Master of Science in Biological and Agricultural Engineering (MSBAE)
Biological and Agricultural Engineering
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.
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.
Faubion, Mindi Huguet, "Photo-Control of Antisense Activity in Cell Culture Using Caged Oligodeoxynucleotides" (2007). LSU Master's Theses. 985.