Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

Abstract

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.

Figure 1

Strategy for the preparation of a double-stranded DNA fragment coupled to an NLS peptide. A functional luciferase gene of 3,380 bp was cut out of pCMVLuc with XmaI and SalI. Further digestion with XmnI and BspHI cut the unwanted restriction fragment into small fragments (970, 875, 768, and 240 bp) that were removed by sucrose gradient centrifugation. The capped CMVLuc-NLS DNA was obtained by ligation of the ³²P-labeled (∗) oligonucleotide-peptide and oligonucleotide-cap hairpins to the restriction fragment.

Figure 2

Reaction scheme for the chemical coupling steps leading to the oligonucleotide-peptide conjugate (oligo-NLS). A hairpin oligonucleotide with a free alkylamino group in the T₄ loop (oligo-NH₂) was reacted with the heterobifunctional crosslinker SMCC to give a thiol-reactive maleimide oligonucleotide (oligo-Mal), which was in turn reacted with the C-terminal cysteinamide residue of the NLS dodecapeptide.

Figure 3

(A) Synthesis of oligonucleotide-NLS. PAGE analysis of the formation of the oligonucleotide-peptide conjugate is shown. Aliquots of the reaction mixture were taken at different times and analyzed on a 20% denaturing gel after radiolabeling of the oligonucleotides. Lanes: 1, oligonucleotide-NH₂; 2, oligonucleotide-NH₂/SMCC reaction mixture after 2 h; 3–5, reaction mixture 1.5 h, 3 h or overnight after addition of the NLS-peptide to the oligonucleotide-NH₂/SMCC mixture, respectively. (B) The presumed oligonucleotide-NLS conjugate is a substrate of proteinase K. Radiolabeled oligonucleotide-NH₂ or the crude oligonucleotide-NLS reaction mixture were digested with proteinase K. Products were analyzed on a 20% denaturing gel and show total conversion of oligonucleotide-NLS into a faster migrating band, presumably oligo-Mal-Cys.

Figure 4

NLS peptide promotes high and sustained transfection levels down to 10 ng DNA. 3T3 cells were transfected with decreasing amounts of DNA complexed in 150 mM NaCl to a cationic lipid (Transfectam, vector nitrogens over DNA phosphate, N/P = 6) or to a cationic polymer (25-kDa PEI, N/P = 10) in the absence of serum.

Figure 5

Sustained luciferase expression levels are due to the nuclear localization peptide. HeLa cells were transfected with various amounts of DNA complexed to ExGen500 PEI (N/P = 5 in a 5% glucose solution) in the presence of 10% serum.

Figure 6

Reporter protein activity appears faster with CMVLuc-NLS. HeLa cells were transfected with 10 ng of DNA complexed to ExGen 500 PEI (N/P = 5 in 5% glucose solution) in the absence of serum. Cells were lysed and luciferase activity was measured at the indicated time after transfection. The absence of error bars indicates that the SEM is smaller than the label on the graph.

Figure 7

Hypothetical scheme of foreign DNA entering the nucleus. The vectorial movement is driven by DNA binding to chromatin-forming basic proteins. A single NLS peptide helps initial threading, whereas too many signals may inhibit full translocation.