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Gene Delivery: A Single Nuclear Localization Signal Peptide Is Sufficient to Carry DNA to the Cell Nucleus
Maria Antonietta Zanta, Pascale Belguise-Valladier and Jean-Paul Behr
Proceedings of the National Academy of Sciences of the United States of America
Vol. 96, No. 1 (Jan. 5, 1999), pp. 91-96
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/47127
Page Count: 6
You can always find the topics here!Topics: DNA, Transfection, HeLa cells, Cell lines, Oligonucleotides, Hepatocytes, Signals, 3T3 cells, Plasmids, Neurons
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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.
Proceedings of the National Academy of Sciences of the United States of America © 1999 National Academy of Sciences