| Popis: |
Many biochemical processes in which DNA and other nucleic acids participate are central to functions in both living cells and in molecular biology assays. While many compounds have been used to regulate the activity of DNA, these strategies are limited to the aqueous-based diffusion of the activator to the target DNA molecule. An improvement to the induction of DNA bioactivity is to move to a light-based modulation. This research demonstrates a light-based technique using a photo-cleavable cage compound to transiently inactivate DNA hybridization. Function can be restored with exposure to near-UV light, allowing for temporal control of DNA oligonucleotide (ODN) activity. This method has demonstrated the control of hybridization in molecular biology assays, and provides the framework for in vivo experimentation. A similar light-activated strategy has been shown useful in controlling expression of plasmid transgenes (Monroe 1999). By adapting this method to DNA oligonucleotides (ODNs), we have partially blocked hybridization with the cage compound (1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) for both phosphodiester and phosphorothioate DNA ODNs. The production and purification of DMNPE-caged DNA ODNs yields products with similar spectrophotometric properties to caged plasmids. In hybridization studies, 20-mer (20 base long) caged DNA ODNs were hybridized with complementary 30-mer molecular beacon probes, and fluorescence measurements were used to assess hybridization of native (non-caged), caged, and caged-light-exposed ODNs. Developments of the molecular beacon assays were studied to improve sensitivity of the assay to caged and caged-flashed ODN hybridization control. Results demonstrated that hybridization can be blocked and subsequently restored by light through the attachment of the DMNPE cage compound, and were further characterized with gel electrophoresis assays. ODN hybridization was restricted to as little as 2% when compared to native (non-caged) ODNs and restored to up to nearly 80% of the native (non-caged) ODN hybridization activity levels. Additional studies on adduction, purification, and characterization of the DMNPE-caged ODNs were performed to optimize their production and efficacy in controlling hybridization. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity, including applications with antisense ODNs as a form of controlled gene therapy. |
