Master of Science in Biological and Agricultural Engineering (MSBAE)
Biological and Agricultural Engineering
To control gene expression in vivo with spatial and temporal precision remains a significant hurdle in laboratory studies of development as well as clinical genetic therapies. Here we demonstrate such control over gene expression by use of photochemistry to reversibly inactivate the hybridization of a nucleic acid analog used for specific protein knockdown. A morpholino oligonucleotide, commonly used for knockdown of protein expression in developmental studies, was “caged” using carbodiimide conjugate chemistry which yielded photocleavable adducts that can be removed with light exposure. Photochemical inactivation approaches to produce caged molecules have been used to control the spatiotemporal activity of biomolecules such as nucleotides, neurotransmitters, proteins and nucleic acids. In this case, the morpholino oligonucleotide was caged through direct alkylation of exocylic amines with a carboxylic acid-based nitrobenzyl cage compound to demonstrate blockade of hybridization. Due to the site of attachment on nucleobases, results indicate that presumably, any nucleic acid antisense molecule could be used in this reaction scheme and thus, effectively caged. The degree of cage alkylation was determined using absorbance spectrophotometry, and the light-induced control over hybridization was characterized with gel-shift and fluorescence-based melting temperature assays. Using a behavioral assay in the zebrafish embryonic model as an endpoint for synthetic molecule assessment, in vivo demonstration of light-induced protein knockdown was shown where caged morpholino oligonucleotides do not possess protein knockdown activity until exposed to near-UV light. Perfect binary on/off behavioral responses with light exposure were not observed in the in vivo studies, presumably due to the statistical, or random-style of cage attachment to the many suitable bases on the oligonucleotide. This investigation should act to expand caged morpholino oligonucleotide technologies, and more generally antisense technologies as a whole due to the ease of synthesis required in caging these compounds, as well as further the understanding of molecular mechanisms governing embryonic development.
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Jarreau, Chad Michael, "Caged Morpholino Oligonucleotide for Control of Gene Expression in Zebrafish" (2010). LSU Master's Theses. 3969.
Monroe, W. Todd