Synthesis and Assembly of Conjugates Bearing Specific Numbers of

Apr 20, 2012 - (23) The conjugates of the synthesized thiolated DNA with nanoparticles bearing a different number of nucleic acid molecules per partic...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/bc

Synthesis and Assembly of Conjugates Bearing Specific Numbers of DNA Strands per Gold Nanoparticle Natalia Borovok,† Elad Gillon,†,‡ and Alexander Kotlyar*,†,‡ †

Department of Biochemistry, George S. Wise Faculty of Life Sciences and ‡The Center of Nanoscience and Nanotechnology, Tel-Aviv University, Ramat Aviv 69978, Israel ABSTRACT: Here, we present a relatively simple, efficient, and highyielding polymerase-based method for the synthesis of 15 nm gold nanoparticle conjugates bearing a specific number of 25 base oligonucleotide strands. We have shown that the conjugates bearing one or two oligonucleotide strands per particle, with the conjugates comprising a single complementary strand, self-assemble into nanoparticle dimers and trimers, respectively. Incubation of fully coated AuNPs, containing tens of oligonucleotide strands, with a conjugate bearing a single complementary strand leads to the formation of flower-shaped structures. The assembly of particles into nanoparticle structures shown here is a prerequisite for more complex controlled assembly of particles into three-dimensional macrostructures.

■

INTRODUCTION Conjugates of various metal nanoparticles including gold nanoparticles (AuNPs) with DNA1−4 and other biomaterials exhibit unique optical properties and have been used as bases for development of various applications in nanobiotechnology,5 nanomedicine, 6−9 material science, 10,11 and nanoelectronics.10−13 The ability of nucleic acids to self-assemble into well-defined supramolecular architectures offers a variety of possibilities for the design and production of novel two- and three-dimensionally organized DNA-based nanostructures.14,15 The use of DNA−nanoparticle conjugates bearing specific numbers of DNA strands as a starting material is of particular importance for the creation of novel nanomaterials. Alivisatos and colleagues demonstrated that binding of single-stranded oligonucleotides produces a significant effect on particle mobility in an electric field and succeeded in separating conjugates bearing different numbers of oligonucleotide strands per particle by electrophoresis.16,17 Increasing the particle size and decreasing the DNA length, however, substantially reduce the separation efficiency. Good separation of the conjugates can be achieved only for relatively small particles (2−10 nm) bearing relatively long (50−100 bases) sequences. Separation of conjugates of bigger AuNPs (diameter more than 10 nm) with relatively short (less than 50 bases) single- or double-stranded DNA molecules is very challenging. To overcome this separation problem and to produce stable DNA−nanoparticle conjugates, several enzyme-based methods have been recently employed.18−23 The polymerase-based approach was used to synthesize long double-stranded DNA, containing a thiol-group at the end of the polymer.23 The conjugates of the synthesized thiolated DNA with nanoparticles bearing a different number of nucleic acid molecules per particle have been efficiently separated from each other by electrophoresis. © 2012 American Chemical Society

The ligase-based method was also used to covalently join the ends of DNA molecules and thus produce a stable connection between the particles in DNA−nanoparticle conjugates.18,21 Another enzyme, the restriction endonuclease, was shown to disassemble the DNA−nanoparticle structures by cleaving the double-stranded DNA connections between particles.19 In this work, we present a novel polymerase-based method for the synthesis of AuNP−DNA conjugates bearing a specific number of oligonucleotide strands. We have demonstrated that the conjugates bearing complementary strands self-assemble into uniform multiparticle structures. The optical properties and the molecular morphology of individual AuNP−DNA conjugates and complexes between them were elucidated by AFM and TEM analysis.

■

EXPERIMENTAL PROCEDURES Unless otherwise stated, reagents and chemicals were obtained from Sigma-Aldrich (USA) and were used without further purification. Klenow fragment exonuclease minus of DNA polymerase I from E. coli lacking the 3′-, 5′-exonuclease activity (Klenow exo−) was purchased from Epicenter Biotechnologies (USA). DNA Samples. All DNA samples were purchased from Alpha DNA (Montreal, Canada). The following sequences were used in this study: 1, 5′-a10A5-3′ oligonucleotide comprising 10 phosphorothioated adenine nucleotides (a10) and 5 regular ones (A5); 2, 5′-GCTGACTATCCATCCTTCATCCTTGaaaaaaaaaa-3′ (R1-a10), containing a 25 base random sequence (R1) and 10 phosphorothioated adenine Received: September 5, 2011 Revised: April 8, 2012 Published: April 20, 2012 916

dx.doi.org/10.1021/bc200485r | Bioconjugate Chem. 2012, 23, 916−922

Bioconjugate Chemistry

Article

time, 10 mM of NaPi was added and the sample was incubated for 30 min at 50 °C. The particles were purified from the excess oligonucleotides and from a minor fraction of multiparticle structures formed during the incubation by size-exclusion chromatography as follows. The sample was loaded onto a Sepharose CL-6B 1.6 × 40 cm column equilibrated with 10 mM NaPi buffer, pH 7.5, at a flow rate of 0.5 mL/min. The particles were eluted in approximately half the column volume and were completely separated from the excess of nonbound a10A5 oligonucleotides. The particles were collected and concentrated by centrifugation at 10 000 rpm for 20 min on an Eppendorf Table 5424 centrifuge (Eppendorf, Germany). Concentration of the particles was calculated using an extinction coefficient of 7.4 × 108 M−1 cm−1 at 520 nm.27,28 TEM Measurements. TEM images were acquired by using carbon-coated grids (400 mesh). 2.5 μL of a sample in 40 mM Tris-Ac, pH 7.8, was dropped onto a grid surface. After incubation for 5 min at ambient temperature, the excess solution was removed by blotting with a filter paper. TEM imaging was performed on a TEM (JEM model 1200 EX instrument) operated at an accelerating voltage of 120 kV. The TEM analysis showed that the particles are uniform and spherical with diameters of 15 ± 2 nm. Atomic Force Microscopy. AFM was performed on the molecules adsorbed onto muscovite mica surfaces. A 20 μL aliquot of 1.0−2.0 nM AuNP−DNA solution, containing 2.0 mM MgCl2 and 25 mM NaCl, were deposited on freshly cleaved 1 cm2 mica plates for 5 min. The surface was then washed with distilled water and dried by nitrogen blow. AFM imaging was performed on a Solver PRO AFM system (NTMDT, Russia), in semicontact (tapping) mode, using 130 μm 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.29 Absorption Spectroscopy. Absorption spectra were recorded with a Jasco V-630 spectrophotometer (Japan).

nucleotides; 3, 5′-CAAGGATGAAGGATGGATAGTCAGCaaaaaaaaaa-3′ (R2-a10), containing a 25 base random sequence (R2) complementary to R1 (see above) and 10 phosphorothioated adenine nucleotides; 4, 5′-GCTGACTATCCATCCTTCATCCTTG-A15-3′ (R1-A15), containing a 25 base random sequence (R1) and 15 regular adenine nucleotides; 5, 5′-CAAGGATGAAGGATGGATAGTCAGCA15-3′ (R2-A15), containing a 25 base random sequence (R2) and 15 regular adenine nucleotides; 6, 15 base deoxythymidine oligonucleotide (dT)15. The oligonucleotides (∼1 mg) were dissolved in ∼200 μL of double-distilled water and subsequently passed through a prepacked Sephadex G-25 DNA-grade column (Amersham Biosciences) equilibrated with 2 mM Tris-Ac, pH 8.5. The oligonucleotide, eluted in the void volume, was collected in 0.4−0.5 mL and purified by ionexchange HPLC to homogeneity as described in our previous studies.24 Concentrations of the oligonucleotides were calculated using extinction coefficients at 260 nm of 356, 407, 475, 424, 140, and 122 mM−1 cm−1 for R1-a10, R2-a10, R2-A15, R1-A15, a10A5, and (dT)15, respectively. DNA Synthesis Reaction. Syntheses of long (hundreds of base pairs) double-stranded poly(dA)-poly(dT) homopolymers, containing either R1 or R2 sequence at 5′-end of the poly(dA) strand, 5′-R1-poly(dA)-poly(dT), and 5′-R2-poly(dA)-poly(dT), respectively, was performed as previously described.24,25 A standard reaction mixture contained the following: 60 mM KPi, pH 6.5, 5 mM MgCl2, 5 mM DTT, 1.5 mM dATP, 1.5 mM dTTP, 0.2 μM Klenow exo−, and HPLC purified template-primers, 5′-R1-(dA)15-(dT)15 or 5′-R2-(dA)15(dT)15. The enzymatic extension of a 15-base-pair doublestranded fragment of the template-primer was conducted for 2 h at 37 °C. The reaction was halted by the addition of EDTA to a final concentration of 20 mM. HPLC Purifications. The separation of synthesized DNA molecules from nucleotides and other reaction components was achieved with a TSK-gel G-5000-PW HPLC column (7.8 × 300 mm) from TosoHaas (Japan) by isocratic elution with 20 mM Tris-Ac, pH 7.5, in the presence of 0.1 M NaCl for 30 min at a flow rate of 0.5 mL/min. HPLC purification of the oligonucleotides and the synthesized DNA was conducted on a Finnigan Surveyor LC (Thermo Electron Corporation, USA) HPLC system with a photodiode array detector. Peaks were identified from their retention times obtained from the absorbance at 260 nm for DNA. Eluted products were concentrated by Amicon Ultra-30K-50K MWCO filter devices (Millipore, USA). The length of synthesized molecules was determined by 1.5% agarose gel electrophoresis. Electrophoresis. The DNA or DNA-nanoparticle samples were loaded onto 1.5% agarose gel 7 × 7 cm, and electrophoresed at 130 V for 40 min in an ice bath. TAE buffer, in addition to being used to prepare the agarose, also served as the running buffer. AuNPs. Spherical gold nanoparticles with a diameter of 15 nm were prepared by HAuCl4 reduction in the presence of citric acid essentially as described.26 The resulting nanoparticles were screened for their size and uniformity by TEM, revealing an average diameter of 15 ± 2 nm. The UV−vis spectra showed a characteristic absorption peak at 520 nm. The particles were coated with a10A5 (or in a few cases with R1-a10 or R2-a10) as follows. The particles with absorption of approximately 30 at 520 nm were incubated with 20 μM a10A5 in the presence of 10 mM NaPi buffer, pH 7.5, for 30 min at 50 °C. The buffer concentration was increased stepwise from 10 to 50 mM. Each

■

RESULTS The procedure of AuNP−DNA conjugate production (see Scheme 1) includes the following steps: 1. Synthesis of the DNA Polymer. The DNA polymer, 5′R1-poly(dA)-poly(dT), contains a 0.8 Kbp poly(dA)-poly(dT) and 25 base overhang fragment, R1 at the 5′-end of the poly(dA) strand (see stage 1 of Scheme 1). The molecules were synthesized using a polymerase-based method that we recently invented.24,25 This enzymatic method enables extension of short (tens of base pairs) blunt-ended double-stranded template primers into long (varying from several nanometers to several micrometers) uniform double-stranded homopolymers, poly(dA)-poly(dT) or poly(dG)-poly(dC), functionalized with oligonucleotide sequences or (and) other moieties at one or both ends of the DNA. 5′-R1-poly(dA)-poly(dT) was synthesized and purified by HPLC as described in the Experimental Procedures section. 2. Annealing of the Overhang End Fragment. The overhang end fragment, R1 of the synthesized polymer was annealed with the complementary sequence flanked by 10 phosphorothioated adenosine residues at its 3′ ends, R2-a10 (see stage 2 of Scheme 1). The synthesized polymer, 5′-R1917

dx.doi.org/10.1021/bc200485r | Bioconjugate Chem. 2012, 23, 916−922

Bioconjugate Chemistry

Article

Scheme 1. AuNP−Oligonicleotide Conjugate Preparation

Figure 1. Electrophoretic separation of AuNP-a10-R2-R1-poly(dA)poly(dT) conjugates. Lane 1: 0.1 μM AuNPs were incubated with 0.1 (lane 2), 0.2 (lane 3), and 0.4 (lane 4) μM of 0.8 Kbp a10-R2-R1poly(dA)-poly(dT) for 72 h at 42 °C in 10 mM NaPi buffer, pH 7.5, containing 0.1 M NaCl. Electrophoresis was conducted on 1.5% agarose gel (7 × 7 cm2) in TEA buffer at 130 V for 40 min in an ice bath.

DNA and the particles in the incubation. At 1 to 1 ratio, the major (most intensive) band corresponds to the conjugate containing one DNA molecule (Figure 1, lane 2); at higher ratios, the relative amounts of conjugates bearing 2, 3, and even larger numbers of DNA molecules are increased (compare lanes 2 and 4). In this study, we were mainly focused on the conjugates bearing one and two DNA molecules per particle. These conjugates were prepared by the incubation of AuNP with DNA at 1 to 2 molar ratio (see Figure 1, lane 3). The gel areas corresponding to mono- and bimolecular conjugates, bearing one and two DNA molecules per particle (two red bands indicated by arrows II and III in Figure 1), were cut out of the gel with a razor blade. Each of the colored gel slices was placed in a dialysis bag containing TAE buffer, and the compounds were electroeluted from gel. The molecular morphology of the conjugates was elucidated by AFM. The molecules were deposited onto freshly cleaved muscovite mica and imaged by AFM in a noncontact mode as described in Experimental Procedures. As seen in Figure 2, the vast majority of conjugates extracted from bands II and III (see Figure 1) are composed of 1 and 2 DNA molecules connected to a nanoparticle, respectively. The average estimated contour length of DNA molecules connected to the particles is equal to 250 ± 10 nm. This value corresponds well with the contour length of 0.8 Kbp poly(dA)-poly(dT) molecules.25 4. Removing R1-poly(dA)-poly(dT) from AuNP-a10-R2R1-poly(dA)-poly(dT). R1-poly(dA)-poly(dT) was separated from AuNP-a10-R2 during incubation of AuNP-a10-R2-R1poly(dA)-poly(dT) with 100-molar excess of R2 oligonucleotides for 10 min at 70 °C in the presence of 50 mM NaCl. This treatment results in a complete dissociation of the doublestranded DNA fragment from the nanoparticle. The sample was then centrifuged at 12 000 rpm for 30 min, and the supernatant containing R1-poly(dA)-poly(dT) was discarded. The pellet containing a10-R2-AuNP was resuspended and analyzed by electrophoresis. As seen in Figure 3, the band corresponding to the conjugate moves in the gel similarly to that of nonmodified AuNP (compare lanes 1 and 3, Figure 3). This clearly shows that the treatment results in dissociation of the DNA polymer from the particle. In order to confirm that a short

1: Synthesis of a 0.8 Kbp R1-poly(dA)-poly(dT), a double-stranded DNA polymer, containing a 25 base random overhang fragment sequence (R1, shown in blue) at the 5′-end of the poly(dA)-strand. 2: Hybridization of R2-oligonucleotide (shown in green), comprising 10 phosphorothioated adenine bases (shown in orange) at the 3′-end of the sequence, with R1-poly(dA)-poly(dT). 3: Formation of a complex between a10-R2-R1-poly(dA)-poly(dT) and AuNPs. 4: Separation of R1-poly(dA)-poly(dT) from AuNP-a10-R2 by heat treatment in the presence of R2.

poly(dA)-poly(dT), was mixed with 10-fold molar excess of R2-a10 and incubated for 0.5 h at room temperature in 20 mM Tris-Ac buffer, pH 7.5, in the presence of 0.1 M NaCl. The excess of nonbound R2-a10 was removed by size-exclusion HPLC as described in the Experimental Procedures. The eluted product, a10-R2-R1-poly(dA)-poly(dT), was collected and concentrated by ultrafiltration (see Experimental Procedures) . 3. Synthesis of AuNP−DNA Conjugates. A a10-R2-R1poly(dA)-poly(dT) polymer prepared as shown above was incubated with AuNPs in 20 mM Tris-Ac buffer, pH 7.5, in the presence of 0.1 M NaCl, for 48 h at 42 °C (see stage 3 of Scheme 1). The incubation yielded a mixture of AuNP−DNA conjugates bearing different numbers of DNA molecules per particle. In contrast to nanoparticles conjugated with short oligonucleotides, the mobility of the AuNP conjugates comprising long (hundreds and thousands of base pairs) DNA is governed by the nucleic acid content. The latter conjugates, bearing different numbers of DNA molecules, can, therefore, be completely and efficiently separated from each other by electrophoresis. Electrophoresis of the conjugate mixture in a 1.5% agarose gel is shown in Figure 1. The redcolored bands in the gel (see Figure 1) correspond to the conjugates bearing distinct number of a10-R2-R1-poly(dA)poly(dT) molecules per AuNP. As seen in the figure (lanes 2− 4), the relative amounts of conjugates comprising different numbers of DNA molecules depend on the ratio between the 918

dx.doi.org/10.1021/bc200485r | Bioconjugate Chem. 2012, 23, 916−922

Bioconjugate Chemistry

Article

Figure 2. AFM images of AuNPa10-R2-R1-poly(dA)-poly(dT) conjugates. The conjugates extracted from the gel (see Figure 1) were deposited on a mica surface in 2 mM MgCl2 and scanned in a semicontact mode as described in Experimental Procedures. Left and right panels present images of the conjugates extracted from bands II and III (see Figure 1), respectively.

AuNP-a10-R2-R1-poly(dA)-poly(dT) into R1-poly(dA)-poly(dT) and AuNP-a10-R2. The same approach was applied for synthesis of AuNP-conjugates bearing two R1, one R2, or two R2 strands. The conjugates containing complementary sequences (R1 and R2) were assembled to form nanoparticle dimers and trimers. We have shown (see Figure 4, lane 1) that mixing equal amounts of AuNP-a10-R1 and AuNP-a10-R2 (prepared as described above) together results in their association. The gel

Figure 3. Electrophoretic analysis of AuNP−DNA conjugates. AuNPs (lane 1) and a conjugate extracted from band II in Figure 1 (lane 2) treated for 10 min at 70 °C in the presence of 100 molar excess of a R2 (lane 3). Lane 4: A conjugate electroeluted from the gel area indicated by the arrow in lane 3 and incubated with 10-fold molar excess of R1poly(dA)-poly(dT) in the presence of 0.1 M NaCl for 1 h at 4 °C. Samples were run in 1.5% agarose gel in TEA buffer at 130 V for 40 min in an ice bath. Figure 4. Electrophoresis of AuNP assemblies. Lane 1: Equimolar amounts of AuNP-a10-R1 and AuNP-a10-R2 conjugates (each bearing a single oligonucleotide strand per particle) were incubated in 10 mM NaPi buffer, pH 7.5, containing 0.1 M NaCl for 16 h at room temperature. Lane 2: AuNP-a10-R2 was incubated with AuNP-(a10R1)2, a conjugate bearing two R2 strands, at a 2 to 1 molar ratio in identical conditions (see above). Lane 3: AuNPs. Samples were run in 1.5% agarose gel at 110 V for 40 min in 2×TAE buffer. The bands corresponding to AuNP assemblies are indicated by arrows.

oligonucleotide fragment (a10-R2) remained bound to the particle we have incubated the pellet fraction, containing the conjugate, with an excess of R1-poly(dA)-poly(dT). As seen in Figure 3 (compare lanes 3 and 4), the incubation results in restoring the band mobility back to the initial level, corresponding to AuNP-a10-R2-R1-poly(dA)-poly(dT). These results confirm that the heat treatment results in dissociation of 919

dx.doi.org/10.1021/bc200485r | Bioconjugate Chem. 2012, 23, 916−922

Bioconjugate Chemistry

Article

Figure 5. TEM images of AuNP-assemblies. The structures were electroeluted from gel areas indicated by arrows in Figure 4. Panel B: lane I. Panel C: lane II. Panel A: AuNPs. Moelcules were deposited on 400 mesh copper carbon grids and visualized by TEM. Below the images are schematic drawings of the structures.

Figure 6. Electrophoresis (panel A) and TEM image (panel B) of complexes between AuNPs fully coated with R1 and a monomolecular AuNP-a10R2 conjugate. The fully coated particles, AuNP-(a10-R1)n containing a large number (tens) of R1-strands were prepared as described in Experimental Procedures; AuNP-a10-R2 conjugate was prepared as described in Figure 3. Panel A: Lane 1, fully coated particles; lane 2, the fully coated particles were incubated with 10-fold excess of AuNP-a10-R2 in 10 mM NaPi buffer, pH 7.5, containing 0.1 M NaCl for 72 h at room temperature. The sample was loaded on 1.5% agarose gel and electrophoresed at 110 V for 40 min in an ice bath in 2×TAE buffer. Panel B: The slice corresponding to a band indicated by the arrow in panel A (lane 2) was cut out, and the molecules were electroeluted from the gel. They were subsequently deposited on 400 mesh copper carbon grids and visualized by TEM. Panel C: Schematic drawings of the structure.

By increasing the number of DNA strands in the conjugate, following the above strategy, one can gradually increase the number of particles in the cluster. We have shown that incubation of AuNPs fully coated with R1-oligonucleotide with more than 10-fold molar excess of the conjugates bearing a single R2-strand leads to the formation of a complex that moves through the gel more slowly than AuNPs (indicated by the arrow, Figure 6A, lane 2). The slice corresponding to the indicated complex was cut out of the gel, and the compound was electroeluted and analyzed by TEM. As clearly seen from the TEM image (Figure 6B), the structures are flower-shaped and consist of a central AuNP surrounded by several (5−6) peripheral particles. The central particle in the structure is connected to each peripheral one via a double-stranded R1-R2 linker.

area corresponding to the complex (indicated by arrow I, Figure 4) was cut out of the gel with a razor blade; the red compound was electroeluted and analyzed by TEM. As clearly seen in the TEM image (see Figure 5B), the structures are composed of a pair of particles. We thus suggest that the two complementary strands (R1 and R2) attached to different particles associate with each other forming a double-stranded DNA bridge between the particles (see drawing below panel B in Figure 5). We have also shown that incubation of a monomolecular AuNP-a10-R1 conjugate with a bimolecular one, AuNP-a10-(R2)2 (bearing two R2 strands), at a 2 to 1 molar ratio also results in the association of the conjugates (see lane 2, Figure 4). The complex was electroeluted from the gel area (indicated by arrow II, Figure 4) and imaged by TEM. As seen in the image (Figure 5, panel C), the structures are composed of three particles. The particles in the trimer are connected to each other by a double-stranded R1-R2 bridge (see drawing below panel C, Figure 5). 920

dx.doi.org/10.1021/bc200485r | Bioconjugate Chem. 2012, 23, 916−922

Bioconjugate Chemistry

■

Article

DISCUSSION In this study, we present a novel method for synthesis of AuNP conjugates bearing a specific number of short (25 base) ssDNA molecules. The method is relatively simple and characterized by high yield of individual conjugates. In the conjugates, DNA is anchored to the particle by phosphorothioated bases at the 5′end of the polynucleotide strand. We have demonstrated in our recent works that phosphorothioated oligonucleotides are capable of efficient covalent binding to silver 30 and gold nanoparticle surfaces.31 The method of NP−oligonucleotide conjugate production described here is not limited to AuNPs. We have shown (data not presented) that an identical approach can be applied for production of conjugates between silver nanoparticles and DNA. One can also vary over a wide range the diameter of NPs, as well as the length and the number of oligonucleotide strands in the conjugate. The efficiency of the DNA−NP conjugate separation in gels decreases as the particle size increases, and increases as the DNA length increases. Therefore, in order to prepare conjugates of bigger (diameter larger than 20 nm) particles, the length of poly(dA)-poly(dT) polymer can be increased to several thousand base pairs. The enzymatic method of extension24,25 allows us to synthesize tens of Kbase pair long dsDNA homopolymers. The use of very long poly(dA)-poly(dT), we believe, will make it possible to achieve good electrophoretic separation of individual conjugates of big (tens or even hundreds of nm) nanoparticles and other nanostructures (nanorods and nanoshells) with DNA. We have demonstrated that mixing of two monomolecular AuNP−DNA conjugates, each bearing a single DNA strand, results in the formation of a dimer (see Figure 5B). The complementary strands attached to different particles recognize each other and form a double-stranded DNA bridge between the particles. We have also shown that incubation of the AuNP conjugate bearing two DNA strands with a 2-fold molar excess of the conjugate functionalized with a single complementary sequence yielded nanoparticle trimers (see Figure 5C). By increasing the number of DNA strands in the conjugate, following the above strategy, one can increase the number of particles in the cluster. Indeed, mixing of the AuNPs bearing a single R2 sequence to AuNPs and the nanoparticle fully coated with R1-strands yields structures composed of a central nanoparticle and 5−6 peripheral ones (see Figure 6B). The results of preliminary experiments (data not presented) show that mixing together equal amounts of AuNP-(R1)2 and AuNP(R2)2 conjugates, each bearing two identical strands, yields chains of nanoparticles. Each particle in the chain is bridged to the two neighboring ones by a 25 bp double-stranded R1-R2 molecule. The results demonstrated here are a prerequisite for complex controlled self-assembly of nanoparticle conjugates bearing a large number of DNA strands into two- and three-dimensional macrostructures. The method of NP−oligonucleotide conjugate production described here allows one to vary the size of nanoparticles, the distance between them (by changing the length of a DNA linker), and a number of connections that each particle establishes with neighboring ones. Nanostructures composed of silver or gold nanoparticles, as well as heterogeneous structures composed of both types of particles can also be synthesized. We believe that these structures will possess interesting plasmonic and conductive properties and

will lead to development of new multiparticle functional materials.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by the Israel Science Foundation, 172/10. REFERENCES

(1) Mirkin, C. A., Letsinger, R. L., Mucic, R. C., and Storhoff, J. J. (1996) A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382, 607−609. (2) Alivisatos, A. P., Johnsson, K. P., Peng, X., Wilson, T. E., Loweth, C. J., Bruchez, M. P., Jr., and Schultz, P. G. (1996) Organization of ‘nanocrystal molecules’ using DNA. Nature 382, 609−611. (3) Loweth, C. J., Caldwell, W. B., Peng, X., Alivisatos, A. P., and Schultz, P. G. (1999) Angew. Chem., Int. Ed. 38, 1808−1812. (4) Dujardin, E., Hsin, L. B., Wang, C. R. C., and Mann, S. (2001) DNA-driven self-assembly of gold nanorods. Chem. Commun. 14, 1264−1265. (5) Goluch, E. D., Nam, J. M., Georganopoulou, D. G., Chiesl, T. N., Shaikh, K. A., Ryu, K. S., Barron, A. E., Mirkin, C. A., and Liu, C. (2006) A bio-barcode assay for on-chip attomolar-sensitivity protein detection. Lab Chip 6, 1293−1299. (6) Huang, X., El-Sayed, I. H., Qian, W., and El-Sayed, M. A. (2006) Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc. 128, 2115−2120. (7) Durr, N. J., Larson, T., Smith, D. K., Korger, B. A., Sokolov, K., and Ben-Yakar, A. (2007) Two-photon luminescence imaging of cancer cells using molecularly targeted gold nanorods. Nano Lett. 7, 941−945. (8) Huang, X., El-Sayed, I. H., Qian, W., and El-Sayed, M. A. (2006) Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc. 128, 2115−2120. (9) Yu Cheng, J. D., Meyers, A. M., Broome, M. E., Kenney, J. P., Basilion, and Burda, C. (2011) Deep penetration of a PDT drug into tumors by noncovalent drug-gold nanoparticle conjugates. J. Am. Chem. Soc. 133, 2583−2591. (10) He, Y., Zhang, S., Zhang, X., Baloda, M., Gurung, S., Xu, H., Zhang, X., and Liu, G. (2011) Ultrasensitive nucleic acid biosensor based on enzyme-gold nanoparticle dual label and lateral flow stop biosensor. Biosens. Bioelectron. 26, 2018−2024. (11) Alivisatos, P. A. (2008) Birth of a nanoscience building block. ACS Nano 2, 1514−1516. (12) Choi, C. L., and Alivisatos, A. P. (2010) From artificial atoms to nanocrystal molecules: preparation and properties of more complex nanostructures. Annu. Rev. Phys. Chem. 61, 369−389. (13) Burda, C., Chen, X. B., Narayanan, R., and El-Sayed, M. A. (2005) Chemistry and properties of nanocrystals of different shapes. Chem. Rev. 105, 1025−1102. (14) Kim, M. R., Kim, J. Y., and Jang, D. J. (2007) Electrochemical fabrication of arrayed alumina nanowires showing strong blue emission. Eur. Phys. J. D 43, 279−282. (15) Kumar, A., Pattarkine, M., Bhadbhade, M., Mandale, A. B., Ganesh, K. N., Datar, S. S., Dharmadhikari, C. V., and Sastry, M. (2001) Linear superclusters of colloidal gold particles by electrostatic assembly on DNA templates. Adv. Mater. 13, 341−344. (16) Zanchet, D., Micheel, C. M., Parak, W. J., Gerion, D., and Alivisatos, A. P. (2001) Electrophoretic isolation of discrete Au nanocrystal/DNA conjugates. Nano Lett. 1, 32−35. (17) Zanchet, D., Micheel, C. M., Parak, W. J., Gerion, D., Williams, S. C., and Alivisatos, A. P. (2002) Electrophoretic and structural 921

dx.doi.org/10.1021/bc200485r | Bioconjugate Chem. 2012, 23, 916−922

Bioconjugate Chemistry

Article

studies of DNA-directed Au nanoparticle groupings. J. Phys. Chem. B 106, 11758−11763. (18) Claridge, S. A., Mastroianni, A. J., Au, Y. B., Liang, H. W., Micheel, C. M., Fréchet, J. M. J., and Alivisatos, A. P. (2008) Enzymatic ligation creates discrete multinanoparticle building blocks for self-assembly. J. Am. Chem. Soc. 130, 9598−9605. (19) Kanaras, A. G., Wang, Z. X., Brust, M., Cosstick, R., and Bates, A. D. (2007) Enzymatic disassembly of DNA−gold nanostructures. Small 3, 590−594. (20) Kanaras, A. G., Wang, Z., Hussain, I., Brust, M., Cosstick, R., and Bates, A. D. (2007) Site-specific ligation of DNA-modified gold nanoparticles activated by the restriction enzyme StyI. Small 3, 67−70. (21) Kanaras, A. G., Wang, Z. X., Bates, A. D., Cosstick, R., and Brust, M. (2003) Towards multistep nanostructure synthesis: programmed enzymatic self-assembly of DNA/gold systems. Angew. Chem., Int. Ed. 42, 191−194. (22) Pena, S. R. N., Raina, S., Goodrich, G. P., Fedoroff, N. V., and Keating, C. D. (2002) Hybridization and enzymatic extension of Au nanoparticle-bound oligonucleotides. J. Am. Chem. Soc. 124, 7314− 7323. (23) Zikich, D., Borovok, N., Molotsky, T., and Kotlyar, A. (2010) Synthesis and AFM characterization of poly(dG)-poly(dC)-gold nanoparticle conjugates. Bioconjugate Chem. 21, 544−547. (24) Kotlyar, A. B., Borovok, N., Molotsky, T., Fadeev, L., and Gozin, M. (2005) In Vitro synthesis of uniform poly(dG)−poly(dC) by Klenow exo− fragment of polymerase I. Nucleic Acids Res. 33, 525−535. (25) Borovok, N., Molotsky, T., Ghabboun, J., Cohen, H., Porath, D., and Kotlyar, A. (2007) Poly(dG)−poly(dC) DNA appears shorter than poly(dA)−poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions. FEBS Lett. 581, 5843−5846. (26) Nasir, S. M., and Nur, H. (2008) Gold nanoparticles embedded on the surface of polyvinyl alcohol layer. J. Fund. Sci. 4, 245−252. (27) Reynolds, R. A., III, Mirkin, C. A., and Letsinger, R. L. (2000) Homogeneous, nanoparticle-based quantitative colorimetric detection of oligonucleotides. J. Am. Chem. Soc. 122, 3795−3796. (28) Jana, N. R., Gearheart, L., and Murphy, C. J. (2001) Seeding growth for size control of 5−40 nm diameter gold nanoparticles. Langmuir 17, 6782−6786. (29) Horcas, I., Fernandez, R., Gomez-Rodriguez, J. M., Colchero, J., Gomez-Herrero, J., and Baro, A. M. (2007) WSXM: a software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 78, 013705. (30) Lubitz, I., and Kotlyar, A. (2011) Self-assembled G4-DNA-silver nanoparticle structures. Bioconjugate Chem. 22, 482−487. (31) Lubitz, I., and Kotlyar, A. (2011) G4-DNA-coated gold nanoparticles: synthesis and assembly. Bioconjugate Chem. 22, 2043− 2047.

922

dx.doi.org/10.1021/bc200485r | Bioconjugate Chem. 2012, 23, 916−922