No Enhancement of Nuclear Entry by Direct Conjugation of a Nuclear

Revised Manuscript Received October 6, 2003. Efficient ... point during the transfection (6), suggesting that DNA could enter ... Hokkaido University...
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Bioconjugate Chem. 2003, 14, 1197−1202

1197

No Enhancement of Nuclear Entry by Direct Conjugation of a Nuclear Localization Signal Peptide to Linearized DNA Mitsuhide Tanimoto,†,‡ Hiroyuki Kamiya,†,‡ Noriaki Minakawa,† Akira Matsuda,† and Hideyoshi Harashima*,†,‡ Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan and CREST, Japan Science and Technology, Japan. Received May 8, 2003; Revised Manuscript Received October 6, 2003

Efficient nuclear entry of exogenous DNA is one of the key factors toward gene therapy success with nonviral vectors. To re-address the effects of a nuclear localization signal (NLS) peptide attached directly to DNA, we prepared three dumbbell-shaped, green fluorescent protein (GFP)-encoding DNAs containing one or two NLS peptides. The peptide was conjugated to the loop-forming oligodeoxyribonucleotides by cross-linking reactions between the peptide and a modified uracil base with a dioxaoctylamino linker, and the oligonucleotides were then ligated to the DNA molecules. The NLSconjugated DNA dumbbells were microinjected into the cytosols and nuclei of simian COS-7 cells. In addition, unconjugated DNA dumbbells, with or without a modified uracil base, were also examined for comparison. The GFP gene was expressed with efficiencies in the order of the unmodified DNA g the NLS-conjugated DNA > the unconjugated DNA with the base modification, with both cytosolic and intranuclear microinjections. Thus, we concluded that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus.

INTRODUCTION

Nonviral vectors are highly attractive in gene therapy due to their excellent safety profile, despite their low transgene expression efficiency in comparison to viral vectors. The intracellular disposition, especially the DNA entry into the nucleus, is a very important issue for high transgene expression (1). Intranuclear microinjections of DNA result in about 100- to 1000-fold more efficient expression than cytoplasmic microinjections (2, 3). Thus, the nuclear entry pathway(s) of exogenous DNA are of great interest. One proposed mechanism is the trafficking of DNA when the nuclear membrane disappears at the M phase of cell division (4). However, the nuclear entry occurs in nondividing cells (5) and at a very early time point during the transfection (6), suggesting that DNA could enter the nucleus in the presence of the nuclear membrane. We and others pointed out the possibility that the cationic liposomal lipid and the nuclear envelope may fuse, and thus the exogenous DNA may enter the nucleus (4, 6). An alternative explanation is the nuclear pore complex (NPC)-mediated import of DNA. Although the contribution of this putative pathway to the nuclear entry of DNA is unclear, it has been proven to be useful for the delivery of a protein conjugated with nuclear localization signal (NLS) peptides (7). Recently, chemical conjugations of the NLS peptide to DNA molecules have been attempted to improve the nuclear entry. Sebestye´n et al. used a cross-linker to conjugate the NLS peptide to the N3-position of adenine bases in double-stranded DNA (8). The introduction of 60-100 NLS peptides/kbp DNA was efficient for nuclear * To whom correspondence should be addressed: Tel +8111-706-3919. Fax +81-11-706-4879. E-mail harasima@ pharm.hokudai.ac.jp. † Hokkaido University. ‡ CREST.

uptake in digitonin-permeabilized cells. However, no increase in the nuclear uptake of the modified DNA was observed after microinjection into the cytoplasm of living cells, and the transgene expression was completely abolished due to the high modification level. In contrast, Zanta et al. synthesized a loop-forming oligodeoxyribonucleotide (ODN) cross-linked with the NLS peptide, and this modified loop-forming ODN was then enzymatically ligated to linearized double-stranded DNA (9). This NLS-DNA was transfected with a cationic polymer, polyethylenimine (PEI), and a single NLS peptide reportedly could enhance transgene expression from ten- to hundreds-fold, depending on the cell lines used. However, the nuclear entry effects of the NLS peptide attached to DNA are still open to dispute (10). Zanta et al. obtained their results using PEI-mediated transfection. However, PEI may enter the nucleus together with DNA (11). In this study, we reexamined the effects of the NLS peptide conjugated to DNA by cytosolic and intranuclear microinjections. In addition, we compared the expression of the transgene on chemically modified and unmodified DNA molecules. Our results suggest that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus. MATERIALS AND METHODS

General. COS-7 cells were from the RIKEN Cell Bank (Tsukuba, Japan). The plasmid pQBI 25-63, containing the cytomegalovirus promoter and the green fluorescent protein (GFP) gene (3), was purified with a Qiagen (Valencia, California) EndoFree Mega kit. The SV40 large T antigen NLS peptide (NH2-PKKKRKVEDPYC) with C-terminal amidation was obtained from Sigma Genosys Japan (Ishikari, Japan) in the purified form. Physical data were measured as follows: The NMR spectra were recorded with a JEOL GX-270 spectrometer,

10.1021/bc034075e CCC: $25.00 © 2003 American Chemical Society Published on Web 10/29/2003

1198 Bioconjugate Chem., Vol. 14, No. 6, 2003 Table 1. Oligodeoxynucleotides Used in This Study ODN

sequence (5′f3′)a

ODN-1 ODN-2 ODN-3 ODN-4 ODN-5

dGATCTGGCTCGCCTGTTTTTCAGGCGAGCCA dAGCTTGGCTCGCCTGTTTTTCAGGCGAGCCA dGATCTGGCTCGCCTGTTXTTCAGGCGAGCCA dGATCTGGCTCGCCTGTXTXTCAGGCGAGCCA dAGCTTGGCTCGCCTGTTXTTCAGGCGAGCCA

a X represents the modified uracil derivative conjugated or unconjugated to the NLS peptide. The italicized and underlined sequences represent sticky ends and loop-forming sites, respectively. Annealed ODN-2 and ODN-5 have an end compatible with Hind III, and the others have that compatible with Bgl II.

with CDCl3 as the solvent and tetramethylsilane as the internal standard. Chemical shifts are reported in parts per million (δ), and signals are expressed as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Mass spectra were measured on a JEOL JMS-D300 spectrometer. Matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF-MS) was conducted with Applied Biosystems Voyager System 1065 (Applera, Norwalk, CT). Edman peptide sequencing analysis was carried out with Procise 492 (Applera, Norwalk, CT). TLC was done on Merck Kieselgel F254 precoated plates. The silica gel used for column chromatography was Merck silica gel 5715. 5-N-(8-Trifluoroacetylamino-3,6-dioxaoctyl)carbamoyl-5′-O-dimethoxytrityl-2′-deoxyuridine (3). To a solution of 1 (12) (2.1 g, 3.2 mmol) in pyridine (30 mL) was added 2,2′-(ethylenedioxy)bis(ethylamine) (1.4 mL, 9.6 mmol), and the mixture was stirred for 1 h at room temperature. The solvent was removed in vacuo, and the residue was coevaporated with toluene to give crude 2. The resulting 2 was dissolved in MeOH (80 mL), and Et3N (2.7 mL, 19.2 mmol) and ethyl trifluoroacetate (2.3 mL, 19.2 mmol) were added to the solution. The reaction mixture was stirred at room temperature until compound 2 was consumed on TLC analysis. The solvent was removed in vacuo, and the residue was purified on a neutral silica gel column with 5-10% EtOH in CHCl3 to give 3 (1.8 g, 70% as white foam): FAB-MS: m/z 800 (M+); FAB-HRMS: calcd for C39H43N4O11NaF3 (MNa+): 823.2747. found: 823.2750; 1H NMR (CDCl3) δ 9.10 (s, 1H), 8.80 (m, 1H), 8.61 (s, 1H), 7.59 (m, 1H), 7.41-6.82 (m, 13H), 6.15 (t, 1H, J ) 6.6 Hz), 4.33 (m, 1H), 4.00 (q, 1H, J ) 4.6 Hz), 3.78 (s, 6H), 3.62-3.52 (m, 12H), 3.47 (dd, 1H, J ) 4.6 and 9.9 Hz), 3.37 (dd, 1H, J ) 4.6 and 9.9 Hz), 2.45 (m, 1H), 2.45 (ddd, 1H, J ) 6.6, 4.0, and 13.9 Hz), 2.21 (ddd, 1H, J ) 6.6, 7.3, and 13.9 Hz). 5-N-(8-Trifluoroacetylamino-3,6-dioxaoctyl)carbamoyl-5′-O-dimethoxytrityl-3′-O-((2-cyanoethoxyl)(N,N-diisopropylamino)phosphynyl)-2′-deoxyuridine (4). After successive coevaporation with pyridine, 3 (80 mg, 0.1 mmol) was dissolved in CH2Cl2 (5 mL) containing N,N-diisopropylethylamine (35 µL, 0.2 mmol). 2-Cyanoethyl N,N-diisopropylphosphoramidochloride (33 µL, 0.15 mmol) was added to the solution, and the reaction mixture was stirred for 1 h at room temperature. The mixture was diluted with CHCl3 and washed with saturated aqueous NaHCO3 and brine. The separated organic layer was dried (Na2SO4) and concentrated in vacuo. The residue was purified on a neutral silica gel column with 60-100% AcOEt in hexane to give 4 (61 mg, 61% as white foam): 31P NMR (CDCl3) δ 149.93, 149.53. Oligodeoxyribonucleotides. ODNs with the modified uracil derivative were designed to incorporate it in the loop consisting of five pyrimidine bases and to make a 5′-protruding end compatible with the Bgl II or Hind III restriction enzyme (ODN-3 to ODN-5, Table 1). These

Tanimoto et al.

ODNs were synthesized using the phosphoramidite derivative 4 on an ABI model 381A DNA/RNA synthesizer. These ODNs were purified essentially as described (13). The NLS peptide was conjugated to an ODN essentially as described (9), using 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC) as a bifunctional cross-linker. Namely, an ODN containing the modified uracil base(s) (16.1 nmol) was dissolved in 10 mM of sodium phosphate buffer (pH 7.5), and a 20 molar excess of SMCC (in DMF) was added. After an incubation for 1 h at room temperature, the same amount of SMCC was added, and the reaction mixture was then incubated at room temperature for another 1 h. The unreacted SMCC was removed with a NICK Column (Amersham Biosciences, Piscataway, NJ). A 20 molar excess of the NLS peptide was added, and the reaction mixture was incubated at room temperature overnight. In the case of ODN-4, containing two modified uracil bases, the amounts of SMCC and the NLS peptide were twice those described above. The conjugated ODNs were purified by anion-exchange HPLC, using a TSKGEL DEAE-2SW column (4.6 × 250 mm, Tosoh, Japan) with a linear gradient of ammonium formate (450 mM to 650 mM) in 20% aqueous acetonitrile. A new peak eluted at 15 min, faster than the unconjugated ODN (starting material, 18 min), and this peak was collected as an NLS-conjugated ODN (Figure 1). When ODN-4 containing two modified uracil bases was used, two peaks (8 and 12 min) that eluted faster than the unconjugated ODN (17 min) were observed. The 8 min peak was collected as the ODN containing two NLS peptides. The faster elution upon anion-exchange HPLC corresponded to the conjugation of the positively charged peptide. The ammonium formate was removed by gel filtration using Sephadex-G-25 (Amersham Biosciences). The unmodified ODNs (ODN-1 and ODN-2, Table 1) were obtained from Hokkaido System Science (Sapporo, Japan) in the purified form. These ODNs were annealed and 5′-phosphorylated by T4 polynucleotide kinase and ATP. Preparation of Linearized DNA Dumbbells. The plasmid pQBI 25-63 was digested with Bgl II and Hind III, according to the manufacturer’s instructions. The digested DNA was loaded onto a low melting point agarose gel. The linearized DNA was recovered from the gel by visualization with UV irradiation in the presence of a thin-layer chromatography plate. The DNA was then purified as described in the literature (14). The purified 2.3-kb DNA was mixed and joined with the corresponding ODNs (DNA:ODN ) 1:100), as reported previously (3). After ethanol precipitation, the ligated DNA was purified by anion-exchange HPLC using a TSK-gel DNA-NPR column (φ4.6 × 75 mm, Tosoh, Japan), essentially as described (15). Microinjection. Cells were microinjected at day 1 postplating. In this procedure, we used a semiautomatic injection system (Eppendorf transjector 5246, Hamburg, Germany) attached to the Eppendorf micromanipulator 5171. Intranuclear and cytosolic microinjections were performed with the Z (depth) limit option using a 0.2-s injection time and a 10-200 hectopascal-injection pressure. The DNA was diluted with an injection buffer solution (0.5% tetramethylrhodamine-labeled dextran in phosphate-buffered saline). At 24 h postinjection, GFP expression was monitored by fluorescence microscopy, and the ratio of cells expressing GFP to tetramethylrhodamine-positive cells was calculated.

Effects of NLS and Base Modification

Bioconjugate Chem., Vol. 14, No. 6, 2003 1199

Figure 1. Conjugation of the NLS peptide and ODN-3. (A-C) Behavior upon anion-exchange HPLC. Unconjugated ODN-3 (A), mixture of the conjugation reaction (B), and conjugated ODN-3 after purification (C) are shown. (D) Polyacrylamide gel electrophoresis. Lane 1, unconjugated ODN-3; lane 2, conjugated ODN-3. (E) Molecular weight analysis by MALDI-TOF-MS. The calculated molecular weight of conjugated ODN-3 is 11364. Scheme 1

RESULTS AND DISCUSSION

Design of DNA Dumbbells. A modified uracil base with a dioxaoctylamino linker (5), for conjugation to the NLS peptide, was incorporated into the ODNs, as shown

in Scheme 1. The conjugation of an ODN containing the modified uracil base and the NLS peptide was conducted using a bifunctional cross-linker, essentially as described (9). The conjugated ODNs were separated from the

1200 Bioconjugate Chem., Vol. 14, No. 6, 2003

Figure 2. Structures of DNA dumbbells containing the GFP gene used in this study. X, the modified uracil derivative; NLS, NLS peptide; CMV, CMV promoter. Bgl II and Hind III represent the restriction enzyme sites used for the ligation of loop-forming ODNs.

unconjugated ODNs by anion-exchange HPLC. Successful conjugation of an ODN and the positively charged peptide was indicated by chromatographic behavior upon anion-exchange HPLC (Figure 1A-C), lower mobility than the unconjugated ODN upon polyacrylamide gel electrophoresis (Figure 1D), molecular weight analysis by matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF-MS, Figure 1E), and sequencing of N-terminal 10 amino acids upon the Edman analysis (data not shown). We prepared three DNA dumbbells with the NLS peptide (Figure 2). The NLS peptide-conjugated ODN-3 and ODN-4 (Table 1) were used to construct Dumbbell1-0 and Dumbbell-2-0, respectively, which contain one and two peptide(s) in the loop near the Bgl II site. These DNA dumbbells contain the unmodified loop-forming ODN-2 near the Hind III site. Dumbbell-1-1 contains NLS peptides in both loops, using ODN-3 and ODN-5 conjugates. As controls, two DNA dumbbells were constructed: Dumbbell-0-0, which was capped with ODN-2 and unconjugated ODN-3, and the unmodified Dumbbell capped with ODN-1 and ODN-2. The unmodified Dumbbell was used to examine the effects of the base modification in comparison with Dumbbell-0-0. These DNA dumbbells were purified by anion-exchange HPLC to remove the unligated ODNs, which could possibly compete with the nuclear entry of NLS-conjugated DNA dumbbells via the NPC. Cytosolic Microinjection. First, we microinjected various DNA dumbbells (10000 copies/cell) into the cytoplasms of simian COS-7 cells. GFP expression was monitored by fluorescence microscopy, and the ratio of cells expressing GFP to those containing the labeled dextran in their nuclei was calculated. Figure 3 shows the percentage of GFP-positive cells at 24 h postinjection. When microinjected into the cytoplasms of simian COS-7 cells, approximately 40% of the cells expressed GFP, in the case of the unmodified Dumbbell. The GFP genes on the NLS-conjugated DNA dumbbells (Dumbbell1-0, -1-1, and -2-0) were expressed with an efficiency similar to or slightly less than the unmodified Dumbbell (∼35%, Figure 3). Thus, no dramatic increase in transgene expression was observed by the conjugation of the NLS peptide to DNA molecules, when we attached one or two peptide(s) per DNA. Interestingly, Dumbbell-0-0 showed decreased GFP expression, as compared with that of the unmodified Dumbbell (∼15%, Figure 3). This result suggests that the presence of the modified base suppressed transgene

Tanimoto et al.

Figure 3. Expression efficiencies of the GFP gene in COS-7 cells by cytoplasmic microinjection of DNAs. The DNA (10000 copies) was injected, and the expression was examined at 24 h postinjection. The data are expressed as means ( standard deviation of two separate experiments. The datum of Dumbbell1-1 is that obtained in a single injection experiment. cc represents closed circular plasmid DNA.

Figure 4. Expression efficiencies of the GFP gene in COS-7 cells by intranuclear microinjection of DNAs. The DNA (100 copies) was injected, and the expression was examined at 24 h postinjection. The data are expressed as means ( standard deviation of two separate experiments. cc represents closed circular plasmid DNA.

expression, for by one or two reasons: (i) the nuclear entry of the modified dumbbell (Dumbbell-0-0) occurred less efficiently than that of unmodified Dumbbell, and/ or (ii) the expression of the gene on the modified Dumbbell-0-0 was reduced in the nucleus by the base modification. Moreover, the fact that the NLS-conjugated DNAs showed better expression than that of Dumbbell-0-0 (and similar expression to the unmodified Dumbbell) suggests the possibilities that the peptide improved the nuclear entry and/or the expression in the nucleus. Intranuclear Microinjection. Next, we microinjected various DNAs (100 copies/nucleus) into the nuclei of simian COS-7 cells. We microinjected only Dumbbell1-0 as the NLS-conjugated DNA, because very similar results were obtained with the three NLS-dumbbells in the cytosolic microinjection experiments described above. As shown in Figure 4, results similar to those obtained with the cytosolic microinjection experiments were observed. Dumbbell-0-0 showed decreased GFP expression as compared with that of the unmodified Dumbbell. The transgene on the NLS-conjugated DNA dumbbell (Dumbbell-1-0) was expressed more efficiently than that on Dumbbell-0-0 (∼20% and ∼15%, respectively), and with less efficiency than that on the unmodified Dumbbell (∼30%). Thus, the efficiencies of GFP expression by the

Effects of NLS and Base Modification

cytosolic microinjections, shown above, mainly depended on the event(s) in the nucleus. Effects of the Modified Base and the NLS Peptide. On the basis of the results obtained with the cytosolic and intranuclear microinjections, we found that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of the DNA decreased the transgene expression. Although the actual reason(s) for the latter is unclear, the transcription efficiency or stability in the nucleus may be reduced by the introduction of the modified base. The uracil derivative used in this study was incorporated in the loop near the Bgl II site (Figure 2), and thereby near the promoter region. Thus, transcriptional factors may load less efficiently than on unmodified DNA. Alternatively, the modified base may be recognized by DNA repair enzyme(s) with endonuclease function(s). The NLS-conjugated DNA dumbbells, in contrast, showed GFP expression similar to that of the unmodified Dumbbell. The NLS-conjugation ‘shielded’ the effects of the base modification in the transcription and/ or excision reactions. The peptide consists of positively charged amino acid residues and may interact with the DNA phosphate backbone. If this putative interaction occurs in the loop region, then the cellular protein(s) may hardly recognize the modification. We showed that one or two NLS peptide(s) conjugated to DNA did not enhance transgene expression dramatically, at least when the naked DNA was microinjected into the cytoplasm. Thus, the nuclear entry of DNA appears to be similar with and without the peptide. The NLS peptide used in this study is positively charged and may interact with the DNA phosphate backbone. Thus, the NLS peptide might not act as a nuclear entry device. This interpretation is in contrast to the results of Zanta et al. (9), who reported ten- to hundreds-fold increased transgene expression by the conjugation of the NLS peptide to the loop region of dumbbell-shaped DNA. Improved transgene expression was also reported with a noncovalently bound NLS peptide. Brande´n et al. combined a peptide nucleic acid (PNA) molecule to the NLS peptide, and this NLS-PNA was attached to plasmid DNA containing a sequence complementary to the PNA molecules (16). They reported that the addition of the NLS-PNA enhanced PEI-mediated transgene expression 3- to 5-fold. Interestingly, both Zanta et al. and Brande´n et al. obtained their results using PEI-mediated transfection. Godbey et al. observed the intracellular trafficking of a PEI-DNA complex by confocal microscopy and found that both PEI and DNA entered the nucleus as a complex (11). Thus, PEI may alter the transcription efficiency of the delivered DNA. The NLS peptide may enhance the transcription, since the transcriptional factors may be loaded more efficiently if the interaction between the PEI and DNA is impaired. It should be emphasized that the combination of cytosolic and intranuclear microinjections enables separate considerations of the nuclear entry and the events inside the nucleus. Recently, attachment of the NLS peptide to plasmid DNA using a triple helix-forming padlock ODN was reported (17). Upon transfection experiments, no enhancement of transgene expression was observed. Very recently, Nagasaki et al. reported the covalent conjugation of the NLS peptide to plasmid DNA, by diazo coupling through a PEG chain (18). They observed by fluorescence microscopy that the NLS peptide did not enhance the nuclear entry of the circular plasmid DNA upon cytosolic microinjection, although the conjugation of 5.1 NLS peptides per DNA increased the transgene expression

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4-fold upon transfection. Their results agree with our present results, on the point that a small number of NLS peptides could not enhance the nuclear entry remarkably. In this study, we observed that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of a linearized DNA dumbbell and that (ii) chemical modification of the DNA reduced the transcription efficiency or stability in the nucleus. These results suggest that the NLS peptide should be attached to a DNAcarrying molecule, not to the DNA itself, to enhance the nuclear entry of DNA via the NPC. ACKNOWLEDGMENT

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas, from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science, and a Grant-in-Aid from the Uehera Memorial Foundation. LITERATURE CITED (1) Kamiya, H., Tsuchiya, H., Yamazaki, J., and Harashima, H. (2001) Intracellular trafficking and transgene expression of viral and nonviral gene vectors. Adv. Drug Del. Rev. 52, 153-164. (2) Pollard, H., Remy, J. S., Loussouarn, G., Demolombe, S., Behr, J. P., and Escande, D. (1998) Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J. Biol. Chem. 273, 7507-7511. (3) Kamiya, H., Yamazaki, J., and Harashima, H. (2002) Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells. Gene Ther. 9, 1500-1507. (4) Tseng, W. C., Haselton, F. R., and Giorgio, T. D. (1999) Mitosis enhances transgene expression of plasmid delivered by cationic liposomes. Biochim. Biophys. Acta 1445, 53-64. (5) Ludtke, J. J., Sebestye´n, M. G., and Wolff, J. A. (2002) The effect of cell division on the cellular dynamics of microinjected DNA and dextran. Mol. Ther. 5, 579-588. (6) Kamiya, H., Fujimura, Y., Matsuoka, I., and Harashima, H. (2002) Visualization of intracellular trafficking of exogenous DNA delivered by cationic liposomes. Biochem. Biophys. Res. Commun. 298, 591-597. (7) Tachibana, R., Harashima, H., Shono, M., Azumano, M., Niwa, M., Futaki, S., and Kiwada, H. (1998) Intracellular regulation of macromolecules using pH-sensitive liposomes and nuclear localization signal: qualitative and quantitative evaluation of intracellular trafficking. Biochem. Biophys. Res. Commun. 251, 538-544. (8) Sebestye´n, M. G., Ludtke, J. J., Bassik, M. C., Zhang, G., Budker, V., Lukhtanov, E. A., Hagstrom, J. E., and Wolff, J. A. (1998) DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA. Nat. Biotechnol. 16, 80-85. (9) Zanta, M. A., Belguise-Valladier, P., and Behr, J. P. (1999) Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc. Natl. Acad. Sci. U.S.A. 96, 91-96. (10) Cartie, R., and Reszka, R. (2002) Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems. Gene Ther. 9, 157-167. (11) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. U.S.A. 96, 5177-5181. (12) Nomura, Y., Ueno, Y., and Matsuda, A. (1997) Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nucleaseresistance properties. Nucleic Acids Res. 25, 2784-2791. (13) Kamiya, H., and Kasai, H. (1997) Substitution and deletion mutations induced by 2-hydroxyadenine in Escherichia coli: Effects of sequence contexts in leading and lagging strands. Nucleic Acids Res. 25, 304-310.

1202 Bioconjugate Chem., Vol. 14, No. 6, 2003 (14) Sambrook, J, Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., pp 6.306.31, Cold Spring Harbor Laboratory Press, Woodbury, NY. (15) Kamiya, H., and Kasai, H. (2000) 2-Hydroxy-dATP is incorporated opposite G by Escherichia coli DNA polymerase III resulting in high mutagenicity. Nucleic Acids Res. 28, 1640-1646. (16) Brande´n, L. J., Mohamed, A. J., and Smith, C. I. (1999) A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA. Nat. Biotechnol. 17, 784787.

Tanimoto et al. (17) Roulon, T., He´le`ne, C., and Escude´, C. (2002) Coupling of a targeting peptide to plasmid DNA using a new type of padlock oligonucleotide. Bioconjugate Chem. 13, 1134-1139. (18) Nagasaki, T., Myohoji, T., Tachibana, T., Futaki, S., and Tamagaki, S. (2003) Can Nuclear Localization Signals Enhance Nuclear Localization of Plasmid DNA? Bioconjugate Chem. 14, 282-286.

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