Targeting expression with light using caged DNA.
In this report, we describe the inactivation and site-specific light induction of plasmid expression using a photosensitive caging compound. Plasmids coding for luciferase were caged with 1-(4, 5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) and transfected into approximately 1-cm diameter sites of the skin of rats with particle bombardment. Skin sites transfected with caged plasmids did not express luciferase. However, subsequent exposure of transfected skin sites to 355-nm laser light induced luciferase expression in proportion to the amount of light. Liposome transfection of HeLa cells with DMNPE-caged green fluorescent protein (GFP) plasmids showed similar results. Caging DNA with DMNPE blocks expression at the level of transcription, since in vitro production of mRNA from linearized GFP plasmid was also blocked by caging and subsequently restored by exposure to light. Under the reaction conditions of these experiments, our absorbance data indicate that each DMNPE-caged GFP plasmid contains approximately 270 caging groups. In addition to inhibition and subsequent restoration of plasmid bioactivity, the presence and photocleavage of this relatively small number of cage groups also alters electrophoretic mobility of plasmids and optical absorption characteristics. This light-induced expression strategy provides a new means to target the expression of genetic material with spatial and temporal specificity.
Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee 37235, USA.
W T
WT
Monroe
M M
MM
McQuain
M S
MS
Chang
J S
JS
Alexander
F R
FR
Haselton
D000818:Animals; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D018390:Gene Targeting; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008156:Luciferases / Q000235:genetics*; D009578:Nitrobenzenes; D010957:Plasmids / Q000235:genetics; D051381:Rats; D014162:Transfection / Q000379:methods*
D009578:Nitrobenzenes; C085061:1-(4,5-dimethoxy-2-nitrophenyl)diazoethane; D004247:DNA; D008156:Luciferases