OnOff Switching of Gene Expression Regulated with

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On-Off Switching of Gene Expression Regulated with Carbohydrate-Lectin Interaction Kazunori Matsuura,*,‡ Katsuhiro Hayashi,† and Kazukiyo Kobayashi† Department of Molecular Design, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan, and Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan Received April 11, 2005; Revised Manuscript Received June 24, 2005

A novel strategy for artificial regulation system of gene expression applying the specific molecular recognition between carbohydrate and lectin is proposed. Plasmid-lactose conjugates (pActin-lactose and pGFPlactose) prepared via diazocoupling maintained the transcription activity with T7 RNA polymerase. Gelshift assay showed that the pActin-lactose conjugates were specifically complexed with galactose-specific lectin RCA120 with a strong binding affinity (Ka ) 7.6 × 105 M-1 per Lac-unit). The complexes were observed to form aggregates of sub-several micrometer size by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM). The activities of transcription and expression of the conjugates were evaluated, respectively, on the basis of the amount of transcript of pActin and the fluorescent intensity of the expressed GFP. These activities were repressed in the presence of an increasing concentration of RCA120, and then recovered by adding lactose, lactosylceramide-containing liposomes, and lactose-carrying polymers to the conjugate-RCA120 complex. Gel-shift assay and TEM observation revealed that the aggregation form of the complex was relaxed partially in the presence of the lactose derivatives, which increased the accessibility of T7 RNA polymerase to result in the recovery of transcription activity. Introduction Cells have smart devices to regulate the level of specific gene expressions responding to the environments by controlling the binding of proteins to DNA in both eukaryotes and prokaryotes.1 In eukaryotes, DNA is packaged in a nucleus as chromatin by incorporation with histone proteins, in which the gene expression is usually repressed. When the chromatin structure is modified by some reaction and/or interaction, for example, acetylation of histones, the gene is expressed, which is known as a chromatin remodeling. It is also known that gene expression can be silenced by methylation of DNA in mammalian cells. Artificial regulation of gene expression is an important subject for post-genome researches, especially for various medical applications. Several molecular systems for artificial regulation of gene expression have been devised.2-9 Dervan et al. reported repression and activation of specific gene expression with pyrrole-imidazole polyamides that are sequence-selective groove binders.2 Interaction of DNA with aminoglycosides3 and cationic gold nanoparticles4 could regulate gene expression. Recently, Asanuma et al. reported photoresponsible artificial regulation of transcription using azobenzene-modified promoter.5 Katayama et al. reported signal-responsive artificial gene regulation systems using polymers carrying oligopeptides, which are substrates for cyclic AMP-dependent protein kinase and caspase-3.6 * To whom correspondence should be addressed. Phone: +81 92 642 3598. Fax: +81 92 642 2011. E-mail: [email protected]. † Nagoya University. ‡ Kyushu University.

Glycosylated DNAs occur in T-even phages,10 Trypanosoma brusei,11 and so on. These glycosylated DNAs may be involved in the protection of the DNAs in the parasites from nucleases in infected host cells12 and also in the regulation of gene expression,13 although their detailed biological roles remain an intriguing question to be answered. Inspired by the interesting structures of these glycosylated DNAs, we14 and other groups15 have investigated the synthesis and functions of various types of artificial DNAcarbohydrate conjugates. In the course of the study, we have found that a plasmid-lactose conjugate prepared via diazocoupling formed a complex with the galactose-specific lectin, RCA120.14c In addition, the conjugate was recognized as a template chain by T7 RNA polymerase to afford the transcript.14c Hence, it is very interesting that the conjugate retained the transcription activity, despite the existence of nucleobases modified with the large lactose substituent. Among other examples, DNAs modified with aminofluorene and pyrene derivatives were reported to retain the transcription activity.16 On the basis of these results, we have proposed a novel strategy for on-off regulation of gene expression applying the specific recognition of the plasmid-lactose conjugate to lectin, as shown in Figure 1.17 When the plasmid-lactose conjugate was complexed strongly with RCA120 lectin, the access of T7-RNA polymerase to the template DNA was hindered and the transcription of DNA was repressed. When an excess amount of lactose was added to the complex, the binding between the conjugate and RCA120 was relaxed or dissociated and the RNA polymerase became accessible to

10.1021/bm050255a CCC: $30.25 © 2005 American Chemical Society Published on Web 08/05/2005

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Figure 1. Schematic illustration of model system for regulation of gene expression utilizing carbohydrate-lectin interaction.

the DNA. As a result, the transcription of DNA was recovered. A similar strategy for artificial regulation of transcription was recently reported by Murata and coworkers.18 The transcription of FITC-labeled plasmid was repressed by anti-FITC monoclonal antibody, although the recovery of transcription was not shown. This paper describes a detailed study of the on-off regulation of gene expression of pTRI-β-Actin-mouse vector plasmid (abbreviated as pActin) and pQBI T7 spGFP vector plasmid (abbreviated as pGFP). The structure and binding affinity of the complex between the pActin-lactose conjugate and the specific RCA120 lectin are evaluated by AFM, TEM, and gel-shift assay. In vitro transcription of pActin and expression of pGFP was evaluated, and discussion is made on the structure of the complexes and the activity of gene expression. Experimental Section General. UV-vis spectra were taken on a JASCO V-530 UV/vis spectrophotometer. Fluorescence spectra were recorded on a JASCO FP-777 spectrophotometer equipped with a thermal controller. Agarose gel electrophoresis was performed on a submerged gel electrophoresis apparatus AE6111 (Atto Co., Ltd.) with 1% agarose in 0.5 × TBE buffer (45 mM Tris, 45 mM boric acid, and 1mM EDTA) at 25 °C at 4.0 V/cm. Polyacrylamide gel electrophoresis was performed on a mini-slab type electrophoresis apparatus AE6520P (Atto Co., Ltd.) with PAGEL AE-6000 SPU-R15S (Atto Co., Ltd.) gel in 1 × TBE buffer at 25 °C at 4.0 V/cm. DNA and RNA bands were stained in a 6 µg/mL ethidium bromide solution or SYBR Gold Nucleic acid gel stains, visualized by transilluminator AB-1500 (Atto Co., Ltd.) with ultraviolet light (310 nm) equipped with a Kodak Digital Science Electrophoresis Documentation and Analysis System, and analyzed with Lane and Spot Analyzer ver. 6.0 (Atto Co., Ltd.). Surface plasmon resonance (SPR) was carried out with a SPR670 (Nippon Laser and Electronics Lab.). Atomic force microscopy (AFM) was conducted on a SPM-9500J2 (Shimadzu Co. Ltd.) instrument. Transmission electron microscopy (TEM) was conducted on a JEOL JEM1200 instrument by the negative staining method. Dynamic light scattering (DLS) was carried out with an Otsuka Electronics DLS-6600HK using vertically polarized light of 488 nm from an Ar ion laser as an incident beam. Materials. Diethylpyrocarbonate (DEPC)-treated Milli-Q water (Millipore) and RNase-free reagents were used to avoid

Chart 1. Structures of PVLac and PNLac

the contamination of RNases through the experiments. Plasmid DNAs, pTRI-β-Actin-mouse (3320 bp, GC content ) 50.7%, linearized for runoff transcription), and pQBI T7 spGFP vector (5115 bp, GC content ) 50.4%) were respectively purchased from Ambion Inc. and Biotechnologies Inc. Lamda DNA-Bst E II Digest marker was purchased from New England Biolabs Inc.; RNA Century Marker template set, MAXIscript in vitro transcription kits, and PROTEINscript-Pro T7 in vitro expression kits were from Ambion Inc.; SYBR Gold Nucleic acid gel stains were from Molecular Probes, Inc.; and Ricinus communis agglutinin (RCA120), concanavalin A (ConA, from ConaValia ensiformis), lactocerebrosides (LacCer), and dipalmitoyl-D-R-phosphatidylcholine (DPPC, C16:0) were from Sigma Co, Ltd. PVLac (poly(N-vinylbenzyl-lactonolactonamide), M h n ) 4.4 × 104, M h w/M h n ) 3.3),19 PNLac (poly(p-vinylbenzamido-βlactose, M h n ) 2.0 × 104, M h w/M h n ) 1.8),20 and N-(ω-(paminobenzamido)hexyloxy)-lactosylamine (1)14b (Chart 1) were synthesized according to the previously described procedure. Gel loading buffer was prepared by dissolving 1 wt % bromophenol blue, 1 wt % xylene cyanol FF, 0.5 M EDTA, and 40 wt % sucrose in deionized water. Typical Procedure for Preparation of Plasmid-Lactose Conjugates. Plasmid-Lac conjugates were prepared by improving the previously described procedure.14c To an aqueous solution of N-(ω-(p-aminobenzamido)hexyloxy)lactosylamine (1) (4.5 mg, 7.8 µmol) in 1 M HCl (81 µL) was added an aqueous solution of NaNO2 (81 µL of 7.7 mg/ mL) at 0 °C. The mixture was incubated at 0 °C for 5 min and then quenched with 1 M NaOH aq (70 µL) to afford the diazonium salt. To the solution were added an aqueous solution of plasmid DNA (pTRI-β-Actin-mouse or pQBI T7 spGFP vector) (20 µg in 40 µL water) and borate buffer (0.2 M, pH 9, 95 µL). The mixture was then incubated at 25 °C for 120 min and quenched with 3 M AcONa (35 µL). The product was purified by centrifugal filtration at 4000g for 15 min (five times) using Microcon-PCR (Millipore Co., Ltd.) and was lyophilized from the filtrate to provide plasmid-Lac conjugate as a yellowish fiber.

On-Off Switching of Gene Expression

These plasmid-lactose conjugates are coded as pActinLac-n and pGFP-Lac-n, where pActin and pGFP stand for pTRI-β-Actin-mouse vector and pQBI T7 spGFP vector, respectively, and n stands for the lactose content (%) in a nucleobase. Gel-Shift Assay. A mixture (10 µL) of pActin-Lac-8.2 (49.8 nM, [base] ) 331 µM) and RCA120 in PBS (10 mM phosphate buffer saline solution, pH 7.4) was incubated at 25 °C for 10 min (for Con A, in PBS containing 1 mM Mn2+ and Ca2+). After addition of gel loading buffer (2 µL), the mixture was loaded onto a 1% agarose gel, and then electrophoresis was carried out in 0.5 × TBE buffer at 25 °C at 4.0 V/cm. Preparation of Lactoside-Carrying Liposome. Each solution of DPPC, cholesterol, and LacCer in chloroform/ methanol (2/1 v/v) was mixed in a flask to a mole ratio of DPPC:Chol:LacCer ) 1:1:0.1 (total lipid ) 10 µmol). A homogeneous thin film of the lipids was obtained by evaporating volatiles. After addition of TBE buffer (0.5 mL), the flask was heated to 50 °C for 10 min, and then sonicated in a bath-type sonicator to obtain liposome. The size of liposome was estimated with DLS to be 209 ( 27 nm. In Vitro Transcription of pActin-Lactose in the Absence and Presence of Lectin and Lactose Derivatives. Transcription of plasmids was carried out with a MAXIscript transcription kit (Ambion Inc.). pActin-Lac-12.2 (0.5 µg, 2.5 pmol (16.5 nmol-base)) and 1 mM NTPs (AMP, GMP, CMP, UMP) were incubated in transcription buffer at 25 °C for 10 min in the absence and presence of lectin (0-5 µM). An aqueous solution of carbohydrates (0-1000 µM of lactose, cellobiose, PVLac, PNLac, and lactose-carrying liposome) was then added to the transcription solution. The solutions were incubated at 25 °C for 10 min, and then T7 RNA polymerase (15 unit) was added to the solution (total 10 µL). The mixture was incubated at 25 °C for 2 h and then quenched by heating to 95 °C. Production of mRNA was monitored on polyacrylamide gel electrophoresis (Atto, PAGEL). The amount of transcription was normalized for that of the native plasmid as 100%. The data were reproducible within 10% error. In Vitro Expression of pGFP-Lactose in the Absence and Presence of Lectin and Lactose Derivatives. Expression of plasmids was carried out with a PROTEINscriptPro T7 kit (Ambion Inc.). A solution of pGFP-Lac-8.7 (0.5 µg, 3.8 pmol (38.9 nmol-base)), 1 × Master Mix minus amino acids, 0.1 mM amino acids (-Met), 0.1 mM amino acids (-Leu), 30 µM methionine, and E. coli S30 extract was incubated in the absence and presence of lectins (05.4 µM) at 25 °C for 10 min. Each aqueous solution of carbohydrates (0-1000 µM of lactose, cellobiose, PVLac, PNLac, and lactose-carrying liposome) was added to the solution. The solution was incubated at 25 °C for 10 min, and then T7 RNA polymerase (15 unit) was added to the solution (total 50 µM). The expression reaction was carried out at 37 °C for 1 h and quenched at 0 °C. The mixture was diluted with 4 volumes of PBS, and then the fluorescence spectra of expressed sgGFP excited at 474 nm were recorded at 37 °C. The data were reproducible within 10% error.

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Figure 2. Preparation of plasmid-Lac conjugates via diazocoupling.

Transmission Electron Microscopy. Each solution of unmodified plasmid and pActin-Lac-8.2 (249 nM, [base] ) 1.65 mM) was incubated with 3.4 µM RCA120 in water at 25 °C for 10 min and diluted with 5 volumes of water. An aliquot (15 µL) of the solution was then dropped on a carboncoated TEM grid. The specimen was post-stained with 1% aqueous uranyl acetate. Atomic Force Microscopy. Each solution of unmodified plasmid and pActin-Lac-9.7 (249 nM, [base] ) 1.65 mM) was incubated with 3.4 µM RCA120 in water at 25 °C for 10 min. The solution was diluted with 100 volumes of water. An aliquot (10 µL) of the solution was mounted on a mica substrate pretreated with 3-aminopropyltrimethylsilane. The substrate was incubated for 10 min, rinsed with Milli-Q water, and dried under reduced pressure for 20 h. The surface was imaged under ambient pressure at room temperature by dynamic mode AFM. Results Preparation of Plasmid-Lactose Conjugates. Figure 2 illustrates the synthetic route of the conjugates of plasmid DNAs (pTRI-β-Actin-mouse and pQBI T7 spGFP vector) with lactose according to our previous reports.14b,c The diazotization of the lactose derivative 1 with HCl/NaNO2 was quenched with 1 M NaOH aqueous solution, and the diazocoupling of the product 2 to the plasmid DNA was carried out in basic borate buffer (pH9).21 The conjugates were purified through centrifugal ultrafiltration using Microcon-PCR, followed by lyophilization, and obtained as yellowish fibers in high yields. The degree of substitution (DS) of the lactose derivative in plasmid nucleobase was determined by calibrating the absorbances at 350 nm (diazo) and at 260 nm (nucleobase), as shown in Figure 3.14c The conjugates with DS ) 8-14% were obtained in 60-100%

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Figure 3. UV spectra of plasmid-Lac conjugates at [base] ) 250 µM in water at 25 °C.

yields. Both yield and DS became higher as compared to the previously reported conjugate (yield, 48%; DS, 2.5% for pTRI-β-Actin-mouse plasmid) that had been purified through ethanol precipitation and anion exchange chromatography. The increase in yield and DS is probably due to the improved purification method of the products. Binding Affinity of pActin-Lactose Conjugates to RCA120 Lectin. Gel-shift assay of the conjugate was carried out in the absence and presence of galactose-specific RCA120 lectin to estimate quantitatively the affinity of the conjugate to the specific lectin (Figure 4A). The electrophoretic mobility of the pActin-Lac-8.2 conjugate was almost the same as that of unmodified plasmid, indicating that the modification little affected the character of plasmid as an anionic polymer. When pActin-Lac-8.2 conjugate was incubated with RCA120, a less-mobile band appeared at the origin, indicating that the aggregation via the multiple plasmid-lectin interaction was too large to migrate through the pores of 1% agarose gel. On the other hand, such a band was not observed when unmodified pActin was mixed with RCA120 and also when pActin-Lac-8.2 was mixed with R-mannnose/R-glucose specific concanavalin A (Con A) lectin. These results suggest that pActin-Lac-8.2 was specifically recognized with the β-galactose-specific lectin. The relative intensity of the bands of plasmids quantified with a densitometer was normalized for the absence of lectins and plotted against the concentration of lectins (Figure 4B). The saturation curve was converted to a linear relationship (Figure 4C) according to eq 1, [RCA120]/∆I ) [RCA120]/∆Imax + 1/∆ImaxKa

(1)

where ∆I stands for the change of the normalized relative intensity of the bands of plasmids. Apparent affinity constant Ka of pActin-Lac-8.2 to RCA120 was calculated from the intercept and slope to be Ka ) 7.6 × 105 M-1 per Lac unit.22 Because this value is comparable to the affinity constant between the lactose-carrying polymer and RCA120,14f we can conclude that the conjugate is strongly bound to the lectin by the glycocluster effect.23

Figure 4. Gel-shift assay for interaction between plasmid-Lac conjugate and lectins. (A) Agarose gel (1%) electrophoresis of pActin and pActin-Lac-8.2 conjugate in the presence or absence of lectins. (B) Change of relative band intensity of free plasmids depending on the concentration of RCA120. (C) Linearized plot according to eq 1 for interaction of pActin-Lac-8.2 with RCA120. The interaction condition: [plasmid] ) 49.8 nM ([base] ) 331 µM) in water at 25 °C.

Microscopic Observation of the Complex between pActin-Lactose Conjugate and RCA120 Lectin. Figure 5A and B compares the respective AFM images of a mixture between pActin-Lac-8.2 and RCA120 and a mixture between unmodified pActin and RCA120 on 3-aminopropyltrimethylsilane-treated mica substrate. There appeared an object of about 0.5-1.0 µm diameter and about 30-50 nm high


Figure 5. Observation of complex of plasmid-Lac conjugate with RCA120: (A) AFM image of pActin-Lac-9.7 with RCA120; (B) AFM image of unmodified pActin with RCA120; (C) TEM image of pActinLac-9.7 with RCA120. The complex solutions ([plasmid] ) 249 nM and [RCA120] ) 3.4 µM in water) were diluted by 100 times for AFM and by 5 times for TEM with water.

(Figure 5A), which is quite different from the individual images of the unmodified pActin and RCA120 (Figure 5B). As shown in Figure 5C, a mixture of pActin-Lac-8.2 and RCA120 gave a TEM image of disordered aggregates with several-micrometers size, where dark indistinct or amorphous objects cover wholly around bright fibrous objects. Because pActin-Lac-8.2 was estimated to have about 540 lactose residues per one molecule, these AFM and TEM images suggest that the conjugate molecules may be cross-linked with the binding sites of RCA120 to afford huge aggregates.


Regulation of in Vitro Transcription of pActin-Lactose with Lectin and Multivalent Lactose Derivative. Transcription of pActin-Lac was carried out with phage-derived robust T7 RNA polymerase and NTPs at 25 °C for 2 h in the absence and presence of lectins using commercial in vitro transcription kits in test tubes (Figure 6A). The amount of transcription was monitored on denatured PAGE with a densitometer and normalized for that of unmodified plasmid as 100%. As shown in Figure 6B, the transcription level of the pActin-Lac-12.2 conjugate was found to be 75% that of the unmodified pActin vector. This high transcription level is worthy of remark because several crucial G bases in T7 promoter region are known to be sensitive to chemical modification.5,9 The probability of modifying the T7 promoter region with at least one lactose residue can be estimated to be about 99%. It suggests that the modification little disturbed the initiation of transcription. The transcription level was decreased with an increase of RCA120 concentration, whereas little decrease of the transcription level was observed for the combinations between the conjugate and nonspecific Con A and also between the unmodified pActin and RCA120. Thus, the specific interaction of RCA120 to the lactose residues along the DNA strand repressed the transcription of the pActin-Lac-12.2. As shown in Figure 6C, addition of various lactose derivatives increased the transcription level of the complex of the conjugate (249 nM) with RCA120 (2.40 µM), whereas cellobiose minimally affected the transcription level. More effective recovery of the transcription level was achieved with lactose-carrying polymers (62% for PVLac19 and 71% for PNLac20), reflecting their strong binding to RCA120 by the glycocluster effect. It is reasonable to assume that the specific binding of excess lactose derivatives to the lectin brought about relaxation of the complex, which resulted in the recovery of transcription. Regulation of in Vitro Expression of pGFP-Lactose with Lectin and Multivalent Lactose Derivative. To verify whether the present strategy can regulate gene expression, we have employed green fluorescent protein (GFP)-coded plasmid (pQBI T7 spGFP vector). In vitro expression of the plasmids was carried out with a PROTEINscript-Pro T7 kit. The expression level of GFP was estimated by the fluorescence intensity at 507 nm of the solution and normalized for that of the unmodified plasmid as 100%. As shown in Figure 7, the expression level of pGFPLac-8.7 was maintained as 93% that of the unmodified plasmid. Addition of an increasing concentration of RCA120 to the pGFP-Lac-8.7 decreased the expression level to be about 6% at [RCA120] ) 5.43 µM. It is probable that the repression of GFP expression results from formation of the complex between the conjugate and lectin. When lactose was added to the solution, the expression was recovered to about 50%, which is in contrast to no effect of cellobiose on the expression (Figure 8). Addition of PVLac or PNLac recovered the expression almost completely to the level of pGFPLac-8.7 in the absence of RCA120. These results indicate that the expression as well as the transcription of the lactosemodified plasmid could be regulated by the carbohydratelectin interaction.

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Figure 7. Repression of in vitro expression activity of pGFP-Lac8.7 ([plasmid] ) 76.4 nM, [base] ) 782 µM) by complexation with RCA120. (A) Fluorescence spectra of GFP expressed in the presence or absence of RCA120 (Ex ) 474 nm). (B) Dependence of RCA120 concentration on the expression level of GFP.

Figure 6. Regulation of in vitro transcription of pActin-Lac-12.2 conjugate by Lac-RCA120 interaction. (A) Polyacrylamide gel electrophoresis of mRNA transcribed from unmodified plasmid and pActin-Lac-12.2 with T7 RNA polymerase in the absence and presence of RCA120. (B) Repression of the transcription activity depending on RCA120 concentration at [plasmid] ) 249 nM ([base] ) 1.65 mM). (C) Recovery of the repressed transcription activity by adding lactose derivatives at [plasmid] ) 249 nM and [RCA120] ) 2.40 µM.

Aggregation Form of Complex and Regulation of Expression. As shown in Figures 6 and 8, the repression in transcription of pActin-Lac and expression of pGFP-Lac

in the presence of lectins were regained by adding the lactose-carrying polymers. To obtain information on structural change of the aggregates between the conjugate and lectin, gel-shift assay and TEM observation of the complex were carried out in the presence of an increasing amount of lactose derivatives (Figure 9). In the electrophoresis of the complex of pActin-Lac-8.2 with RCA120 (2.7 µM), the band of pActin-Lac conjugate free from RCA120 appeared when the lactose derivatives were added. The relative band intensity was increased, but only about 40% recovery was regained even in the presence of 1 mM lactose derivatives (Figure 9A and B). The TEM (Figure 9C) shows that linear plasmids (bright images) were exposed partially to solvent, but large aggregates of several-micrometers size still remained even in the presence of 1 mM lactose derivatives. This is in contrast to the TEM images (Figure 5C) of the complex between pActin-Lac-9.7 and RCA120 in the absence of lactose derivatives. The aggregation form of the complex was not completely dissociated, but relaxed partially in the presence of the lactose derivatives. We assume that the partial relaxation of the complex increased the accessibility of T7



Figure 8. Recovery of the repressed expression activity of pGFPLac-8.7 ([plasmid] ) 76.4 nM, [base] ) 782 µM) by adding lactose derivatives. (A) Fluorescence spectra of GFP expressed in the presence of RCA120 (2.72 µM) and recovering signals (1 mM) (Ex ) 474 nm). (B) The effect of recovering signals on the expression level of GFP.

RNA polymerase to result in the recovery of transcription activity. Conclusion We have proposed a novel strategy for artificial regulation of gene expression applying carbohydrate-lectin interaction. Plasmid-lactose conjugates (pActin-Lac and pGFP-Lac) were strongly interacted with the specific lectin RCA120 to form complexes with the size of sub-several micrometers. Their activities in transcription of pActin-Lac and expression of pGFP-Lac were repressed with RCA120 and then recovered with lactose-carrying polymers. It was suggested that the multivalent lactose derivatives interacted with the lectin to relax the aggregate of the complexes and cause the recovery of the activities. The glycosylated DNA of Trypanosoma brusei was suggested13 to be involved in the regulation of the gene expression. Although the regulation in natural biological

Figure 9. Effect of addition of lactose-carrying polymer on the structure of complex of conjugate with RCA120. (A) Agarose gel (1%) electrophoresis of complex of pActin-Lac-8.2 and RCA120 (2.7 µM) in the absence and presence of PVLac. (B) Change of relative band intensity of free plasmids depending on the concentration of Lac. The interaction condition: [plasmid] ) 49.8 nM ([base] ) 331 µM) in water at 25 °C. (C) TEM image of pActin-Lac-8.2 with RCA120 and PVLac. The complex solutions ([plasmid] ) 249 nM, [RCA120] ) 3.4 µM, and [PVLac] ) 1 mM-Lac in water) were diluted by 5 times for TEM observation.

systems is much more complicated, the system proposed in this report may be a simple model for regulation of gene expression using glycosylated DNA.

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The present system can be widely applied to the tailormade design of various ligand-receptor responding geneexpression systems. We envisage that the proposed strategy will contribute to the development of expression systems responding to environments of cells. Acknowledgment. This work was supported by a Grantin-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture. We are grateful to Prof. A. Mizuno, Dr. Y. Matsuzawa, and Mr. J. Komatsu of Toyohashi University of Technology for the measurement of TEM and AFM, and to Dr. T. Akasaka of Hokkaido University for his useful suggestions. References and Notes (1) Watson, J. D.; Baker, T. A.; Bell, S. P.; Gann, A.; Levine, M.; Losick, R. Molecular Biology of the Gene, 5th ed.; The Benjamin/Cummings Publishing Co.: USA, 2003. (2) (a) Gottesfeld, J. M.; Neely, L.; Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1997, 387, 202. (b) Mapp, A. K.; Ansari, A. Z.; Ptashne, M.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3930. (c) Arora, P. S.; Ansari, A. Z.; Best, T. P.; Ptashne, M.; Dervan, P. B. J. Am. Chem. Soc. 2002, 124, 13067. (d) Fechter, E. J.; Dervan, P. B. J. Am. Chem. Soc. 2003, 125, 8476. (e) Arndt, H. D.; Hauschild, K. E.; Sullivan, D. P.; Lake, K.; Dervan, P. B.; Ansari, A. Z. J. Am. Chem. Soc. 2003, 125, 13322. (3) Werstuck, G.; Green, M. R. Science 1998, 282, 296. (4) McIntosh, C. M.; Esposito, E. A., III; Boal, A. K.; Simard, J. M.; Martin, C. T.; Rotello, V. M. J. Am. Chem. Soc. 2001, 123, 7626. (5) Asanuma, H.; Tamaru, D.; Yamazawa, A.; Liu, M.; Komiyama, M. ChemBioChem 2002, 786. (6) Katayama, Y.; Fujii, K.; Ito, E.; Sakakihara, S.; Sonoda, T.; Murata, M.; Maeda, M. Biomacromolecules 2002, 3, 905. (7) Lin, H.; Abida, W. M.; Sauer, R. T.; Cornish, V. W. J. Am. Chem. Soc. 2000, 122, 4247. (8) Cruz, F. G.; Koh, J. T.; Link, K. H. J. Am. Chem. Soc. 2000, 122, 8777. (9) Kro¨ck, L.; Heckel, A. Angew. Chem., Int. Ed. 2005, 44, 471. (10) (a) Lehman, I. R.; Pratt, E. A. J. Biol. Chem. 1960, 235, 3254. (b) Lichtenstein, J.; Cohen, S. S. J. Biol. Chem. 1960, 235, 1134. (11) (a) Gommers-Ampt, J.; van Leeuwen, F.; de Beer, A. L. J.; Viliegenthart, J. F. G.; Dizdarouglu, M.; Kowalak, J. A.; Crain, P. F.; Borst, P. Cell 1993, 75, 1129. (b) van Leeuwen, F.; Taylor, M. C.; Mondragon, A.; Moreau, H.; Gibson, W.; Kieft, R.; Borst, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2366. (12) Wiberg, J. S. J. Biol. Chem. 1967, 242, 5824. (13) (a) Borst, P.; van Leeuwen, F. Mol. Biochem. Parasitol. 1997, 90, 1. (b) van Leeuwen, F.; Kieft, R.; Cross, M.; Borst, P. Mol. Biochem. Parasitol. 2000, 109, 133.

Matsuura et al. (14) (a) Matsuura, K.; Akasaka, T.; Hibino, M.; Kobayashi, K. Chem. Lett. 1999, 247. (b) Matsuura, K.; Akasaka, T.; Hibino, M.; Kobayashi, K. Bioconjugate Chem. 2000, 11, 202. (c) Akasaka, T.; Matsuura, K.; Emi, N.; Kobayashi, K. Biochem. Biophys. Res. Commun. 1999, 260, 323. (d) Matsuura, K.; Hibino, M.; Kataoka, M.; Hayakawa, Y.; Kobayashi, K. Tetrahedron Lett. 2000, 41, 7529. (e) Matsuura, K.; Hibino, M.; Yamada, Y.; Kobayashi, K. J. Am. Chem. Soc. 2001, 123, 357. (f) Akasaka, T.; Matsuura, K.; Kobayashi, K. Bioconjugate Chem. 2001, 12, 776. (g) Matsuura, K.; Hibino, M.; Ikeda, T.; Yamada, Y.; Kobayashi, K. Chem.-Eur. J. 2004, 10, 352. (15) (a) Akhtar, S.; Routledge, A.; Patel, R.; Gardine, J. M. Tetrahedron Lett. 1995, 36, 7333. (b) de Kort, M.; Edwin, E.; Wijisman, E. R.; van der Marel, G. A.; van Boom, J. H. Eur. J. Org. Chem. 1999, 2337. (c) Adinolfi, M.; Barone, G.; Napoli, L. D.; Guariniello, L.; Iadonisi, A.; Piccialli, G. Tetrahedron Lett. 1999, 40, 2607. (d) Hunziker, J. Bioorg. Med. Chem. Lett. 1999, 9, 201. (e) Sheppard, T. L.; Wong, C.-H.; Joyce, G. F. Angew. Chem., Int. Ed. 2000, 39, 3660. (f) Tona, R.; Bertolini, R.; Hunziker, J. Org. Lett. 2000, 2, 1693. (g) Forget, D.; Renaudet, O.; Defrancq, E.; Dumy, P. Tetrahedron Lett. 2001, 42, 7829. (h) Dubber, M.; Fre´chet, J. M. J. Bioconjugate Chem. 2003, 14, 239. (i) Sando, S.; Matsui, K.; Niinomi, Y.; Sato, N.; Aoyama, Y. Bioorg. Med. Chem. Lett. 2003, 13, 2633. (j) Wang, Y.; Sheppard, T. L. Bioconjugate Chem. 2003, 14, 1314. (16) (a) Choi, D.-J.; Roth, R. B.; Liu, T.; Geacintov, N. E.; Scicchitano, A. J. Mol. Biol. 1996, 264, 213. (b) Chen, Y.-H.; Bongenhagen, D. F. J. Biol. Chem. 1993, 268, 5849. (17) Preliminary report: Matsuura, K.; Hayashi, K.; Kobayashi, K. Chem. Commun. 2002, 1140. (18) Murata, M.; Yamasaki, T.; Maeda, M.; Katayama, Y. Chem. Lett. 2004, 33, 4. (19) Kobayashi, K.; Sumitomo, H.; Ina, Y. Polym. J. 1985, 17, 567. (20) Kobayashi, K.; Tsuchida, A.; Usui, T.; Akaike, T. Macromolecules 1997, 30, 2016. (21) (a) Modudrianakis, E. N.; Beer, M. Proc. Natl. Acad. Sci. U.S.A. 1965, 53, 564. (b) Modudrianakis, E. N.; Beer, M. Biochim. Biophys. Acta 1965, 95, 23. (22) We have estimated the apparent affinity constant using also surface plasmon resonance (SPR) to be 4.1 × 105 M-1 per Lac-unit.17 We consider that the apparent affinity constant calculated from gel-shift assay is more accurate than that from SPR, because the SPR experiments observed the binding of plasmid-Lac conjugates to RCA120 immobilized on gold substrate via covalent bonds. (23) (a) Lee, Y. C. FASEB J. 1992, 6, 3193. (b) Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71. (c) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754. (d) Lundquist, J. J.; Toone, E. J. Chem. ReV. 2002, 102, 555.

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OnOff Switching of Gene Expression Regulated with

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