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dc.contributor.advisorDesaulniers, Jean-Paul
dc.contributor.authorHammill, Matthew
dc.date.accessioned2021-10-01T15:18:27Z
dc.date.accessioned2022-03-29T19:06:42Z
dc.date.available2021-10-01T15:18:27Z
dc.date.available2022-03-29T19:06:42Z
dc.date.issued2021-08-01
dc.identifier.urihttps://hdl.handle.net/10155/1346
dc.description.abstractShort interfering RNAs (siRNAs) are biopolymers that are used for post-transcriptional gene regulation and act as endogenous defenses against attack from viruses and dsRNA parasites. The siRNA field is making advances for use as pharmaceuticals and biomolecular research tools for the study of gene silencing in uncontrollably upregulated genes. Through chemical modification of the RNA structure, inherent limitations found in nature can be eliminated to make better pharmaceuticals and research tools. This dissertation investigated the incorporation of azobenzene derivative spacers into RNA backbones by replacing two base pairs. Several siRNAs were synthesized, and then characterized bio-physically through several means. After bio-physical characterization, these siRNAs were tested in vitro against firefly luciferase and endogenous BCL-2 to determine gene silencing efficacy, as well as nuclease resistance when azobenzenes were at the 3’ end of the passenger strand. The ability of these azobenzene siRNAs to be reversibly controlled with UV (ultraviolet) light after transfection into the cells was investigated. Next, utilization of tetra-halogenation at the ortho position on the azobenzene to red-shift the π→π* of the N=N double bond out of the UV portion of the electromagnetic spectrum into the visible region, allowing the siRNA to be inactivated with red light, which is less toxic and through constant exposure inactivates the siRNA for up to 24 h. Finally, we developed an ortho tetra-fluorinated azobenzene derivative to further improve the design of these siRNAs. This tetra-fluorinated derivative maintains the desirable red-shifting property of azobenzene, while having a greatly improved cis conformer half-life over the tetra-chlorinated azobenzene derivative. Furthermore, the tetra-fluorinated azobenzene containing siRNAs were able to remain inactive for up to 72 h with minimal green light exposure. These results in cell-based assays have shown that azobenzenes embedded within siRNAs are well tolerated in the RNA induced silencing complex (RISC). This introduces new functionality into the pathway through photo-switchable azobenzenes which can be controlled with light in real time. We further show that the properties of the siRNAs can be fine tuned through the use of different azobenzene derivatives, and can also show differences in nuclease resistance between the trans and cis isomers.en
dc.description.sponsorshipUniversity of Ontario Institute of Technologyen
dc.language.isoenen
dc.subjectOrganic chemistryen
dc.subjectAzobenzeneen
dc.subjectPhoto-switchen
dc.subjectChemical biologyen
dc.subjectsiRNAen
dc.titleThe synthesis and in vivo evaluation of chemically modified siRNAs that contain red-shifted internal azobenzene derivative spacers for selective and tunable photocontrol of activity without UV lighten
dc.typeDissertationen
dc.degree.levelDoctor of Philosophy (PhD)en
dc.degree.disciplineApplied Bioscienceen


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