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 α. 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.
|Number of pages||5|
|State||Published - 18 Jun 1999|
Bibliographical noteFunding Information:
This work was supported by a grant from the French Ministry of Research. We thank Dr. François F. Clerc and his staff for peptide synthesis and Dr. Marc Vuilhorgne and his staff from the Structural Analysis Department of Rhône-Poulenc Rorer for mass spectroscopy analysis. We thank Dr. Y. Yoneda for the gift of the pGEX-2T plasmid. We thank D. Bisch, Dr. F. Blanche and Dr. V. Thuillier for importin α-GST purification.
- Gene therapy
- Gene transfer
- Nuclear localization signal
- Triple helix