Identifier

etd-11142007-115425

Degree

Doctor of Philosophy (PhD)

Department

Engineering Science (Interdepartmental Program)

Document Type

Dissertation

Abstract

RNA interference (RNAi) is a phenomenon in which RNA molecules elicit potent and sequence-specific post-transcriptional gene silencing. Nucleic acid chemical modifications have been incorporated to improve their pharmacological properties. Despite numerous developments in chemical modifications for increased stability, safety, and efficiency of these therapeutic agents, they still face challenges of spatial and temporal targeting. One potential targeting strategy uses photocaging techniques, which involves the covalent attachment of photolabile compounds to the effector NA species that blocks bioactivity until exposed to near UV-light. This work demonstrates that fully-2’-fluorinated nucleic acids (FNAs) can elicit RNAi and that these effectors are resistant to sugar-specific enzymatic digestion. 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) was used to cage NA oligonucleotides, including FNAs for controlling RNAi. Photo-control over RNAi was demonstrated in cell culture and developing zebrafish embryos. Caging also afforded additional protection against nuclease digestion. High doses of siFNAs were toxic to fish embryos, as characterized by a developmental delay and increased mortality rate. Caging siFNAs rendered the effectors inert and eliminated the observed toxicity in the developing embryos. Building on the success of caging chemically-modified siFNAs through random phosphate alkylation, a strategy for the site-specific incorporation of an amine-based cage post-synthetically was developed. To demonstrate the feasibility of directed post-synthetic base caging, a base-caged ATP was generated and tested for photo-activation capacity. A novel cage group, 4,5-dimethoxy-2-nitrobenzylamine, was synthesized and used for the conversion of chloropurine riboside triphosphate and the corresponding free nucleoside. Base-caged ATPs were inactive until photoexposure to 365nm light. Additionally, these compounds did not demonstrate competitive inhibition of protein-ATP interactions, as previously described for γ-phosphate caged ATP. For the site-specific incorporation of caged nucleobase derivatives, a strategy relying on amine-reactive convertible nucleotides was developed for positional substitution using 2-nitrobenzyl amine cage compounds. These convertible nucleotides can be incorporated during custom solid-phase synthesis, and may provide a reactive target for the presented amine-based cage compounds. Caging RNAi effectors will allow for spatial and temporal targeting of a controlled dose release of gene silencing agents, which has potential applications for wet-bench assays, probing developing biological systems, and potentially targeted therapeutics.

Date

2007

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

W. Todd Monroe

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