Targeting DNA expression and hybridization with light: Application to plasmid transfections and antisense oligodeoxynucleotides.

摘要

To demonstrate a targeting strategy for genetic therapies, we describe the inactivation and site-directed light induction of plasmid expression and oligodeoxynucleotide (ODN) hybridization using the photosensitive caging compound 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE). The technology is based on the covalent attachment of the caging compound, which disrupts DNA bioactivity until light photocleaves the cage and restores DNA to its native bioactive form.; In vivo particle-mediated delivery of caged luciferase plasmids into rat skin showed no expression. However, subsequent exposure of transfected skin sites to 355 nm laser light induced luciferase expression in proportion to the amount of light. Similar results were observed with the transfection of caged plasmids coding for green fluorescent protein (GFP) in HeLa cultures. Linear plasmid expression of GFP mRNA in an in vitro transcription assay was also blocked by caging, but was subsequently restored by exposure to light, suggesting that caging plasmid DNA with DMNPE blocks expression at the level of transcription.; Similar to the light-based control over plasmid bioactivity, the use of this biotechnology to block ODN hybridization and antisense activity was studied. In hybridization studies, DMNPE-caged 20-mer ODNs were hybridized with complementary 30-mer molecular beacons. DNA gel banding patterns and molecular beacon fluorescence measurements were used to assess hybridization of native (non-caged) ODNs, caged ODNs, and caged-light-exposed ODNs. Antisense blockade studies followed a similar design. Caged ODNs designed to block expression of Intracellular Adhesion Molecule-1 (ICAM-1) were transfected into HeLa cells. After light exposure to selected culture dishes, cells were harvested and ICAM-1 levels assayed by immunostaining and flow cytometry. Both experimental designs showed a reduction in these DNA bioactivities which could subsequently be restored by light. The complete restoration of intracellular antisense activity was not achieved. Our data indicate that this is due to the low tolerance of the cell culture system to light doses required for complete photoactivation of caged antisense ODNs.; Together these results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of DNA expression and hybridization. With further developments, this light-inducible strategy may provide a new means to target gene and antisense therapies.