Intracellular fate and nuclear targeting of plasmid DNA

One of the major steps limiting nonviral gene transfer efficiency is the en try of plasmid DNA from the cytoplasm into the nucleus of the transfected c ells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have develope d two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formati on of a triple helix with an oligonucleotide-NLS peptide conjugate. After s uch modification with one NLS peptide per plasmid molecule, plasmid DNA rem ained fully active in cationic lipid-mediated transfection. In the other me thod, we randomly coupled 5-115 p-azidotetrafluorobenzyllissamine-NLS pepti de molecules per plasmid DNA by photoactivation. Oligonucleotide-NLS and pl asmid-lissamine-NLS conjugates interacted specifically with the NLS-recepto r importin alpha. Plasmid-lissamine-NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from t he site of injection and plasmid-lissamine-NLS conjugates appeared to be pr ogressively degraded in the cytoplasm. The process of plasmid DNA sequestra tion/degradation stressed in this study might be as important in limiting t he efficiency of nonviral gene transfer as the generally recognized entry s tep of plasmid DNA from the cytoplasm into the nucleus.