Synthesis and AFM Characterization of Poly (dG)-poly (dC)-gold

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Bioconjugate Chem. 2010, 21, 544–547

Synthesis and AFM Characterization of Poly(dG)-poly(dC)-gold Nanoparticle Conjugates Dragoslav Zikich, Natalia Borovok, Tatiana Molotsky, and Alexander Kotlyar* Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, The Center for Nanoscience and Nanotechnology, Tel Aviv University, Ramat Aviv, 69978. Received December 1, 2009; Revised Manuscript Received January 29, 2010

In the present work, we have synthesized conjugates between the 5 nm gold nanoparticles (Au-NP) and 5′ thiolfunctionalized, 700 bp poly(dG)-poly(dC). We have completely separated and purified to homogeneity conjugates bearing different number of poly(dG)-poly(dC) molecules per Au-NP by electrophoresis and HPLC. The conjugates were directly visualized by atomic force microscopy. We have demonstrated that Au NP-bound poly(dG)-poly(dC) can be considerably extended by Klenow exo- polymerase in the presence of dCTP and dGTP.

INTRODUCTION Since the pioneering works by Alivisatos and Mirkin (1, 2), interaction of Au-NP with thiol-modified single-stranded (ss) DNA has become a subject of extensive investigation by chemists, biologists, and physicists. Because of their small size, Au-NP-DNA conjugates exhibit unique physical, chemical, and optical properties and have been used as bases for development of applications in various aspects of nanoassembly (3, 4), biodiagnostics (5-7), and nanoelectronics (8, 9). The ability of DNA to form well-defined secondary and tertiary structures offers a variety of possibilities for nanostructure design in a bottom-up approach (10). The vast majority of studies in this field were focused on the interaction of ssDNA with metal nanoparticles; much less emphasis has been placed on formation of conjugates between long, functionalized dsDNA and the particles. It has been shown that DNA molecules connected to a particle retain their functional activity and can be processed by restriction enzymes (10-12), polymerases (13), and ligases (14). Tsai and co-workers have successfully used a ligase-based approach for the formation of complexes between long (1714 base pairs) random-sequence thiolated dsDNA and gold nanoparticles (15). The conjugates bearing a different number of DNA molecules per particle have been efficiently separated from each other by electrophoresis and seen by AFM (15). To the best of our knowledge, no nanoparticle conjugates with homogeneous dsDNA polymers have been reported so far. The results of theoretical calculations show that poly(dG)-poly(dC) exhibits better conductance than other homopolymeric DNA form, poly(dA)-poly(dT) (16). Therefore, poly(dG)-poly(dC) homopolymer seems to be a promising new candidate for use in DNA-based nanodevices. We have recently invented a new enzymatic method for the synthesis of homogeneous dsDNA polymers (17). This method enables us to synthesize poly(dG)-poly(dC) molecules, ranging from tens to tens of thousands base pairs that are characterized by a narrow length distribution. In addition, these molecules can be functionalized at one or at both 5′-ends with SH-groups or other residues. The ability to introduce SH-groups into the synthesized molecules provides a tool for their covalent attachment to gold surfaces. Attachment of metal particles might provide good electric communication between the electrode and * To whom correspondence should be addressed. Phone: 972 3 6407138; Fax: 972 3 6406834; E-mail: [email protected].

the DNA-NP conjugate via metal/metal contact interactions, thus promoting charge migration though the DNA. Here, we describe an efficient method for production of poly(dG)-poly(dC) Au-NPs conjugates bearing a different number of DNA molecules per Au-NP. The conjugates were efficiently separated by agarose gel electrophoresis and visualized by AFM. We demonstrate that poly(dG)-poly(dC) molecules connected to a Au-NP can be extended by Klenow exofragment of DNA polymerase.

EXPERIMENTAL PROCEDURES Synthesis of Au Nanoparticles. Au-NPs of 5 nm in diameter were prepared by the modified citrate reduction method (18, 19). 5.9 mg of HAuCl4 was dissolved in 1 mL of double distilled water and added into 47 mL of H2O to the final concentration of 0.3 mM. The solution was incubated with constant stirring at 25 °C for 10 min. 0.5 mL of 50 mM sodium citrate solution was added to the reaction mixture to a final concentration of 0.5 mM, and the solution was incubated under constant stirring for another 10 min. 0.67 mL of cooled 0.1 M NaBH4 was added into the stirred mixture; the solution developed an orange-red color. The resulting nanoparticles were screened for their size and uniformity by TEM, revealing an average diameter of 5 ( 1.5 nm. The UV-vis spectra showed a characteristic absorption peak at 520 nm. The particles were stabilized with BSPP (bis sulfonatophenylphosphine dehydrate dipotassium salt, Strem Chemicals, USA) as follows. The Au-NPs above were incubated with 0.1 mM BSPP in the dark for 20 h at RT and concentrated using Microcon device YM-50 (Millipore, USA). The excess of BSPP was removed by passing the particles though G-25 Sephadex column (15 × 50 mm, Healthcare, USA) in 20 mM Tris-acetate buffer, pH 7.5. The Au-NPs eluted in the void volume of the column were concentrated using YM-50 Microcon device (Millipore, USA). Concentration of the particles was calculated using an extinction coefficient (ε) of 9.3 × 106 M-1 cm-1 at 517 nm. DNA Samples. The (dC)12 and SH-(dG)12 oligonucleotides were purchased from Alpha DNA (Montreal, Canada). The SHgroup in SH-(dG)12 was linked to the terminal base at the 5′ end of G-12mer (dG)12 oligonucleotides via a six-carbon linker. The oligonucleotides were purified to homogeneity essentially as described (20). Purified SH-(dG)12 and (dC)12 oligomers were incubated in 0.1 M NaOH at an equimolar ratio for 15 min at room temperature and further dialyzed against 20 mM Trisacetate buffer, pH 7.5, containing 2 mM DTT for 1 h. The

10.1021/bc900527a  2010 American Chemical Society Published on Web 02/18/2010

Poly(dG)-poly(dC)-gold Nanoparticle Conjugates

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Figure 1. Electrophoretic separation of Au-NP poly(dG)-poly(dC) conjugates.700 bp 5′-SH-poly(dG)-poly(dC) molecules were incubated with 5 nm Au-NPs at a 4 to 1 molar ratio for 2 h at 60 °C in 20 mM Tris-acetate pH 7.5, containing 0.1 M NaCl. (A) Separation of the sample was conducted on 1.2% agarose gel (7 × 7 cm) at 130 V for 25 min at RT in TEA buffer. The wine-colored bands marked with the numbers correspond to the conjugates bearing one, two, three, and four poly(dG)-poly(dC) molecules, correspondingly. The band in the lower right corner of the gel corresponds to 5 nm Au-NPs. (B) Normalized absorption spectra of samples extracted from gel slices corresponding to bands 2, 3, and 4 (see left panel). Two distinct absorption maxima at 260 and 520 nm correspond to DNA and Au nanoparticle, respectively. The amount of individual conjugates extracted from the gel was quantified by measuring absorption at 520 nm. The relative yield of conjugates was calculated by multiplying the volume by absorption at 520 nm for each individual fraction and dividing the resulting value with that obtained for the initial mixture of conjugates (before electrophoresis). The calculated yields are correspondingly equal to 0.15, 0.37, 0.31, and 0.09 for conjugates bearing one, two, three, and four poly(dG)-poly(dC) molecules.

dialysis was continued against the same buffer not containing DTT for one more hour. Concentrations of G- and C-oligonucleotides were calculated using extinction coefficients at 260 nm of 11.7 and 7.5 mM-1 cm-1 for G and C bases (21), respectively. DNA Synthesis Reactions. Synthesis of 5′SH-poly(dG)poly(dC) was performed as described previously (20) in a standard reaction assay containing: 60 mM KPi, pH 7.4, 5 mM MgCl2, 5 mM DTT, 1.5 mM dCTP, 1.5 mM dGTP, 0.2 µM Klenow exo-, and 1 µM HPLC purified (see above) templateprimer, 5′-SH-(dG)12-(dC)12. The synthesis was conducted for 2 h at 37 °C. The reaction was terminated by the addition of EDTA to a final concentration of 10 mM. HPLC Purifications. The separation of synthesized DNA molecules from nucleotides, template-primer, and other reaction components was achieved with a TSK-gel G-DNA-PW HPLC column (7.8 × 300 mm) from TosoHaas (Japan) by isocratic elution with 20 mM Tris-acetate, pH 7.0, for 30 min at a flow rate of 0.5 mL/min. HPLC purification of the oligonucleotides and the synthesized DNA was conducted on an Agilent 1100 HPLC system with a photodiode array detector. Peaks were identified from their retention times obtained from the absorbance at 260 nm for DNA. Electrophoresis. The poly(dG)-poly(dC) and Au NP-poly(dG)poly(dC) conjugates were loaded onto 1.2% agarose gel 7 × 7 cm, and then electrophoresed at room temperature at 130 V for 25 min. TAE buffer, in addition to being used to prepare the agarose, also served as the running buffer. Atomic Force Microscopy. Atomic force microscopy was performed on the molecules adsorbed onto muscovite mica surfaces. A 20 µL aliquot of 1.0-2.0 nM Au NP-DNA sample in 2.0 mM MgCl2 or 2.0 mM NiCl2 was deposited on freshly cleaved mica plates for 2 min, then washed with distilled water and dried by nitrogen blow. AFM imaging was performed on a Solver PRO AFM system (NT-MDT, Russia), in a semicontact (tapping) mode, using 130-µm-long Si-gold-coated cantilevers (NT-MDT, Russia) with resonance frequency of 119-180 kHz. The images were “flattened” (each line of the image was fitted to a second-order polynomial, and the polynomial was then subtracted from the image line) by the Nova image processing software (NT-MDT, Russia). The images were analyzed and

visualized using a Nanotec Electronica S.L (Madrid) WSxM imaging software (22).

RESULTS To produce the Au-NP-DNA conjugates, 700 bp 5′-thiolated poly(dG)-poly(dC) was mixed with Au-NPs at 4 to 1 molar ratio in 20 mM Tris buffer, pH 7.5, in the presence of 0.1 M NaCl, for 2 h at 60 °C. The incubation resulted in the formation of Au-NP-DNA conjugates bearing a different number of DNA molecules per nanoparticle. The conjugates were separated by electrophoresis in a 1.2% agarose gel, as shown in the Figure 1. The wine-colored bands in the gel (see Figure 1A) correspond to the conjugate products containing distinct number of poly(dG)poly(dC) molecules per Au-NP. Each band was cut out of the gel with a razor blade. Colored gel slices were placed in dialysis bags containing TAE buffer; each sample was then electroeluted into the bag and collected. Absorption spectra of the eluted AuNP-DNA conjugates are shown in Figure 1B. The spectra are characterized by absorption maxima at 260 and 520 nm. The absorption at 260 nm is mainly due to DNA, while absorption at 520 nm is due to Au-NPs. Thus, the 260 to 520 nm absorption ratio in the spectrum reflects a relative amount of the DNA molecule per particle (see Figure 1B). Fractions extracted from the gel slices were further purified and characterized by size-exclusion HPLC. Figure 2A displays the superimposed size-exclusion chromatograms of Au-NP DNA conjugates extracted from the four individual bands in the gel (see Figure 1A). As seen in Figure 2, the conjugates are eluted from the column as single and symmetrical peaks, indicating their high-purity complexes. The HPLC, in addition to confirming their purity, completely separates the conjugates from relatively low molecular weight agarose oligomers, extracted from the gel slices together with the conjugates. The molecular morphology of HPLC purified Au-NP-poly(dG)poly(dC) conjugates was elucidated by AFM. The molecules eluted from the HPLC column were deposited onto freshly cleaved, 1 × 1 cm mica and imaged by AFM in a tapping mode. The images of gel-extracted fraction are shown in Figure 3. The vast majority of conjugates seen on A, B, C, and D panels of Figure 3 are composed of 1, 2, 3, or 4 poly(dG)-poly(dC) molecules bound to a single nanoparticle (seen as bright spots

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Figure 2. Size-dependent HPLC separation of Au-NP poly(dG)poly(dC) conjugates. The individual conjugates extracted from the gel (see Figure 1) were loaded onto the size-exclusion column equilibrated with 20 mM Tris-acetate, pH 7.5. Elution profiles of conjugates corresponding to bands 1, 2, 3, and 4 in Figure 1 are indicated with numbers 1 through 4, respectively. The elution was followed at 260 nm. Figure 4. Enzymatic extension of poly(dG)-poly(dC) molecules bound to a Au-NP. The conjugates comprising two (A and B) and three (C and D) poly(dG)-poly(dC) molecules per Au-NP before (A and C) and after (B and D) enzymatic extension. The conjugates were extracted from the gel (Figure 1) and purified by HPLC as shown in Figure 2. The conjugates were incubated in an assay mixture containing 60 mM KPi buffer, pH 7.4, 5 mM MgCl2, 5 mM DTT, 1.5 mM dCTP, 1.5 mM dGTP, and 0.2 µM Klenow exo-, for 20 h at 37 °C, and deposited on a mica surface in 2 mM NiCl2, as described in Experimental Procedures.

contour length of DNA molecules attached to the particle is increased from 170 nm to approximately 450 nm (compare DNA lengths in right and left panels of Figure 4). This clearly demonstrates that Klenow exo- DNA polymerase is capable of elongating poly(dG)-poly(dC) molecules connected to the AuNP. The rate of particle-bound poly(dG)-poly(dC) extension is, however, two orders in magnitude lower than that of nonbound DNA (data not shown).

DISCUSSION

Figure 3. AFM images of Au-NP poly(dG)-poly(dC) conjugates. The conjugates were extracted from the gel (see Figure 1) and purified by size-exclusion HPLC as shown in Figure 2. All conjugates were deposited on a mica surface in 2 mM NiCl2 and scanned in a tapping mode as described in Experimental Procedures. Panels A, B, C, and D present images of conjugates extracted from bands 1, 2, 3, and 4 (see Figure 1) correspondingly.

on the images), respectively. The average estimated contour length of bound DNA molecules is equal to 170 ( 22 nm. This value corresponds well with the contour length of 700 bp poly(dG)-poly(dC) molecules deposited on mica (23). We have investigated the ability of DNA polymerase to extend poly(dG)-poly(dC) molecules attached to a gold nanopaticle. The conjugates comprising two or three poly(dG)poly(dC) molecules per Au-NP (see Figure 4A,B, respectively) were subjected to the enzymatic reaction with Klenow exoDNA polymerase, in the presence of dGTP, dCTP nucleotides, for 20 h at 37 °C. The products of the synthesis were subsequently purified by size-exclusion HPLC and analyzed using AFM. Figure 4 shows that incubation with the enzyme causes considerable increase of the DNA length; the average

The ability to control the number and the length of DNA molecules attached to a single nanoparticle is essential for assembly of DNA-NP conjugates into new functional nanomaterials. Incubation of functionalized oligonucleotides and DNA species with NPs, however, results in a mixture of DNA-NP conjugates bearing different numbers of DNA molecules. Alivisatos and colleagues demonstrated that binding of single-stranded oligonucleotides produces a significant effect on the particle mobility in the electrical field and succeeded to separate conjugates bearing different numbers of oligonucleotides per particle by electrophoresis (24, 25). The efficiency of the DNA-NP conjugate separation in gels decreases as the particle size increase and increases as the DNA length increases. Indeed, an excellent separation of the NP conjugates with long random sequence dsDNA was demonstrated by Tsai and co-workers (15). In the latter study, random sequence dsDNA molecules were attached to Au-NPs through the SH-group introduced into the DNA, using a ligase-dependent reaction. Here, we employed a polymerase-based method (17) to produce the AuNP conjugates with a homogeneous dsDNA polymer, poly(dG)poly(dC). This method enables us to synthesize uniform doublestranded poly(dG)-poly(dC) or poly(dA)-poly(dT) wires of desired length (varied from several nanometers to several micrometers), as well as the DNA functionalized with SH groups or/and random oligonucleotide sequences. We have shown that incubation of the

Poly(dG)-poly(dC)-gold Nanoparticle Conjugates

synthesized 700 bp SH-poly(dG)-poly(dC) with Au-NPs yielded a mixture of Au-NP-DNA conjugates bearing different number of DNA molecules per particle that can be easily and completely separated from each other using electrophoresis. The AFM visualization of these products (see Figure 2) confirms that the conjugates extracted from the discrete bands in the gel contain the exact number of poly(dG)-poly(dC) molecules per nanoparticle. We have demonstrated that Klenow exo- catalyzes extension of poly(dG)-poly(dC) molecules connected to a Ag-NP, in the presence of dGTP and dCTP. This result corresponds nicely with earlier observations showing that Au-NP-bound DNA molecules canbemanipulatedwithvariousenzymes,suchasligases(14,15,26), polymerases (13), and restriction enzymes (10-12). The formation of conjugates between long (hundreds of base pairs) homogeneous dsDNA polymers and metal NPs has never been shown before. Due to its regular structure, a doublestranded poly(dG)-poly(dC) composed of G- and C-homopolymers provides better conditions for π overlap compared to random sequence DNA. In addition, guanines, which have the lowest ionization potential among DNA bases, promote charge migration through the DNA. This property is especially useful for application of the conjugates in nanoelectronics. Moreover, the polymerase-based method for synthesis used in this work can be effectively employed with the range of functionalities attached to the DNA terminals. In addition to the SH group used for attaching Au-NP, one can functionalize the DNA, for instance, with a random oligonucleotide sequence. The NP-DNA conjugates containing random single-stranded overhang sequences will be recognized by the complementary ones attached to different DNA-NP conjugates, thus connecting DNA wires to one another. These functionalized conjugates may serve a basis for the design and construction of two- and threedimensional DNA-NP structures. Such NP-DNA structures might possess interesting electrical and plasmonic properties and can be used in the fields of nanoelectronics and nano-optics.

ACKNOWLEDGMENT This work was supported by European Commission FP6 Information Society Technologies program, grant “DNABased Nanodevices”, and by the ISF Converging Technologies program, grant 1714/07.

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