Gene Chemistry: Functionally and Conformationally Intact Fluorescent Plasmid DNA

Abstract

We describe an effective approach using a peptide nucleic acid (PNA) "clamp" to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. To demonstrate this approach a highly fluorescent preparation of plasmid DNA was generated by hybridizing a fluorescently labeled PNA to the plasmid. Fluorescent plasmid prepared in this way was neither functionally nor conformationally altered. PNA binding was sequence specific, saturable, extremely stable, and did not influence the nucleic acid intracellular distribution. This method was utilized for the first time to study the biodistribution of conformationally and functionally intact plasmid DNA in living cells after cationic lipid-mediated transfection. A fluorescent plasmid expressing green fluorescent protein (GFP) enabled simultaneous colocalization of both plasmid and expressed protein in living cells and in real time. GFP was shown to be expressed in cells containing detectable nuclear fluorescent plasmid. The fluorescent PNA-labeled plasmid revealed a marked difference in the nuclear uptake between oligonucleotide and plasmid, suggesting that nuclear entry of plasmid may require cell division. This detection method provides a way to simultaneously monitor the intracellular localization and expression of plasmid DNA in living cells, and to elucidate the mechanism of plasmid delivery and its nuclear import with synthetic gene delivery systems.