemically modified siRNAs and AONs were conjugated with maleimide, alkyne, and cyclooctyne functionalities to allow straightforward and robust coupling to the exterior of an antibody-targeted nanoparticle delivery system (Her-NP). This novel delivery system features an enzyme-mediated ON release approach, and an antibody-targeted cellular uptake mechanism allowing specific cell targeting. Results demonstrate that siRNAs and AONs delivered on Her-NPs enact gene silencing as efficiently as siRNAs delivered with Lipofectamine, but without the associated cellular toxicity. The first synthesis of novel difluorinated nucleoside analogues 2′,4′-difluorouridine (DiF-U) and 2′,4′-difluorocytidine (DiF-C) is described. In-depth NMR-based structural characterization reveals that these nucleoside analogues adopt rigid North sugar conformations. DiF-U was incorporated in a variety of oligonucleotide duplexes, demonstrating several interesting effects on duplex stability. DiF-U is highly destabilizing in the DNA strand of DNA:RNA and DNA:DNA duplexes, mildly destabilizing in the RNA strand of a DNA:RNA duplex, and neutral in the RNA strand of an RNA:RNA duplex. / Les petits ARN interférents (siARN) et les oligonucléotides antisens (OA) se servent de mécanismes cellulaires naturels pour inhiber l'expression de l'ARN messager (ARNm) de manière spécifique à la séquence. Le développement des oligonucléotides synthétiques (ON) comme agents efficaces et spécifiques de l'inhibition de l'expression de gènes cibles a permis de fournir des outils d'importance majeure dans les domaines de la biologie moléculaire, de la génomique fonctionnelle et pour la recherche en biochimie. Les acides nucléiques inhibiteurs de l'expression de gènes font l'objet de nombreux tests cliniques en vue d'être utilisés pour le traitement d'un grand nombre de maladies. Cependant, ces résultats encourageants doivent être contrebalancés par plusieurs difficultés auxquelles les thérapies à ON doivent faire face. siARN et OA sont rapidement métabolisés dans le corps, ils peuvent déclencher des effets secondaires indésirables et le transport d'ON actifs dans les cellules cibles demeure un important problème. Modifier chimiquement la structure naturelle d'un acide nucléique peut permettre de résoudre certaines de ces difficultés. Cette thèse décrit la réalisation d'un ensemble d'études visant à développer des analogues fluorés d'acides nucléiques aux propriétés chimiques et biologiques améliorées et à les employer comme agents inhibiteurs de l'expression de gènes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.121363 |
| Date | January 2014 |
| Creators | Deleavey, Glen |
| Contributors | Masad J Damha (Supervisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | Electronically-submitted theses |
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