Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation

The nuclear localization signal (NLS) of the SV40 large T antigen efficient ly induces nuclear entry of proteins, We have developed a strategy for cova lent coupling of one or a controlled number of NLS peptides to plasmid DNA at a specific site by triple helix formation. A psoralen-oligonucleotide-NL S peptide conjugate was synthesized and characterized by proteolysis with t rypsin, This conjugate was used to covalently associate one NLS peptide to plasmid DNA by triple helix formation and photoactivation. The oligonuclcot ide-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 o f 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 . (C) 1999 Federation of European Biochemical Societies.