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G4-DNA-Coated Gold Nanoparticles: Synthesis and Assembly Irit Lubitz 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 describe the preparation of stable 15 nm gold nanoparticles (Au-NPs) coated with parallel-stranded G-quadruplexes (G4-DNA), comprising phosphorothioate residues on both sides of the DNA. Phosphorothioate residues located on the surface of the coated particles can anchor them to noncoated ones. Their incubation with more than 20-fold excess of 15 nm citrate-stabilized Au-NPs leads to the formation of flower-shaped structures comprising a central noncoated particle and five to six G-quadruplex-coated ones at the periphery, as revealed by TEM imaging analysis. The absorption band of the structures is shifted toward long wavelengths compared to individual particles not connected to each other. We show a strong dependence of plasmon coupling strength on the length of the DNA connecting Au-NPs.
’ INTRODUCTION NanoparticleDNA conjugates1,2 are widely used in nanoassembly,3,4 bionanotechnologies,57 and nanoelectronics.8,9 In a classical approach, thiol-containing DNA oligonucleotides are used to stabilize the Au-NPs. Covalent interaction between the thiol attached to the end of the DNA and noble metal atoms results in coating of the particle with DNA. These particles are stable and can bind particles coated with complementary sequences via hybridization. This reaction guided by DNADNA bimolecular recognition leads to the formation of various multiparticle structures.2,10,11 We have recently reported the synthesis of stable conjugates between silver nanoparticles and four-stranded G-quadruplexes.12 The conjugates were formed during incubation of silver particles with G-quadruplexes, containing phosphorothioate anchor residues at both ends of the DNA, at near-equimolar concentrations. In the conjugates, each pair of particles was connected by a G-quadruplex. Increasing the DNA-to-particle ratio in the incubation from 1 to 35 resulted in the formation of aggregates that fall out of solution. Here, we report that incubation of 15 nm gold nanoparticles (Au-NPs) with more than 15-fold molar excess of the G-quadruplexes, comprising phosphorothioate residues at both ends of the DNA, unexpectedly yields stable nonaggregated DNAcoated Au-NPs. Phosphorothioate residues located on their surface can, however, anchor them to citrate-stabilized Au-NPs. Incubation of a 30-fold excess of the latter particles with former ones leads to the formation of flower-shaped structures in which a central citrate-stabilized Au-NP is surrounded by several (56) DNA-coated ones. Interparticle interactions in the structure lead to a shift of the surface plasmon absorption resonance, which strongly depends on the length of the DNA molecules connecting Au-NPs in the structure. r 2011 American Chemical Society
’ EXPERIMENTAL PROCEDURES Unless otherwise stated, reagents were obtained from SigmaAldrich (USA) and were used without further purification. DNA Samples. The deoxyoligonucleotides, a5G5a5, a5G10a5, a5G20a5, were purchased from Alpha DNA (Montreal, Canada). The four-stranded G-quadruplexes composed of 5, 10, and 20 tetrad were prepared as described in our recent work.12 Synthesis of NPs. 15 nm Gold Nanoparticles. Au-NPs with a diameter of 15 nm were prepared by the reduction of HAuCl4 with sodium citrate essentially as described.13 100 mL of 1 mM HAuCl4 solution was heated in a 250 mL round-bottom flask to boiling on the hot plate under reflux. Then, 10 mL of a 38.8 mM sodium citrate solution was added quickly under vigorously stirring. The solution turns deep red in approximately 5 min after the addition of citrate. The solution was refluxed with stirring for an additional 15 min. The mixture was cooled down to room temperature and centrifuged at 10 000 rpm for 20 min at 20 °C in a Sorval SS-34 Rotor. A clear supernatant was carefully discarded, and the pellet was suspended in 45 mL of residual supernatant. The resulting nanoparticles were screened for their size and uniformity by TEM, revealing an average diameter of 15 ( 2 nm. The UVvis spectra showed a characteristic absorption peak at 520 nm. Concentration of the particles was calculated using an extinction coefficient (ε) of 4.2 108 M1 cm1 at 520 nm.14 60 nm Gold Nanoparticles. Au-NPs with a diameter of 60 nm were also prepared by the reduction of HAuCl4 with sodium citrate.13 50 mL of 0.01% HAuCl4 solution were heated in a 250 mL round-bottom flack to boiling on a hot plate under reflux. Received: May 17, 2011 Revised: August 21, 2011 Published: September 07, 2011 2043
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Bioconjugate Chemistry Then, 260 μL of 1% sodium citrate solution was added quickly under vigorously stirring. The solution turns purple in approximately 5 min after the addition of sodium citrate. The mixture was refluxed with stirring for additional 10 min and cooled down to room temperature. The particles were centrifuged at 4500 rpm for 5 min at 20 °C on a table Eppendorf centrifuge (model 5424). A clear supernatant was carefully discarded, and the pellet was suspended in 2 mL of residual supernatant. The resulting nanoparticles were screened for their size and uniformity by TEM, revealing an average diameter of 60 ( 5 nm. The UVvis spectra showed a characteristic absorption peak at 536 nm. Concentration of the particles was calculated using an extinction coefficient (ε) of 5.3 1010 M1 cm1 at 536 nm.15 Gel Electrophoresis. The DNA-NP samples were loaded onto 1.5% agarose gel 7 7 cm2 and electrophoresed at 4 °C at 130 V for 30 min. Tris-Acetate-EDTA (TAE) buffer, containing 40 mM Tris-Acetate and 1 mM EDTA, in addition to being used to prepare the agarose, also served as the running buffer. TEM Measurement. A 5 μL aliquot of a sample solution in 20 mM TAE (pH 8.8) was dropped onto a carbon-coated copper grid (400 mesh). The grids (before depositing) were negatively glow-discharged using an Emitech K100X glow discharger. After incubation for 5 min at ambient temperature, the excess solution was removed by blotting with filter paper. TEM imaging was performed on a TEM JEM model 1200 EX instrument operated at an accelerating voltage of 120 kV. Absorption Spectroscopy. Absorption spectra were recorded with a Jasco V-630 spectrophotometer (Japan).
’ RESULTS Here, we used a four-stranded G-quadruplex DNA containing a central fragment flanked by phosphorothioated adenine residues on either side to coat Au-NPs. The parallel-stranded tetramolecular G-quadruplexes were prepared using oligonucleotides containing 5, 10, or 20 central G-base fragment flanked by two runs of 5 phosphorothioated adenines on either side as described in our recent publication.12 Stable G-guadruplexes coated particles were obtained by gradually increasing NaCL concentration in a mixture of citrate-stabilized Au-NPs with 15-fold molar excess of the 10 tetrad quadruplexes as follows. The 15 nm particles (absorption is approximately equal to 30 at 520 nm) prepared as shown in the Experimental Procedures section were incubated at 42 °C with G-quadruplexes in the presence of 25 mM NaCL for 1 h. Then, the salt concentration in the incubation was increased to 100 mM, and then one hour later to 200 mM. The sample was incubated for another hour at 42 °C. No precipitate was formed during the incubation; the sample remained clear and red. In contrast, the incubation at DNA to particle ratios lying in the range from 2 to 5 leads to spontaneous aggregation of the particles and their precipitation out of the solution. These aggregates did not enter the gel (see Figure 1, lanes 1 and 2) in contrast to the particles obtained during the incubation at high (15 or higher) DNA to Au-NP ratios. The latter particles move as a relatively narrow single band in the gel (see Figure 1, lanes 3 and 4). We thus suggest that at high DNA concentrations (DNA to NP ratios) complete surface coverage is achieved before the particles collide and stick together (see schematic drawing in Scheme. 1). Due to steric reasons, the reactive phosphorothioate residues on the surface of a fully coated particle cannot bind to the metal core of another coated particle. No noticeable changes in the absorption spectrum were
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Figure 1. Electrophoresis of G-quadruplex-coated Au-NPs. 15 nm citrate-stabilized Au-NPs (absorption at 520 nm is equal to 30) were incubated with 2 μM (lane 1), 5 (lane 2), 15 (lane 3), and 20 μM (lane 4) 10 tetrad G-quadruplexes functionalized with phosphorothioated adenine residues at either side of the DNA molecule in the presence of 25 mM NaCL for 1 h at 42 °C. Then, the salt concentration was increased to 100 mM and one hour later to 200 mM. The samples were incubated for another hour at 42 °C and loaded onto a 1.5% agarose gel and electrophoresed at 130 V for 30 min at 4 °C.
Scheme 1. Schematic Drawing of Au-NPs (Red Spheres) Interaction with G-Quadruplexes (Four Closely Spaced Black Parallel Lines) Functionalized with Phosphorothioate Residues (Short Red Fragments at Each Sides of Each Black Line)a
a
(A) At near-stoichiometric concentrations, interaction of Au-NPs with the DNA molecules results in the formation of long nanoparticle chains. In the chain, the particles are connected by G-quadruplex molecules. (B) At high DNA concentrations (DNA to NP ratios), a complete surface coverage is achieved before the particles collide and stick together.
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Figure 2. Electrophoretic purification of NP-flowers. Citrate-stabilized Au-NPs (lane 1) coated with 5 tetrad G-quadruplexes (lane 2) and products of 2 h incubation of citrate-stabilized Au-NPs with 30-fold excess of 5 (lane 3) and 10 tetrad (lane 4) coated Au-NPs in 50 mM NaPi buffer (pH = 7.4) at 42 °C. 20 μL aliquots of each sample were loaded onto a 1.5% agarose gel and electrophoresed at 130 V for 30 min at 4 °C.
observed during incubation of the particles for 14 h at 42 °C in the presence of 0.2 M NaCL. These particles can be chromatographed in contrast to citrate-stabilized ones, which precipitate in columns. High stability enabled us to use size-exclusion chromatography to purify the particles from nonbound DNA and from a minor fraction of multiparticle structures formed during the incubation. The size exclusion chromatography also enabled us to prepare particles with narrow size distribution. The particles were eluted from Sepharose CL-6B column (16 350 mm) in 10 mM Na-Pi (pH 7.4) in two peaks (data not presented). The first peak was eluted close to the void volume of the column and contained violet-colored multiparticle complexes. The second peak was eluted approximately 10 mL after the first one and contained individual red Au-NPs. The latter peak was collected and the particles were concentrated by centrifugation at 10 000 rpm for 20 min on a table centrifuge. The morphology of the particles was elucidated by TEM. As seen in Figure 3A, the particles are uniform and spherical with diameters of 15 ( 0.5 nm. Despite their inability to interact with each other, the G-quadruplex-coated particles can efficiently bind to citrate-stabilized ones. Incubation of these two types of particles at near-equal concentrations yielded large multiparticle aggregates which do not enter the electrophoretic gel (data not shown). Incubation of 30-fold excess of the quadruplex-coated particles with citratestabilized ones, however, resulted in the formation of uniform structures that move as a narrow violet band through the gel (see Figure 2, lanes 3 and 4). The slice corresponding to the band was cut out of the gel with a razor blade. After electroelution into a dialysis bag, the sample was subjected to TEM analysis. As seen in the TEM image (see Figure 3B), the structures comprise a central particle and 56 ones in the periphery. They resemble
Figure 3. TEM images of (A) 15 nm G-quadruplex coated particles and (B) NP-flowers. The particles were prepared by incubation of 2 μM citrate-stabilized Au-NPs with 20 μM 10 tetrad G-quadruplexes functionalized with phosphorothioated adenine residues as shown in Figure 1. NP-flowers were prepared by incubation of citrate-stabilized Au-NPs with 30-fold excess of Au-NPs coated with 10 tetrad G-quadruplexes as shown in Figure 2. The slice corresponding to a violet band (see Figure 2, lane 4) was cut out of the gel with a razor blade, electroeluted into a dialysis bag, deposited on 400 mesh copper carbon grids, and visualized by TEM. The insert is an enlarged image of one of the flowers.
blue wildflowers, so we will refer to them as “NP-flowers” throughout the work. We have shown that the absorption spectrum of NP-flowers is red-shifted with respect to that of Au-NPs not connected to each other (compare blue and black curves in Figure 4). The red-shifted absorption is due to dipolar electromagnetic coupling between the plasmons of closely spaced nanoparticles in the flower. To investigate the dependence of the coupling strength on the distance between particles, we prepared Au-NPs coated with 5, 10, and 20 tetrad G-quadruplexes functionalized with phosphorothioated residues at both ends as described above (see Scheme 1). We have shown that the stability and the reactivity of Au-NPs are independent of the length of G-quadruplex molecules. To prepare NP-flowers, 15 nm citrate-stabilized Au-NPs were incubated with 30-fold excess of the 5, 10, or 20 tetrad quadruplex-coated 15 nm 2045
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Bioconjugate Chemistry Au-NPs. The products were separated by the electrophoresis. As clearly seen in Figure 2 (lanes 34), incubation of particles coated with 5 or 10 tetrad G-quadruplexes results in the appearance of a violet band corresponding to NP-flowers. We have shown by TEM (data not presented) that the average number of particles is independent of the length of DNA and is approximately equal to 6. The length of G-quadruplex molecules has, however, a noticeable effect on the shape of the absorption spectrum of the flowers. As seen in Figure 4, the spectrum of NP-flowers made of 20 tetrad G-quadruplexes is similar to that of Au-NPs not connected to each other (compare red and black curves). The spectrum of the 10 tetrad-based NP-flowers is, however, noticeably red-shifted compared to that of individual Au-NPs (compare green and black curves in Figure 4). The shift is even more pronounced for the 5 tetrad-based structures (blue curve in Figure 4). These results are in line with the dependence
Figure 4. Absorption spectra of NP-flowers. 15 nm Au-NPs (black curve) and 20 (red curve), 10 (green curve), and 5 (blue curve) tetradbased NP-flowers. The particles and the flowers were prepared as shown in Figures 1 and 2, respectively. The samples were electroeluted from the gel and measured.
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of plasmon coupling strength on the distance between metal particles (for review, see refs 1618). We have also synthesized NP-flowers composed of a big (60 nm) central Au-NP particle and smaller (15 nm) peripheral ones. These flowers were prepared by incubation of a 30-fold molar excess of the 10 tetrad quadruplex-coated 15 nm Au-NPs with citrate-stabilized 60 nm ones using the experimental strategy described above. The reaction mixture was electrophoresed, the area of the gel corresponding to the blue band (see Figure 5A) was cut out of the gel, and the structures were electroeluted and analyzed by TEM. As can be clearly seen in the TEM image (see Figure 5B), the structures are composed of many peripheral particles surrounding a bigger central one. Some of peripheral particles are seen as dark spots on top of a central one. Image analysis of more than hundred individual structures revealed that the average number of particles in these NP-flowers is equal to 10 ( 5. As expected, the absorption plasmon band of the flowers is broader and red-shifted compared to that of the particles not connected to each other (compare red curve with blue and black ones in Figure 5C).
’ DISCUSSION We have demonstrated that incubation of Au-NPs with a strong excess of G-quadruplexes, comprising phosphorothioate residues, yields stable nanoparticles. These particles do not aggregate even at relatively high (0.2 M) salt concentrations and can be purified and analyzed by electrophoresis and chromatography. Reactive phosphorothioate residues situated on the particle surface covalently bind to gold and silver atoms and can covalently attach the particles to metal electrodes, and other noncoated or weakly coated metal particles. This property can be used for covering metal surfaces and metal electrodes with densely packed particles. The particles can also be used to anchor long DNA molecules to metal surfaces. The method of nanoparticle coating described here is not limited to gold nanoparticles. We produce a similar coating protocol for coating silver particles as well as quantum nanodots. These particles can be assembled into NP-flowers. We have shown that addition of a small amount (less than 10%) of citrate
Figure 5. Preparation and properties of big NP-flowers: (A) Electrophoretic separation of NP-flowers from individual Au-NPs. 15 nm Au-NPs coated with 10 tetrad G-quadruplexes (lane 1) and products of 60 nm citrate-stabilized Au-NPs incubation with 30-fold excess of 15 nm Au-NPs coated with 10 tetrad G-quadruplexes (lane 2) in 50 mM Na-Pi buffer (pH = 7.4) for 2 h at 42 °C. 20 μL aliquots of each sample were loaded onto a 1.5% agarose gel and electrophoresed at 130 V for 30 min at 4 °C. (B) TEM image of the flowers. A slice corresponding to the violet band (A, lane 2) was cut out of the gel, and the structures were electroeluted, deposited on 400 mesh copper carbon grids, and visualized by TEM. (C) Absorption spectra of 15 nm Au-NPs coated with 10 tetrad G-quadruplexes (black curve), 60 nm citrate-stabilized Au-NPs (blue curve), and NP-flowers (red curve) electroeluted from the gel (see panel A, lane 2). 2046
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Bioconjugate Chemistry Au-NPs to G-quadruplex-coated particles results in the formation of NP-flowers. In the flower, the central citrate Au-NP is surrounded by several quadruplex-coated ones (see Figures 3B and 5B). The flowers composed of identical 15 nm particles are seen in the TEM image (see Figure 3) as planar 2D structures. We have analyzed hundreds of TEM images, but no structures containing additional particles on top of a central one have been found. In contrast, the “flowers” composed of 60 and 15 nm Au-NPs contain many smaller particles on top of a central bigger one (see Figure 5B). We can speculate that a planar arrangement is thermodynamically most favorable due to the repulsion of negatively charged peripheral particles in the NP-flower. We also cannot exclude the possibility that the preference of planar arrangement is governed by interaction of the NP-flower with the surface and that in aqueous solutions the flowers are shaped differently. Further theoretical and experimental studies are needed to address this issue. We have demonstrated that the absorption spectrum of NP-flowers is red-shifted with respect to that of Au-NPs not connected to each other (see Figures 4 and 5C). The red shift is due to a strong dipolar electromagnetic coupling between the plasmons of closely spaced nanoparticles in the NP-flower. It is well-known that the coupling strength and extent of the coupling-induced red shift increases with decreasing interparticle distance within an individual structure.19,20 Indeed, we have shown that the degree of the red shift strongly depends on the length of the G4-DNA linker connecting Au-NPs in the flower. G4-DNA structures are much more stable and rigid compared to canonical ds DNA. Even short G-quadruplexes composed of 510 tetrads are stable at room temperature in contrast to corresponding double-helical DNA molecules that dissociate into single strands. This enabled us to bring the particles very close together and to investigate plasmon coupling in NP-flowers. The shift is very pronounced for NP-flowers composed of the particles coated with 5 tetrad G-quadruplexes, and is negligible for those composed of 20 tetrad G-quadruplex-coated ones (see Figure 4). This suggests that the strength of coupling in the flower drops with increasing interparticle separation distance from 1.6 (the length of the 5 tetrad G-quadruplex)21 to 6.4 nm. The shift in the plasmon absorption of the NP-flowers from 520 nm (absorption maximum of Au-NPs) to the direction of the biological window along with high stability of the structures make them potentially useful for laser photothermal diagnostics and therapy.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by European Commission FP6 Information Society Technologies program, grant “DNA- Based Nanodevices’’, and by the Israel Science Foundation, grant 172/10. ’ REFERENCES
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