The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear entry of proteins. We have developed a strategy for covalent coupling of one or a controlled number of NLS peptides to plasmid DNA at a specific site by triple helix formation. A psoralen-oligonucleotide-NLS peptide conjugate was synthesized and characterized by proteolysis with trypsin. This conjugate was used to covalently associate one NLS peptide to plasmid DNA by triple helix formation and photoactivation. The oligonucleotide-NLS peptide conjugate interacted with the NLS-receptor importin alpha. The reporter gene was expressed after transfection of the modified plasmid in NIH 3T3 cells, indicating no loss of the gene expression functionality of the plasmid. On the other hand, no increase in expression was observed as a result of the NLS peptide. This site-specific coupling technology can be used to couple to a plasmid other ligands targeting to a specific receptor.
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