Master of Science in Biological and Agricultural Engineering (MSBAE)


Biological and Agricultural Engineering

Document Type



Antisense oligodeoxynucleotides (AS ODNs) are short single-stranded pieces of DNA that are designed to hybridize with messenger RNA and thus offer a powerful technique for controlling gene expression. Phosphorothioate AS ODNs are a well-established modification of the traditional phosphodiester structure that increase the in vitro half-life of AS ODN activity. In this work, we describe a method for controlling the hybridization activity of PS ODNs in cell culture with a photo-reversible mechanism. The influence of a photocaging compound, 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) on hybridization of ISIS 2302, a 20-mer PS ODN, was modeled using a molecular beacon assay. Based on this model, a relationship between the number of attached cage groups and the inactivation of hybridization activity was established. In addition, the light-dose effects on cell morphology and intracellular adhesion molecule-1 (ICAM-1) target protein expression levels were used to determine that 40 J/cm&178; of UVA light was the maximum dose that could be used for photorestoration of antisense activity in cell based studies. Active and DMNPE-inactivated forms of ISIS 2302 were delivered to human epithelial carcinoma (HeLa) cells. 78 &177; 2.9% of cells exposed to active ISIS 2302 showed decreases in cytokine-stimulated ICAM-1 protein expression as measured by flow cytometry. However, only 25 &177; 1% of cells exposed to DMNPE-inactivated ISIS 2302 experienced antisense effects. Subsequent exposure of HeLa cells to 40 J/cm&178; of 365 nm light resulted in photo-restoration of cage-inactivated ISIS 2302 antisense activity in 56.7 &177; 5.9% of HeLa cells. There was no significant difference in ICAM-1 expression between cells treated with 40%-caged antisense and cells treated with no antisense. Subsequent exposure to 40 J/cm&178; 365 nm light resulted in a significant increase in antisense activity from the cells treated with 40%-caged antisense. This work demonstrates the in vitro use of a light inducible system for achieving the spatio-termporal control of antisense specific activity.



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Committee Chair

Todd Monroe



Included in

Engineering Commons