Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus.
Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of the cationic vector or the cell type used. A lysine to threonine mutation of the third NLS amino acid completely abolished these remarkable features, suggesting importin-mediated translocation. Our hypothesis is that the 3-nm-wide DNA present in the cytoplasm is initially docked to and translocated through a nuclear pore by the nuclear import machinery. As DNA enters the nucleus, it is quickly condensed into a chromatin-like structure, which provides a mechanism for threading the remaining worm-like molecule through the pore. A single NLS signal is thus sufficient, whereas many signals on a gene would actually inhibit entry, the same DNA molecule being threaded through adjacent pores.
Zanta
M A
MA
Laboratoire de Chimie Génétique associé Centre National de la Recherche Scientifique/Université Louis Pasteur (Unité Mixte de Recherche 7514), Faculté de Pharmacie de Strasbourg, France.
Belguise-Valladier
P
P
Behr
J P
JP
D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008957:Models, Genetic; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010455:Peptides; D004247:DNA; D008156:Luciferases