Phototriggered DNA Complexation and Compaction Using Poly(vinyl

Apr 18, 2012 - Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan. •S Supportin...
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Phototriggered DNA Complexation and Compaction Using Poly(vinyl alcohol) Carrying a Malachite Green Moiety Ryoko M. Uda* and Minami Ohshita Department of Chemical Engineering, Nara National College of Technology, Yata 22, Yamato-koriyama, Nara 639-1080, Japan S Supporting Information *

ABSTRACT: Photoinduced DNA compaction was performed using the interaction of DNA with a photoresponsive random copolymer of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG). Although PVAMG does not have any affinity for DNA under dark conditions, it undergoes photoionization upon exposure to UV light, consequently resulting in a cationic binding site for DNA. Electrophoresis results demonstrated that irradiation of PVAMG retarded the DNA bands due to their complexation, whereas the bands remained unchanged under dark conditions. The binding of PVAMG to DNA occurs at a cationic site/DNA phosphate ratio of approximately 0.036. Single-molecule observations of DNA by fluorescence microscopy revealed that irradiation of PVAMG induced a coil−globule transition in the DNA molecule. Complete compaction of DNA has been accomplished at a cationic site/DNA phosphate ratio >8.0, indicating that PVAMG offers an effective system to photochemically trigger DNA compaction.

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INTRODUCTION The compaction of DNA induced by cationic substances has attracted a large amount of interest due to its importance for technological and biomedical applications, particularly for its potential use in gene delivery and gene transfection.1−3 DNA is a highly charged polyanion that is repelled by negatively charged cell membranes. In complexation between DNA and cationic substances, the effective negative charge of DNA is lowered, allowing the complex to enter cells. In addition, DNA in the compacted form is protected against digestion by enzymes.4,5 Because delivery through viral vectors in vivo poses safety concerns unlikely to be resolved soon, cationic substances such as cationic lipids or cationic polymers have been studied as DNA compaction agents in the context of nonviral gene delivery.6−8 Copolymers composed of a hydrophilic neutral part and a cationic part seem to be promising,9−11 because cationic polymers with a high charge density, exhibiting enhanced DNA compaction, contribute to higher cytotoxicity as observed for poly(L-lysine).12 In the present study, we designed a random copolymer of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG) that undergoes photoionization (Scheme 1). Under dark conditions, PVAMG does not have a positive charge on the malachite green moiety and exhibits no affinity for DNA. In contrast, UV irradiation affords a cationic site, resulting in PVAMG binding to DNA. Simple light illumination enables spatial and temporal control; thus, it is an attractive method for controlling DNA compaction that can be used in diverse fields such as biotechnology and DNAbased chemistry. Lee and coworker have reported photocontrolled DNA condensation by a cationic azobenzene surfactant that exhibits © 2012 American Chemical Society

Scheme 1. Photoionization of Poly(vinyl alcohol) Carrying a Malachite Green Moiety (PVAMG)

cis−trans isomerization.13 Complete compaction of DNA requires a high concentration of the photoresponsive compound at a molar ratio of azobenzene surfactant to DNA phosphate = 125.13 However, a change in the net charge of the structure’s building blocks should be more effective than cis− Received: February 6, 2012 Revised: April 5, 2012 Published: April 18, 2012 1510

dx.doi.org/10.1021/bm3001952 | Biomacromolecules 2012, 13, 1510−1514

Biomacromolecules

Article

where AMG and AMGO are the molar absorptivity at 625 nm of the malachite green unit of PVAMG after UV irradiation and that of malachite green oxalate in 0.1 M phosphate buffer solution at pH 7.0, respectively. When the solution of PVAMG contained DNA, AMGO was determined in the presence of DNA at the corresponding concentration for each case. Fluorescence Emission Spectroscopy. Fluorescence was recorded at λex = 590 nm in an RF-5300PC spectrofluorometer (Shimazu, Kyoto, Japan). The fluorescence intensity scale at 657 nm was calibrated with the fluorescence 3.0 μM of malachite green oxalate in buffer containing 1.0 g L−1 of poly(vinyl alcohol) (Mw = 89 000−98 000). The fluorescence intensity was calibrated for each case. Experimental errors in the fluorescence intensity were within 5%. Polyacrylamide Gel Electrophoresis. The electrophoretic mobility of DNA complexes in Tris-glycine buffer solution (pH 8.4) was determined by gel electrophoresis using 10% polyacrylamide gels. DNA samples (10 μL) were loaded into each sample well. The concentration of DNA was 6 μM. Controls for free DNA and PVAMG were also applied to the gel. Experiments were run at 100 V for 30 min. DNA was visualized by silver staining. Fluorescence Microscopy. Fluorescence microscopy with T4 DNA, which is larger than calf thymus DNA, was used to detect single molecules of DNA. To prevent aggregation, the final concentration of DNA used was 0.11 μM. YOYO-1 (0.4 μM) was used as the fluorescent dye. The sample solution depth was ∼120 μm to avoid the surface effect of the glass plate. Samples were observed with a Nikon 80i fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 100× oil-immersed objective lens and recorded with a CCD camera (Nikon DS-Fi1-L2). To reduce further light-induced damage, an 8 or 16% neutral density filter was placed in front of the excitation source.

trans isomerization because the binding affinity for negatively charged DNA would be directly affected. Moreover, cationic triphenylmethane dyes, for example, crystal violet and malachite green, have been found to bind to DNA externally and to have a general nonspecific affinity for double-stranded DNA,14−16 except for a preference for AT-rich DNA over GC-rich DNA.17−19 Therefore, effective photocontrol of binding to DNA is expected for PVAMG. Here we report phototriggered DNA complexation with PVAMG, investigated using fluorescence emission spectroscopy and polyacrylamide gel electrophoresis. We used a fluorescence microscopic technique that enables single-molecule detection to examine conformational changes of DNA in solution. The results clearly showed that PVAMG affords efficient DNA compaction with light.

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EXPERIMENTAL SECTION

Synthesis of PVAMG. Bis(4-(dimethylamino)phenyl)(4vinylphenyl)methyl cyanide was synthesized as previously reported.20 Vinyl acetate was purified by distillation before use. Radical polymerization of bis(4-(dimethylamino)phenyl)(4-vinylphenyl)methyl cyanide with vinyl acetate was carried out in benzene at 60 °C for 24 h. α-α′-Azobis(isobutylonitrile) was used as the initiator (0.5 mol % to the total monomer). After polymerization, the solvent and the other volatile material were vacuum-evaporated to dryness. Acetone was added to the polymer mixture, precipitated in excess petroleum ether and vacuum-dried. The number of average molar mass (Mn) and weight-average molar mass (Mw) determined by gel permeation chromatography using poly(methyl methacrylate) calibration standards and tetrahydrofuran as eluent were 277 000 and 586 000 g mol−1, respectively. The calculated polydispersity index (Mw/ Mn) was 2.12. The hydrolysis of the obtained poly(vinyl acetate) copolymer was carried out in methanol-aqueous solution containing 1 wt % NaOH. The product, white powder, was solubilized into an aqueous solution, precipitated in excess methanol, and vacuum-dried. The hydrolysis of the copolymer was confirmed by NMR. After hydrolysis, the proton signal arising from the methyl of acetyl group at 2.03 ppm in the poly(vinyl acetate) unit had disappeared completely, and a new peak at 4.60 ppm, which was attributed to hydroxyl groups, appeared. The molar fraction of the malachite green unit, which corresponds to bis(4-(dimethylamino)phenyl)(4-vinylphenyl)methyl cyanide, for the copolymer was also determined by NMR by comparing the peak area of the methyl of the 4-(dimethylamino)phenyl group in the malachite green unit (2.88 ppm) with that of the methine in the poly(vinyl alcohol) unit (3.81 ppm) and was found to be 0.003. PVAMG concentrations were expressed as malachite green units. Other Materials. Calf thymus DNA, type I, was purchased from Sigma (St. Louis, MO). The purity of calf thymus DNA was verified by monitoring the ratio of absorbance at 260 nm to that at 280 nm, which was in the range of 1.8 to 1.9. DNA concentrations, expressed as DNA phosphate, were determined by UV absorbance at 260 nm using the molar absorption coefficient ε260 (6600 M−1cm−1).21 T4 DNA composed of 166 kbp was purchased from Nippon Gene (Toyama, Japan) and used for fluorescence microscopy. YOYO-1 was obtained from Molecular Probes (Eugene, OR). Water used was deionized water. Other materials were of analytical grade and used without further purification. Preparation of Sample Solutions. The mixture of calf thymus DNA and PVAMG was prepared in 0.1 M phosphate buffer solution at pH 7.0 for absorption spectroscopy, fluorescence emission spectroscopy, and electrophoresis. For fluorescence microscopy, T4 DNA and PVAMG were dissolved in 0.1 M phosphate buffer solution (pH 7.0). UV irradiation was carried out for 20 min. The UV light source (260− 390 nm) was a xenon lamp (500 W) equipped with a photoguide tube and a Toshiba UV-D33S filter. After preparation, the samples were equilibrated for 2 h at 25 °C. The ionization ratio of PVAMG was obtained by the following equation: ionization ratio = AMG/AMGO,

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RESULTS AND DISCUSSION Photoionization of PVAMG. Figure 1a shows the typical absorption-spectral change of the PVAMG solution before and

Figure 1. Photoionization of PVAMG. (a) Absorption-spectral changes of 0.1 M phosphate buffer solution containing 0.13 mM of PVAMG before (dark) and after (UV) UV irradiation for 20 min. The arrows denote peaks changed by irradiation. (b) Absorbance at 625 nm after irradiation of 0.1 M phosphate buffer solution containing PVAMG and calf thymus DNA. The concentration of PVAMG is 0.13 mM.

after UV irradiation. An absorption peak around 260 nm, which was decreased by UV irradiation, was assigned to the electrically neutral form of the malachite green moiety of PVAMG. Its ionized form was confirmed by the appearance of a peak at 620 nm after UV irradiation, indicating the photoinduced ionization of the malachite green moiety. The addition of increasing amounts of DNA increased the peak (Figure 1b), whereas the photoionization ratio was independent of the PVAMG concentration. (See the Supporting Information.) The observed 1511

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by restricting such vibrations.25−27 We have checked the viscosity of DNA solutions in separate experiments and found that the increase in viscosity due to the addition of DNA produces a negligible effect on the fluorescence of malachite green. Therefore, the increase in emission intensity by mixing in DNA, as shown in Figure 2a, was attributed to the binding of cationic malachite green of PVAMG to DNA. In contrast, the emission spectrum of the mixture of PVAMG and DNA under dark conditions (+ DNA (dark)) affords an emission intensity at 657 nm, which is smaller than that after ionization. Because the neutral PVAMG does not have a binding site and showed no peak without DNA (data not shown), the small peak observed under dark conditions (+ DNA (dark)) is likely related to DNA. To obtain the binding ratio of PVAMG to DNA, the differences of emission intensity at 657 nm between dark and UV irradiated conditions (FUV − Fdark) were plotted against the charge ratio of ionized malachite green to DNA phosphate (Figure 2b). The value of FUV − Fdark increased linearly with the ratio up to 0.036, above which the slope of the FUV − Fdark curve changes abruptly. The turning point of 0.036 corresponds to the binding ratio of PVAMG to DNA and is close to the value of 0.04 reported for the malachite green−calf thymus DNA complex determined by using linear dichromism.28 The photoresponsive binding to DNA was also analyzed using polyacrylamide gel electrophoresis (Figure 3). Lane 2

hyperchromicity is in contrast with the absorbance changes usually seen for cationic triphenylmethane dyes such as pararosaniline, methyl green, and malachite green, whose major absorption band in the visible light region shows a hypochromic change upon binding to DNA.22−24 This hyperchromic change shown in Figure 1b was explained by the coexistence of DNA, which can stabilize the ionized state of the malachite green moiety of PVAMG owing to the negative charge of phosphate, and consequently prevents the backward reaction of malachite green from the ionized form to the neutral one.19 Because the peak intensity of ionized malachite green depends on the DNA concentration, the concentration of ionized malachite green units [MG+] was determined by the absorbance at 625 nm using calibration curves prepared for the mixture of malachite green oxalate in the presence of DNA at the corresponding concentration for each case. DNA also shifts the maximum absorption wavelength of the ionized malachite green peak to 624.5 nm. (See the Supporting Information.) The bathochromic shift of 4.5 nm was smaller than the reported value of 10 nm for triphenylmethane dyes such as methyl green.19 Photoinduced Complexation of PVAMG with Calf Thymus DNA. From the results of Figure 1, it is difficult to conclude that ionized PVAMG binds to DNA because the absorption peak of ionized malachite green did not exhibit a hypochromic change, and its bathochromic shift was not as significant as that of other triphenylmethane dyes reported.19,22−24 To obtain evidence of PVAMG binding to DNA, we used fluorescence spectroscopy, which is more sensitive than absorption spectroscopy. Figure 2a shows the

Figure 3. Polyacrylamide gel electrophoresis results for calf thymus DNA. (Lane 1) Free DNA. (Lanes 2 and 3) Mixture of DNA and PVAMG under dark conditions (Lane 2) and after UV irradiation (Lane 3) at a ratio of [MG+]/[DNA] = 0.04. (Lane 4) Mixture of DNA and PVAMG after UV irradiation at a ratio of [MG+]/[DNA] = 0.16. (Lanes 5 and 6) Mixture of DNA and malachite green oxalate at a ratio of [malachite green oxalate]/[DNA] = 0.12 (Lane 5) and 0.51 (Lane 6). The concentration of DNA was 6 μM.

Figure 2. Calf thymus DNA enhances fluorescence of ionized PVAMG. (a) Fluorescence emission spectra of 0.065 mM PVAMG solutions with (+ DNA) and without (− DNA) 0.16 mM DNA. (b) Dependence of FUV − Fdark on the ratio of [MG+]/[DNA] (= charge ratio). FUV and Fdark are emission intensity at 657 nm under dark and after UV irradiated conditions, respectively. The concentration of DNA was maintained at a constant of 0.19 mM. The fluorescence intensity was calibrated with the fluorescence 3.0 μM of malachite green oxalate in buffer solution containing 1.0 g L−1 of poly(vinyl alcohol).

represents the mixture of DNA and PVAMG under dark conditions, indicating no interaction between them. In contrast, irradiation of PVAMG retarded the migration of the DNA band, which is clearly demonstrated by comparing Lanes 2 and 3. Lane 4 shows that the DNA band is retarded as the charge ratio of [MG+]/[DNA] is increased. When the ratio of [MG+]/ [DNA] is significantly smaller than the binding ratio of 0.036, no remarkable change was observed in the irradiated sample. (See the Supporting Information.) Accordingly, the retarded DNA bands in the irradiated samples are likely due to PVAMG binding to DNA. There was no band detected for the control

fluorescence emission spectra of PVAMG solutions. Although there was no emission peak for the irradiated PVAMG sample (− DNA (UV)), a new peak appeared at ∼657 nm after the addition of DNA (+ DNA (UV)). Triphenylmethane dye cations normally afford rather weak fluorescence due to easy vibrational deexcitation, but viscous, cold environments or aptamers have been demonstrated to enhance the fluorescence 1512

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4a,b, we concluded that photoionization of PVAMG-induced DNA compaction. To examine the behavior of DNA compaction, we observed T4 DNA by altering the concentrations of PVAMG after UV irradiation; those under dark conditions remained in a coil state. Figure 4c−e illustrates the conformational changes of T4 DNA molecules due to irradiation of PVAMG at various [MG+]/[DNA] ratios. DNA molecules displayed an extended conformation (coil) at a ratio of [MG+]/[DNA] < 0.04 (Figure 4c). With an increase in the concentration of PVAMG, partially globular DNA appears at a ratio of [MG+]/[DNA] = 0.4, in which a compacted part and an elongated part coexist in a single chain, as shown in Figure 4d. Finally, at a ratio of [MG+]/[DNA] = 8.0, all of the DNA exhibited a completely compacted structure (Figure 4e). This stepwise compaction observed for DNA-PVAMG complexation was similar to that reported for DNA-PEG with a pendant amino group, which is distinguished from the all-or-none-type compaction induced by PEG.31 Full compaction with no extended coils detected was obtained at [MG+]/[DNA] = 8.0, which is significantly higher than the binding ratio of 0.036. Because triphenylmethane dyes have been found to have a general nonspecific affinity for double-stranded DNA,14−16 the higher ratio for complete compaction was not related to the difference between T4 DNA and calf thymus DNA. Many studies demonstrated that cationic substance interaction with DNA is a two-stage process, the first attributed to cation binding and the second attributed to DNA compaction.32−35 During the first stage, ionized malachite green of PVAMG binds to DNA because of its affinity for DNA.14−16 In the second stage, DNA chains in a coexistence region can be considered to be in a kind of equilibrium between the elongated and compact states. Therefore, the binding ratio of 0.036 is for the first stage, and the ratio of 8.0, which is higher than that for binding of PVAMG to DNA, is required for the complete compaction. The effect of polymerized malachite green on DNA compaction was investigated by comparing it with the results of malachite green oxalate (Figure 4f). DNA molecules complexing malachite green oxalate displayed extended conformations even at a high concentration of malachite green ([malachite green oxalate]/[DNA] = 86.4). Yoshikawa and coworkers have reported DNA transition to a compacted state due to poly(L-lysine) with different numbers of monomer units.36 They found that the transition concentration decreased by one order of magnitude with an increase in the number of monomers and that short poly(L-lysine) induced all-or-none type compaction. Therefore, whereas the concentration of the malachite green moiety shown in Figure 4f was significantly higher than that for PVAMG (Figure 4d), it was not sufficient to cause a discrete transition that differs from the gradual compaction due to the malachite green moiety on the PVA chain.

sample of PVAMG under both dark and irradiated conditions (data not shown). There exist two possible explanations for the retarded DNA band due to binding to PVAMG. One is that the reduction of negative charges of the DNA molecules, when it is complexed by cationic malachite green, reduces the DNA progression. The other is that the size of the DNA complexed with PVAMG was too big to migrate through the gel pores. To investigate the possibility of the reduced negative charge of DNA, we analyzed malachite green oxalate because it also has affinity for DNA and reduces the negative charge of DNA, and it is considered to be a model compound for the monomeric part of ionized malachite green in PVAMG. The results shown in Lanes 5 and 6 indicate that malachite green oxalate has a negligible effect on the migration of the DNA, whereas the charge ratio was remarkably higher than its binding ratio of 0.04. As such, the increase in DNA size after complexing with PVAMG was likely the main driving force in retarding the DNA band. Phototriggered Compaction of T4 DNA Using PVAMG. Fluorescence microscopy was used to observe visually conformational changes of T4 DNA induced by irradiation of PVAMG. Figure 4 shows fluorescence microscopic images of

Figure 4. Fluorescence images of T4 DNA molecules. (a) Elongated coil in 0.11 mM PVAMG under dark conditions, (b) compact globule induced by irradiation of 0.11 mM PVAMG, (c) coil state in the presence of PVAMG at [MG+]/[DNA] = 0.04, (d) partial globule in the presence of PVAMG at [MG+]/[DNA] = 0.4, (e) compact state in the presence of PVAMG at [MG+]/[DNA] = 8.0, and (f) coil in the presence of 9.5 μM malachite green oxalate. Bars = 2 μm.

T4 DNA molecules moving freely in aqueous solution. Individual T4 DNA molecules were observed as independent elongated coils under dark conditions in the presence of PVAMG (Figure 4a). Although nonionic surfactants, for example, Triton X-100 and poly(ethylene glycol) (PEG), induce a collapse of individual DNA chains,29,30 neutral PVAMG under dark conditions did not cause the DNA compaction. This is because DNA compaction due to a nonionic surfactant requires a drastic increase in internal compressing osmotic pressure and is accomplished by extremely high concentrations of nonionic surfactants, such as 45 to 50 wt %.30 The concentration of PVAMG shown in Figure 4 is too low (