Novel Method for Covalent Fluorescent Labeling of Plasmid DNA That Maintains Structural Integrity of the Plasmid

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Abstract
We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido−tetrafluoro-benzyl-lissamine conjugate was synthesized and purified. This conjugate was used to covalently associate fluorescent molecules to plasmid DNA by photoactivation. In contrast to nick-translated plasmid DNA, plasmid−lissamine conjugates appeared on gel as supercoiled DNA. Reporter gene was expressed after transfection of the plasmid−lissamine conjugates in NIH 3T3 cells, although gene transfer efficiency was decreased by 60% as compared with unlabeled DNA. Intracellular traffic of plasmid−lissamine conjugates was studied in transfected cells. After cytoplasmic microinjection, fluorescent plasmid did not diffuse from the site of injection and appeared to be progressively degraded in the cytoplasm.