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 conc ern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene con taining a single nuclear localization signal peptide (PKKKRKVEDPYC). Transf ection of cells with the tagged gene remained effective down to nanogram am ounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of t he signal peptide was observed irrespective of the cationic vector or the c ell type used. A lysine to threonine mutation of the third NLS amino acid c ompletely abolished these remarkable features, suggesting importin-mediated translocation. Our hypothesis is that the 3-nm-wide DNA present in the cyt oplasm is initially docked to and translocated through a nuclear pore by th e nuclear import machinery. As DNA enters the nucleus, it is quickly conden sed into a chromatin-like structure, which provides a mechanism for threadi ng the remaining worm-like molecule through the pore. A single NLS signal i s thus sufficient, whereas many signals on a gene would actually inhibit en try, the same DNA molecule being threaded through adjacent pores.