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ARTICLES Can Nuclear Localization Signals Enhance Nuclear Localization of Plasmid DNA? Takeshi Nagasaki,*,† Teruhiko Myohoji,† Taro Tachibana,† Shiroh Futaki,‡ and Seizo Tamagaki† Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, SORST (JST), Osaka, 558-8585, Japan, and Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Received September 9, 2002; Revised Manuscript Received November 22, 2002
Nonviral vectors are safer and more cost-effective than viral vectors but are significantly less efficient, and thus, increasing the efficiency of nonviral vectors remains an important objective. One way to overcome this problem is by stimulating the nuclear localization of exogenous genes. Nuclear localization signals (NLSs) are known to be involved in the active transport of exogenous proteins and probes into the nucleus. However, stimulation of nuclear localization of plasmid DNA has yet to be confirmed completely. In the present study, we prepared plasmid DNA-NLS peptide conjugates and adjusted spacer length and number introduced in an attempt to increase transfection efficiency. In comparison to conjugates with unmodified plasmid DNA and short spacers, we found that NLSplasmid DNA conjugates with covalent bonding by diazo coupling through PEG chain (MW 3400) stimulated complexation with the nuclear transport proteins importin R and importin β. Evaluation of transfection showed higher expression efficiency with plasmid DNA-NLS peptide conjugates than with unmodified plasmids. However, evaluation of intracellular trafficking after microinjection into the cytoplasm showed plasmid DNA-NLS peptide conjugates only within the cytoplasm; there was no NLS-plasmid stimulation of nuclear localization. Our findings suggest that stimulation of plasmid nuclear localization cannot be achieved merely by changing spacer length or chemically modifying plasmid DNA-NLS peptide conjugates. An additional mechanism must be involved.
INTRODUCTION
Gene therapy has been seriously investigated in the United States as a clinical treatment for genetic diseases such as adenosine deaminase (ADA) deficiency since 1990. Numerous studies have subsequently investigated the use of gene therapy for treatment of cancer, infections (such as AIDS), and other lifestyle-related diseases. The key to efficient transfection and expression of exogenous genes into cells is the use of “vectors” (gene carriers). Retrovirus and adenovirus vectors have conventionally been used because of their high efficiency. However, reports of a patient death due to an adenovirus vector in the United States in 1999 led to renewed interest in developing nonviral vectors to further increase safety (1). The increasing use of gene therapy in clinical practice also makes the development of nonviral vectors important with regard to cost and availability. Nonviral vectors can broadly be divided into lipoplexes (lipid/DNA complexes) and polyplexes (polymer/DNA complexes) (2). However, the transfection and expression efficiencies of these nonviral vectors are much lower than those of viral vectors. The low efficiency of these vectors is also the most significant problem when they are used * To whom correspondence should be addressed. Phone: 81 6 6605 2696. Fax: 81 6 6605 2785. E-mail: nagasaki@ bioa.eng.osaka-cu.ac.jp. † Osaka City University. ‡ Kyoto University.
for gene therapy. This necessitates research and development of nonviral vectors with higher efficiency by using nanoparticles consisting of complexes of nucleic acids and polycations such as dendrimer polymers or lipid aggregates (liposomes). Nonviral vectors must overcome several intracellular barriers before mRNA transcription (3-5). The greatest barrier facing both polyplexes and lipoplexes is the nuclear membrane (6-9). Nuclear localization efficiency significantly influences expression efficiency. Recent studies have shown that intranuclear transport of large molecules such as proteins and nucleic acids is a selective process through nuclear pore complexes that involves energy dependent carrier proteins (10-15). Several investigations have focused on the nuclearcytoplasm transport system used by biological organisms as a means of stimulating nuclear localization of exogenous genes (16, 17). Some studies have reported dramatic increases in expression efficiency using NLS modified linear DNA (18, 19), but similar results with plasmid DNA have not been reported to date (20-22). The clinical application of gene therapy requires the development of highly efficient plasmid DNA delivery systems that can be synthesized both easily and in large quantities. The availability of plasmid DNA-NLS peptide conjugates providing reliable intranuclear transport would be of enormous benefit. In this regard, we must first consider why previous research has failed to achieve these goals. In plasmid DNA-NLS peptide conjugates
10.1021/bc025602h CCC: $25.00 © 2003 American Chemical Society Published on Web 01/22/2003
Can NLS Enhance Nuclear Localization of Plasmid?
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Scheme 1. Syntheses of Plasmid DNA-NLS Peptide Conjugatesa
a
The Spacer (X) Contains Either the Long PEG or Short Pentamethylene Chain.
designed to date (20-22), suboptimal spacer lengths between the cationic NLS and anionic DNA have resulted in a strong interaction between the NLS and plasmid and burying of the NLS within the negative charges. This interferes with recognition of transport proteins. Furthermore, the localization of large plasmid molecules may require insertion of multiple NLSs, whereas overfull chemical modification cause the inhibition of the transcriptional process. Therefore, our research has focused on developing a convenient method to produce plasmid DNA-NLS peptide conjugates with a more optimum spacer length and where the number of insertions can be regulated. The objective is the development of a highly efficient nonviral gene delivery system. EXPERIMENTAL METHODS
Materials. 5(and 6)-Carboxyltetramethylrhodamine N-hydroxysuccinimide ester was purchased from Molecular Probes (Eugene, OR). N-(6-Maleimidocaproyloxy)succinimide was purchased from Dojindo Lab. (Kumamoto, Japan). Maleimido-peg-carboxylic acid N-hydroxysuccinimide1 ester was purchased from Shearwaer (Huntsville, AL). Di-tert-Butyl dicarbonate and N-(9-fluorenylmethyloxy-carbonyloxy)succinimide were purchased from Peptide Lab. (Osaka, Japan). Recombinant GST fusion Importin R and importin β were prepared as reported previously (12). Lipofectin was purchased from Invitrogen Corp. (Carlsbad, CA). Other reagents and solvents were purchased from Aldrich. Plasmids. pGL3-control (pGL3) was purchased from Promega (Madison, WI). pEGFP-N1 (pGFP) was purchased from Clontech (Palo Alto, CA). Plasmid DNAs were amplified in Escherichia coli and purified by EndoFree Plasmid Kit (Qiagen, Hilden, Germany). Rhodamine-Labeled NLS Peptide. The NLS peptide, from SV-40 large T antigen, used in this study was manually synthesized by the Fmoc solid-phase method on a Rink amide resin as reported previously (23). After the elongation of the NLS sequence PKKKRKVDEPYC, 4-amino butyric acid was introduced as a spacer between the NLS and a fluorescent residue. Finally, the amino 1 Abbreviations: PEG, poly(ethylene glycol); GST, glutathione S-transferase; GFP, green fluorescent protein; SV, simian virus; Fmoc, 9-fluorenylmethoxycarbonyl; TFA, trifluoroacetic acid; RP-HPLC, reversed-phase high performance liquid chromatography; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate buffered saline; DAPI, 4′,6diamidino-2-phenylindole.
group was labeled with 5(and 6)-carboxyltetramethylrhodamine succinimidyl ester. The protecting groups and the resin were removed with TFA in the presence of H2O (10%), phenol (5%), ethandithiol (5%), and thioanisole (5%) at room temperature for 3 h. After addition of diethyl ether, the crude product was precipitated and then purified by RP-HPLC. Preparation of Plasmid DNA-NLS Peptide Conjugates. Diazocoupling (24-26) and photoactiviation (27-32) have been reported as the covalent modifications of DNA. In this study plasmid DNA-NLS peptide conjugates were prepared by the diazo coupling method according with Scheme 1. Synthesized conjugates were purified with ultrafiltration (Centricon Plus-10; nominal molecular weight limit ) 10 kDa, Millipore, Bedford, MA). The number of NLS linked to one plasmid was determined by absorbance at 260 nm and fluorescent intensity (ex ) 546 nm, em ) 576 nm). The fluorescence polarization of rhodamine linked to the NLS was measured by BEACON 2000 spectrometer (Pan Vera, Madison, WI) in order to estimate the type of binding of the NLS to plasmid. PEGylated NLS (NLS-peg) prepared with the NLS peptide and PEG-maleimide (Mw. 5000, Shearwater, Huntsville, AL) was also examined for the fluorescence polarization method as a comparison. Importin Binding Assay. Binding to importin R in the presence of importin β was performed according to Wils et al. (22), with the following method: all assays were performed in binding buffer (20 mM HEPES, pH 7.0, 110 mM potassium acetate, 2 mM magnesium acetate, and 0.1 mg/mL bovine serum albumin). The GST fusion importin R with and without importin β (1 mg per 10 mL of beads, respectively) was incubated with 0.25 mL of glutathione-sepharose beads (Amasham, Piscataway, NJ) in binding buffer (0.25 mL) for 30 min at room temperature. The beads were collected by centrifugation at 2000g for 30 s and washed five times with binding buffer (0.25 mL). The beads slurry (80 µL) was incubated with 2 µg of plasmid DNA-NLS peptide conjugate 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 unbound DNA (this fraction is called the unbound fraction). The beads were washed five times with 0.25 mL of binding buffer and centrifuged at 2000g for 30 s. Finally, beads were resuspended with 50 µL of the dissociation solution (0.1 M EDTA, 10% sodium lauryl sulfate) and vortexed deliberately at roobm temperature
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Table 1. Synthesized NLS-Plasmid DNA Conjugates
conjugate
used plasmid
2.8 NLS-peg-pGFP 3.8 NLS-peg-pGFP 1.9 NLS-C5-pGFP 2.4 NLS-peg-pGL3 5.1 NLS-peg-pGL3 1.7 NLS-C5-pGL3
pEGFP-N1 pEGFP-N1 pEGFP-N1 pGL3-control pGL3-control pGL3-control
ratio of reactants diazonium salt/ no. of plasmid (mol/mol) insertions 3.0 10 3.0 3.0 15 3.0
2.8 3.8 1.9 2.4 5.1 1.7
for 1 h. After centrifugation at 2000g for 30 s, the supernatant was removed as the bound fraction. The bound and unbound fractions were analyzed by electrophoresis on 0.6% agarose gel, and DNA was stained with ethidium bromide. Transfection. COS-1 cells (3-4 × 104 cells for an each well) were grown to just before confluence in a 24-wells culture plates in DMEM with 10% FBS and 100 U/mL penicillin and 100 µg/mL streptomycin in an atmosphere of 5% CO2 at 37 °C and washed twice with 0.5 mL of PBS. Complexation and transfection were performed as described in the protocol of Lipofectin with 1 µg of plasmid DNA (pGL3-control)-NLS peptide conjugate for each well. Luciferase assays were performed as described in the protocol of Steady-Glo Luciferase Assay System (Promega, Madison, WI). Luciferase relative light units (RLU) were analyzed by luminometer (Fluoroskan AscentFL, Thermo Labsystems, Finland). The protein concentrations of the cell lysates were measured as described in the protocol of NanoOrange Protein Quantitation Kit (Molecular Probes, Eugene, OR) using bovine serum albumin as a standard. The light unit values shown in the table represent the specific luciferase activity (RLU/ mg protein) which is standardized for total protein content of the cell lysate. The measurement of transfection efficiency was performed in triplicate. Microinjection. COS-7 cells were grown at 37 °C in a 5% CO2 incubator on coverslips. The solutions of 1 mg/ mL plasmid DNA-NLS peptide conjugates were injected into the cytoplasm via glass micropipets. Injections were carried out under visual control on a fixed stage of an inverted phase contrast microscope (Diaphoto, Nikon, Tokyo, Japan) using a micromanupilator MMO-220N and a microinjector IM-16 (Narishige, Tokyo, Japan). Following injection, the cells were incubated for 20 min to 6 h at 37 °C in a 5% CO2 incubator. The cells were then washed with PBS, fixed with 5% HCHO, and examined by fluorescence microscopy. Images of the samples were collected by fluorescence microscopy on an Olympus IX70 (Tokyo, Japan) attaching a confocal scan unit CSU10 (Yokogawa, Tokyo, Japan) with ORCA-ER CCD Camera (Hamamatsu Photonics, Hamamatsu, Japan) using a 40 × Uapo/340 objective with NA 0.9. RESULT
Preparation of Plasmid DNA-NLS Conjugates. For synthesis of the conjugates, we used a C5 alkyl chain as a short spacer and poly(ethylene glycol) (PEG; 71 units, 3400 Da) as a long spacer. We also adjusted the equivalents of diazonium salt reacted per molecule of plasmid. Table 1 lists the synthesized plasmid DNANLS peptide conjugates with their spacers and number of insertions. The number introduced could easily be adjusted by changing the ratio of reactants. Appended NLSs were limited to a maximum of five introductions to prevent decreased expression due to interference with transcription.
Figure 1. Measurement of fluorescence polarization of plasmid DNA-NLS peptide conjugates in the absence (gray bar) and presence (white bar) of excess PEI. To estimate the type of binding of the NLS to plasmid, the fluorescence polarization (ex ) 546 nm, em ) 576 nm) of rhodamine linked to the NLS was measured in 10 mM Tris-HCl buffer (pH 7.5) at room temperature.
Characterization of Conjugates. The introduced NLSs were basic peptides, and thus, in addition to conjugation by covalent bonding, it was also possible for complexes to be formed by noncovalent bonding through polyionic complexation (33). To confirm the type of bonding, we measured fluorescence polarization of rhodamine linked to the NLS upon addition of PEI (polyethylenimine), which has a higher affinity for plasmid DNA. Figure 1 shows the fluorescence polarization in the presence and absence of PEI. The parameter utilized in this method is the fluorescence polarization value (P). The P value, being a ratio of light intensities, is a dimensionless number. Polarization values are also expressed in millipolarization units (1 polarization unit ) 1000 mp units). The P value was greatest for rhodamine-linked 1.9 NLS-C5-pGFP. The difference of the P value between 1.9 NLS-C5-pGFP and 2.8 NLSpeg-pGFP was nonsignificantly small. Even in the presence of PEI, there was almost no change in fluorescence polarization of these NLS-conjugated plasmids (decrease of
