Novel method for covalent fluorescent labeling of plasmid DNA that maintains structural integrity of the plasmid

We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido-tetrafluoro-benzyl-lissamine conjugate was synth esized and purified. This conjugate was used to covalently associate fluore scent molecules to plasmid DNA by photoactivation. In contrast to nick-tran slated plasmid DNA, plasmid-lissamine conjugates appeared on gel as superco iled DNA. Reporter gene was expressed after transfection of the plasmid-lis samine conjugates in NIH 3T3 cells, although gene transfer efficiency was d ecreased by 60% as compared with unlabeled DNA. Intracellular traffic of pl asmid-lissamine conjugates was studied in transfected cells. After cytoplas mic microinjection, fluorescent plasmid did not diffuse from the site of in jection and appeared to be progressively degraded in the cytoplasm.