Coupling of Nuclear Localization Signals to Plasmid DNA and Specific

Specific Interaction of the Conjugates with Importin r ... specifically with the NLS-receptor importin R, but plasmid-NLS conjugates were not detected...
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Bioconjugate Chem. 1999, 10, 49−55

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Coupling of Nuclear Localization Signals to Plasmid DNA and Specific Interaction of the Conjugates with Importin r Carole Ciolina, Gerardo Byk, Francis Blanche,† Vincent Thuillier,† Daniel Scherman, and Pierre Wils* UMR 133 CNRS/Rhoˆne-Poulenc Rorer and Rhoˆne-Poulenc Rorer Gencell, Centre de Recherche de Vitry Alfortville, B.P.14, 13 quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France. Received June 5, 1998; Revised Manuscript Received October 23, 1998

The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear targeting of proteins. We have developed a chemical strategy for covalent coupling of NLS peptides to plasmid DNA. A p-azido-tetrafluoro-benzyl-NLS peptide conjugate was synthesized. This conjugate was used to covalently associate NLS peptides to plasmid DNA by photoactivation. Reporter gene was expressed after transfection of the plasmid-NLS conjugates in NIH 3T3 cells. The conjugates interacted specifically with the NLS-receptor importin R, but plasmid-NLS conjugates were not detected in the nucleus, by fluorescence microscopy, after cytoplasmic microinjection.

INTRODUCTION

Plasmids used for nonviral gene therapy are generally delivered as a complex with a variety of chemical or biochemical vectors to obtain maximal transfection efficiency. Among the various chemical vectors developed for gene transfer, cationic lipids are the most widely used (1). Nonviral gene transfer with cationic lipids can be divided into several steps (2): internalization of the DNA could occur via fusion at the cellular membrane or via endocytosis (3), then the plasmid diffuses to the nuclear enveloppe and enters the nucleus probably through the pore complexes. One of the steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus (2, 4-6). The role of signal sequences for protein import into the nucleus is well documented (7, 8). Proteins larger than 60 kDa are excluded from the nucleus unless they harbor a nuclear localization signal (NLS).1 The NLSs usually comprise highly basic stretches of amino acids (7). Among the different NLSs, the NLS of the simian virus 40 (SV40) large T antigen has been extensively studied. This short peptide possesses five basic amino acids and efficiently induces nuclear targeting when conjugated to a nonkaryophilic protein of molecular mass up to 465 kDa (9). Moreover, nuclear import is increased when several independent SV40 large T antigen NLS peptides are conjugated to a protein (10). Karyopherin R is involved in nuclear protein import through association with nuclear localization signals in the cytoplasm, then binding to the nuclear pore complex (11, 12). The 58 kDa mouse karyopherin R (called importin R or m-importin) binds to NLS sequence and then interacts with karyopherin β (also called importin β). The resulting complex binds to the nuclear pore (13) and is translocated through the pore in a mechanism involving the small GTPase Ran (14) and other proteins. * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 33 1 55 71 37 33. Fax: 33 1 55 71 37 96. † Rho ˆ ne-Poulenc Rorer Gencell. 1 Abbreviations: NLS, nuclear localization signal; SV40, Simian virus 40; TFPAM-6, N-(4-azido-2,3,5,6-tetrafluorobenzyl)-6-maleimidylhexanamide; GST, glutathione S-transferase.

We developed a new methodology to covalently associate NLS peptides to DNA in which cationic NLS peptides are covalently bound to plasmid DNA by photoactivation. We report here the synthesis and characterization of these conjugates and demonstrate that they specifically interact with the NLS-receptor importin R. EXPERIMENTAL PROCEDURES

Analytical and Semipreparative HPLC. Analytical and semipreparative HPLC were performed on a MerckHitachi gradient pump equipped with a AS-2000A autosampler, an L-6200A intelligent pump, and a UV-vis detector L-4000. Method 1. For analytical separations, we used a column C18 Vydac 218 TP54 (Interchim, Montluc¸ on, France) using a gradient of acetonitrile/water 0.1% TFA (from 0 to 41% acetonitrile in 25 min) with a flow rate of 1 mL/ min during 25 min. For semipreparative separation, we used a column C18 Vydac-218 TP 1022 (10 mm) using a gradient of acetonitrile/water 0.1% TFA (from 5 to 50% acetonitrile in 40 min) with a flow rate of 7 mL/min during 40 min. Detection was by UV absorbance at 254 nm. Method 2. The HPLC system used a 250 × 4.6 mm C8 Vydac column and a gradient of 5 to 50% acetonitrile over 40 min in 0.1 M triethylammonium acetate, pH 7.5 (flow rate ) 1 mL/min). Detection was by UV absorbance at 250 nm. Peptide Synthesis. The following peptides were synthesized: peptide βACGAGPKKKRKV containing wild-type SV40 large T NLS (NLS) or peptide βACGAGPKNKRKV containing mutant SV40 large T NLS (mNLS) or peptide CYGGPDEVKRKKKP containing SV40 large T NLS reverse sequence. Peptides were synthesized on Applied Biosystems 431A automatic synthesizer using a Rink amide resin as solid support and Fmoc strategy for amino acid assembly. A fluorescent molecular probe, Lissamine rhodamine B, was coupled manually in a separate flask at the N-terminal position of the NLS or mNLS peptides with a solution of dichloromethane containing 10 mol/equiv excess of Lissamine rhodamine B sulfonyl chloride (mixture of two isomers purchased from Aldrich), diisopropylethylamine (DIEA-pH 10), and

10.1021/bc980061a CCC: $18.00 © 1999 American Chemical Society Published on Web 12/17/1998

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a catalytic amount of dimethylaminopyridine (DMAP). The peptides were cleaved from the Rink resin as usual and purified by HPLC. Analytical and semipreparative HPLC were performed as described above (method 1). The final purified products were analyzed by mass spectroscopy. Lissamine-SO2-βACGAGPKKKRKVNH2 (HPLC, analytical, Rt ) 26.9 min; semipreparative, Rt ) 28-31 min; MH+, 1782); Lissamine-SO2-βACGAGPKNKRKVNH2 (HPLC, analytical, Rt ) 25 min; semipreparative, Rt ) 26-34 min; MH+, 1767). Preparation of p-Azido-tetrafluoro-benzyl-peptide Conjugates. Lyophilized N-(4-azido-2,3,5,6-tetrafluorobenzyl)-6-maleimidylhexanamide [TFPAM-6 (Molecular Probes, Eugene, OR)] was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL. Lyophilized peptides were dissolved in 100 mM triethylammonium acetate, pH 7.5, at a concentration of 1 mg/mL. Equimolar quantities of TFPAM-6 and peptide were mixed at room temperature. The reaction was monitored by HPLC, and, after 2 h, the conjugate was purified by HPLC as described above (method 2). The conjugate was analyzed by mass spectroscopy. p-Azido-tetrafluoro-benzyl-lissamine-SO2-βACGAGPKKKRKVNH2 (HPLC, Rt )36 min; MH+, 2195); p-azido-tetrafluoro-benzyl-lissamine-SO2-βACGAGPKNKRKVNH2 (HPLC, Rt ) 38.9 min; MH+, 2181); p-azido-tetrafluoro-benzyl-CYGGPDEVKRKKKP (HPLC, Rt ) 26 min; MH+, 2018). Covalent Coupling of Peptide Conjugates to dsDNA. Plasmid pCMVLacZ (7257 bp) carries a cassette containing the enhancer-promoter from the immediateearly gene of cytomegalovirus (CMV) and the gene coding β-galactosidase. Plasmid DNA was purified using Wizard Megaprep kit (Promega, Madison, WI). p-Azido-tetrafluorobenzyl-peptide conjugate was mixed with dsDNA (final concentration 0.3 µg/µL) at various molar excess depending on the assay. The mixture was cooled in an ice-water bath below a sun lamp (Rad Free UV lamp-365 nm, Schleicher and Schuell, Ecquevilly, France) and illuminated for 15 min in the dark. Excess p-azido-tetrafluorobenzyl-peptide conjugate was removed by ion-exchange chromatography (PCR Purification kit, Qiagen, Hilden, Germany). The average number of fluorescent peptides attached to the DNA was determined with a Titertek Fluoroskan II Spectrofluorimeter with free and illuminated p-azido-tetrafluoro-benzyl-peptide conjugate for calibration curve (excitation, 544 nm; emission, 590 nm). Preparation of Recombinant Importin r-GST Protein. Mouse importin R was cloned into pGEX-2T to produce a GST fusion protein: importin R-GST (11). The plasmid encoding the fusion protein was kindly provided by Dr. Y. Yoneda (Osaka University Medical School, Osaka, Japan). Importin R-GST was overexpressed in Escherichia coli strain BL21 grown in 1 L of LB medium, as previously described (11). Cells were harvested by centrifugation, washed with 100 mL of PBS and resuspended in 20 mL of buffer A [50 mM Tris-HCl, pH 8.3, 1 mM EDTA, 2 mM dithiotreitol, 0.5 M NaCl, and 200 µM Pefabloc (Boehringer, Manheim, Germany)]. Cells were disrupted by sonication at 4 °C during 30 min. The extract was clarified by centrifugation (15000g, 1 h). The recombinant protein was purified by affinity chromatography on glutathione-Sepharose 4B (Pharmacia, Uppsala, Sweden) equilibrated in buffer A. Glutathione-Sepharose 4B (8 mL) was cast in a HR10/10 column (Pharmacia, Uppsala, Sweden) connected to an HPLC system (Gilson 305). The clarified cell extract was loaded on the column at 0.5 mL/min. The column was washed with buffer B (20 mM HEPES-NaOH pH 7.3, 2 mM dithiotreitol, 1

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mg/L leupeptin, 1 mg/L pepstatin, and 1 mg/L aprotinin). The column was incubated 10 min in buffer B containing 20 mM glutathione, after which the recombinant protein was eluted at 0.5 mL/min. The protein fractions are desalted on disposable Sephadex G-25 M columns (PD10, Pharmacia, Uppsala, Sweeden) and eluted with 50 mM HEPES-NaOH, pH 7. Protein concentration was 0.6 mg/mL. The purity of the recombinant protein was assessed by denaturing polyacrylamide gel electrophoresis and Coomasie staining. Its identity was checked by Western blotting and probing with a polyclonal antiserum raised against the internal peptide: SEGYTFQVQDGAPGTFBF. The N-terminus of importin R-GST was also sequenced. Importin r Binding Assay. All assays were performed using importin R-GST recombinant protein. Binding to importin R was performed according to Rexach and Blobel (15), with the following modifications: all assays were performed in binding buffer (20 mM HEPES, pH 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, and 2 mM and 100 µg/mL bovine serum albumin). The GST fusion protein was incubated with glutathione-sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Importin R-GST fusion protein (1 µg per 10 µL of beads) was incubated with beads in 0.5 mL of binding buffer for 30 min at room temperature. The beads were collected by centrifugation at 2000g for 30 s and were washed five times with 0.5 mL of binding buffer. Washed beads were resuspended in binding buffer with 1 vol of buffer/vol of beads. The bead slurry (80 µL) was incubated with 2 µg of peptide-DNA conjugate, in 0.5 mL of binding buffer, for 30 min at room temperature. The beads were collected by centrifugation at 2000g for 30 s. The supernatant was removed and 30 µL was used to analyze the presence of the unbound DNA (this fraction is called the unbound fraction). The beads were washed five times with 0.5 mL of binding buffer and centrifugated at 2000g for 30 s. Finally, beads, corresponding to the bound fraction, were resuspended with 15 µL of electrophoresis loading buffer (0.05% bromophenol blue, 40% sucrose, 0.1 M EDTA pH 8, 0.5% sodium lauryl sulfate). The bound and unbound fractions were analyzed by electrophoresis on 0.8% agarose gel and DNA was stained with ethidium bromide for qualitative determination. Quantitative DNA evaluation in the unbound and bound fractions was obtained by resuspending beads in 160 µL of binding buffer and peptide-plasmid conjugate and dsDNA were measured according to the Picogreen ds DNA quantitation kit (Molecular Probes, Eugene, OR). Transgene Expression Studies. NIH 3T3 cells were obtained from the American Type Culture Collection (ATCC CRL-1658). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY) containing 4.5 g/L glucose, supplemented with 2 mM glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% foetal bovine serum. Cells were grown at 37 °C in a 5% CO2/air incubator. One day before the transfection assay, 24-well culture plates were seeded with 50 000 cells/well. Cationic lipid RPR 120535 (16) was used for the transfection assays. Plasmids were diluted in 150 mM NaCl at 20 ng/µL concentration, then mixed with a cationic lipid solution (0.12 mM). The DNA/cationic lipid complex was diluted in culture medium (Dulbecco’s modified Eagle’s medium) in the absence of serum and then added to the cells (0.5 µg of DNA and 3 nmol lipid/well). After 2 h at 37 °C in a 5% CO2/air incubator, 10% foetal bovine serum was added to the cells. The cells were incubated for 48 h at 37 °C in a 5% CO2/air incu-

Interaction of Plasmid−NLS Conjugates with Importin R

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Scheme 1: Synthesis of p-Azido-tetrafluoro-benzyl-peptide Conjugates. The Peptide Contains either the NLS Sequence (X ) K) or the Mutant Sequence (X ) N)

bator. The cells were washed twice with PBS and lysed with 250 µL of cell culture lysis reagent (Promega, Madison, WI). β-Galactosidase expression was measured according to the Lumigal β-Galactosidase genetic reporter system (Clontech, Palo Alto, CA). Light emission was measured by integration over 10 s using a Lumat LB9501 luminometer (EG and G, Berthold, Evry, France). Light emission was normalized to the protein concentration determined using the Pierce BCA assay (Pierce, Rockford, IL). Microinjection Studies. CV-1 cells were kindly provided by Dr. S. Koutouzov (INSERM U25, Paris, France). Cells were cultured in Minimum Essential medium (MEM, Gibco, Grand Island, NY) supplemented with 2 mM glutamine, 100 units/mL penicillin, 100 µg/ mL streptomycin, and 10% foetal bovine serum. Cells were grown at 37 °C in a 5% CO2/air incubator on CELLocate microgrid coverslips (Eppendorf, Hamburg, Germany). The solution of DNA was injected into the cells via glass micropipets having tip diameters ranging from 0.3 to 0.7 µm (Femtotips, Eppendorf, Hamburg, Germany) at 1 µg/µL concentration. Injections were carried out under visual control on a fixed stage of an inverted phase contrast microscope (Axiovert 135, Zeiss, Le Pecq, France) using a Micromanipulator 5171 and Microinjector 5242 (Eppendorf, Hamburg, Germany). The average volume (0.1-0.2 pL) injected into each cell was determined by injecting [35S]dATPRS into cells and measuring the radioactive content (6). These corresponded to an average of 6000-12000 plasmid molecules/cell. Following injection, the cells were incubated for 30 min to 6 h at 37 °C in a 5% CO2/air incubator. The cells were washed three times with PBS, fixed for 20 min in 3% paraformaldehyde in PBS, and washed three times with PBS. Nuclei were then stained with 4,6-diamidino-2-phenylindole (DAPI), at a concentration of 0.1 µg/mL, for 15 min, and washed with PBS. The cells were mounted in Mowiol for examination. Slides were examined with a Zeiss Axiophot fluorescence microscope, with a 100× objective lens, coupled to a cooled CCD camera (Hamamatsu) and a Samba imaging software (Unilog, Meylan, France). RESULTS

Preparation of p-Azido-tetrafluoro-benzyl-peptide Conjugates. Peptides containing wild-type SV40 large T NLS sequence (NLS) or mutant SV40 large T NLS (mNLS) were used. Introduction of a point mutation in the NLS sequence (asparagine instead of lysine) induces loss of NLS targeting activity (9). Scheme 1 illustrates the chemistry used to prepare p-azido-tetrafluoro-benzyl-peptide conjugates. The coupling step involves reaction of TFPAM-6 bearing a maleimide group

Figure 1. HPLC chromatograms of p-azido-tetrafluoro-benzylpeptide conjugates. HPLC profiles of starting material and conjugate obtained after 2 h of reaction are shown. The molecules were analyzed by reversed phase HPLC using method 2 described in the experimental procedures (absorption wavelength ) 250 nm). (A) TFPAM-6; (B) NLS peptide; (C) p-azidotetrafluoro-benzyl-NLS peptide conjugate after 2 h of incubation.

with a peptide bearing a nucleophilic thiol residue (17). Equimolar quantities of the two reagents were used. Since TFPAM-6 absorbs at 250 nm, the conjugation chemistry was easily monitored by reversed-phase HPLC. After 2 h, the reaction was complete as shown by the disappearance of TFPAM-6 and of the starting peptide (Figure 1). The final product was analyzed by mass spectroscopy, which confirmed the disappearance of starting reagents, and the experimental mass was similar to the calculated mass. The yield of this reaction was

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90%. The peptides used were labeled with a fluorescent molecule, lissamine rhodamine B. The conjugate had the same fluorescent properties as the starting peptide (data not shown). Covalent Attachment of Peptides to dsDNA. The p-azido-tetrafluoro-benzyl-peptide conjugates bearing either NLS or mNLS sequence were used to covalently modify plasmids by photoactivation. p-Azido-tetrafluorobenzyl-peptide conjugates were mixed with dsDNA at peptide/DNA molar ratios varying between 5 and 1000 and illuminated at 365 nm. After purification on ionexchange columns, the average number of peptide linked to the DNA could be measured by taking advantage of the peptide fluorescent labeling: by varying the p-azidotetrafluoro-benzyl-peptide/DNA molar ratio during the coupling step, conjugates bearing an average number of 1 peptide/plasmid molecule to 300 peptides/plasmid (7257 bp) were obtained. During the last coupling step, 20% of the added p-azido-tetrafluoro-benzyl-peptide conjugates were found covalently linked with plasmid DNA after purification. For example, with a molar excess (NLS peptide/plasmid) of 500, the average number of NLS peptides linked per plasmid DNA was 100 ( 28 (six experiments). Plasmids modified with NLS and mNLS peptides are referred to as plasmid-NLS and plasmidmNLS, respectively. Characterization of Peptide-Plasmid Conjugates. Peptide-plasmid conjugates were analyzed by agarose gel electrophoresis as described in the Experimental Procedures. With 1-100 peptides/plasmid DNA molecule, there was no visible modification of plasmid DNA migration, and peptide-plasmid conjugates appeared on gels as supercoiled DNA. With higher numbers of peptides per plasmid DNA molecule, plasmid DNA migration delay was observed. No delay was observed when plasmid DNA was mixed with nonilluminated NLS peptides (data not shown). The reporter gene expression was studied after transfection of NIH3T3 cells. Modified plasmids with 3-43 NLS peptides/plasmid DNA molecule expressed β-galactosidase. The increase of expression obtained with 3 NLS peptides/plasmid DNA is not significant. The expression was decreased by 60% with plasmids bearing 43 NLS peptides (Figure 2). Similar results were obtained with mNLS peptides (data not shown). Importin r Binding Assay. Binding of the modified plasmids to importin R was studied on a importin R-GST fusion protein, using glutathione-sepharose beads, followed by agarose gel electrophoresis. Plasmid-NLS was detected in the bound fraction (interacting with the importin R-GST-beads) whereas nonmodified plasmid was all recovered in the supernatant (Figure 3). The same result was obtained with plasmid-mNLS instead of nonmodified plasmid (data not shown). Double-stranded DNA in the fraction bound to importin R-GST was quantified using either the intrinsic fluorescence of the lissamine rhodamine B or, for higher sensitivity, using an intercalating fluorescent molecule, Picogreen reagent. Plasmid-NLS bound significantly more to importin R-GST beads than either plasmidmNLS, nonmodified plasmid, or plasmid conjugated to reverse NLS peptide (VKRKKKP) (Figure 4A and data not shown). Moreover, no specific binding to importin R-GST was detected using neither plasmid prepared by mixing with p-azido-tetrafluoro-benzyl-NLS peptide conjugates, without further UV irradiation, nor plasmid prepared by mixing with a p-azido-tetrafluoro-benzylNLS peptide that had been irradiated prior to contact with DNA (Figure 4B). Hence, the binding of plasmid-

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Figure 2. Transgene expression after transfection of plasmidNLS conjugates. Nonmodified plasmid and plasmid-NLS conjugates were used for transfecting NIH3T3 cells with cationic RPR 120535. The β-galactosidase expression was evaluated after 24 h of incubation. (1) Nonmodified plasmid; plasmid-NLS with the following NLS peptide/plasmid molar ratio: (2) 3; (3) 8; (4) 16; (5) 43; (6) no plasmid. Values are means ( SD (n ) 3).

Figure 3. Interaction between importin R-GST and plasmidNLS conjugates. Beads-immobilized importin R-GST (2 µg) was incubated for 30 min at room temperature with a plasmid conjugated to 66 NLS peptides (1 µg) (1 and 3) or nonmodified plasmid (1 µg) (2 and 4). Bound (1 and 2) and unbound (3 and 4) fractions were analyzed by 0.8% agarose gel electrophoresis and ethidium bromide staining. L ) molecular weight marker for linear DNA fragments (1 kb ladder). S ) molecular weight marker for supercoiled DNA fragments (1 kb ladder).

NLS to importin R is mediated by the specific interaction of nonmodified NLS peptide covalently bound to DNA and its receptor. By increasing the ratio of p-azido-tetrafluoro-benzylNLS peptide, 2-26 NLS peptides were covalently attached to dsDNA. Plasmid interaction with importin R-GST increased with increasing number of peptides per plasmid (Figure 4C). By increasing this number up to 45 and 100, the quantity of dsDNA interacting with 2 µg importin R-GST fusion protein was 30 and 60 ng, respectively (data not shown). Microinjection Studies. NLS-modified plasmids were microinjected into the cytoplasm of CV-1 cells. Plasmids with 100 NLS peptides/plasmid molecule were used. Cells

Interaction of Plasmid−NLS Conjugates with Importin R

Figure 4. Quantitative analysis of plasmid-NLS conjugates binding to importin R-GST. Immobilized importin R-GST (2 µg) was incubated for 30 min at room temperature with the different conjugates (1 µg). DNA in the bound fractions was measured as described in the Experimental Procedures. (A) Nonmodified plasmid (1); plasmid conjugated to 18 NLS peptides (2); plasmid conjugated to 26 mNLS peptides (3). (B) Nonmodified plasmid (1); plasmid conjugated to 18 NLS peptides (2); plasmid prepared with p-azido-tetrafluoro-benzyl-NLS peptide, without UV illumination (3); plasmid mixed with p-azido-tetrafluoro-benzylNLS peptide UV illuminated before coupling (4). (C) Nonmodified plasmid (1); plasmid-NLS conjugate with 2 NLS/plasmid (2); plasmid-NLS conjugate with 6 NLS/plasmid (3); plasmidNLS conjugate with 12 NLS/plasmid (4); plasmid-NLS conjugate with 26 NLS/plasmid (5). Data represent means of two determinations, with error bars indicating the maximal experimental value.

were fixed after 30 min, 2 or 6 h, and nuclei were stained with DAPI. An intense cytoplasmic fluorescence was observed 30 min after microinjection into the cytoplasm, indicating that DNA could only be detected in the cytoplasm (Figure 5A). Cytoplasmic microinjection of NLS-conjugated BSA in the same conditions gave a high nuclear staining (data not shown). The DNA fluorescence pattern evolved 2 h after microinjection into the cytoplasm (Figure 5C): half of the stained cells showed the same intense fluorescent pattern observed at 30 min and the rest exhibited a single fluorescent spot in the cytoplasm. After 6 h, nearly all cells had only one residual fluorescent spot in the cytoplasm (Figure 5E).

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Figure 5. Cytoplasmic microinjection of plasmid-NLS conjugate. CV-1 cells were cytoplasmically microinjected with plasmid-NLS (100 NLS peptides/plasmid DNA molecule) and fixed after 30 min (A and B), 2 h (C and D), or 6 h (E and F). Fluorescent microscopy of plasmid-NLS (A, C, and E) and nuclei staining with 4,6-diamidino-2-phenylindole (B, D, and F) are shown. DISCUSSION

Several methods for covalent attachment of molecules to nucleic acids have been developed and reported so far (18-20). Among them, the use of photoactive molecules, such as tetrafluorophenylazido groups, leads to an efficient photomodification of DNA (21). We have conjugated a cationic peptide, NLS from SV40 large T antigen, to a photoactive tetrafluorophenylazido-containing molecule. The NLS peptide had a sulfhydryl reactive group and the photoactive molecule, TFPAM-6, a maleimide group which reacted to give a photoactive NLS peptide. The peptide used had a fluorescent moiety, making it possible to follow the conjugation to plasmid DNA, to quantify the average number of peptides bound to DNA and to study the intracellular fate of the conjugates. Moreover, we found that the molecules labeled with lissamine rhodamine B are easier to purify and more chemically stable than those labeled with another fluorophore such as tetramethylrhodamine. Covalent coupling of ligands to the DNA could cause transcriptional inactivation. According to a study on the expression of a biotinylated plasmid prepared with a similar photomodification chemistry, a plasmid with up

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to 40 biotins coupled/plasmid retained 40% biological activity (19). Plasmids conjugated to NLS peptides by covalent coupling using a cyclopropapyrroloindole group were modified with 24-101 peptides/1 kb (22). With this number of peptides per plasmid, transgene expression was abolished. With the photoactive p-azido-tetrafluorobenzyl-peptide conjugates, lower levels of modification were obtained and the modified plasmids were still biologically active. With up to 43 peptides per plasmid DNA (7 kb), modified plasmids remained biologically functional. This result is in agreement with transgene expression data previously reported (19). It is also important to notice that plasmid-NLS conjugates appeared on gels as supercoiled DNA. Using a binding assay, we showed that NLS peptides attached to dsDNA interacted with the NLS-binding protein, importin R. This interaction was specific and depended on the number of peptides conjugated to dsDNA. Our results show that NLS peptides are recognized despite the plasmid electrostatic properties. Plasmids associated to NLS peptides by covalent coupling were studied in digitonin-permeabilized cells (22). Nuclear accumulation of the plasmid DNA was observed and was dependent on the number of NLS peptides: peptideplasmid conjugates bearing less than approximately 40 NLS/1 kb did not accumulate in the nucleus. In our experiments, we chose to couple a low number of peptides (less than 10 peptides/1 kb) in order to maintain a certain amount of transgene expression. Nuclear accumulation of plasmid DNA was also obtained in isolated male pronuclei with NLS peptides associated to plasmid DNA by electrostatic interactions (23).We demonstrate here that NLS peptides bound to DNA interact with their receptor: this recognition step is a prerequisite for import via the classical NLS-dependent pathway. However, after microinjection in the cytoplasm, plasmid-NLS conjugates were not detected in the nucleus. These results are in agreement with microinjection data previously reported (22). Our microscopic studies suggest that microinjected plasmid-NLS conjugates are progressively degraded in the cytoplasm, since the staining observed gradually decreased with time. After 6 h, only one spot of fluorescent plasmid-NLS was detected, which probably corresponded to the site of injection. This suggests that the bulk of microinjected plasmid did not readily diffuse from the site of injection. It is possible that part of plasmid-NLS molecules could be sequestered in the cytoplasm as observed for naked DNA injected in myotubes (5). In conclusion, we have designed a novel simple chemistry for coupling by photoactivation peptides to plasmid DNA. This technology allows the control of the number of peptides linked to DNA which is determinant for maintaining plasmid integrity and expression. Moreover, we show in the present work that a conjugate between a targeting NLS-peptide and plasmid-DNA is recognized by importin R NLS receptor. The present strategy could thus be used for coupling other ligands to plasmid DNA, study plasmid-ligand conjugate interaction with receptors. Since a fluorescent peptide could be coupled, the technology also allows to follow intracellular fate of the peptide-DNA conjugate. ACKNOWLEDGMENT

This work was supported by a grant from the French Ministry of Research. We thank Dr Franc¸ ois F. Clerc and his staff for peptide synthesis and Dr. Marc Vuilhorgne and his staff from the Structural Analysis Department

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