Light-mediated Directed Placement of Different DNA Sequences on Single Gold Nanoparticles

Abstract

This paper describes the light-directed functionalization of anisotropic gold nanoparticles with different thiolated-DNA oligomer (oligo) sequences. The starting nanoconstruct are gold nanostars (AuNS) uniformly grafted with one oligo sequence that are then exposed to fs-laser pulses at the plasmon resonance of the branches. The excitation selectively cleaves Au-S bonds at the tips of the branches to create vacant areas for functionalization with a different thiolated oligo sequence. Nanoconstructs synthesized by this approach present one oligo sequence on the AuNS body and branches and a different sequence at the tips. Furthermore, this architecture enables the formation of nanoparticle superstructures consisting of AuNS cores and small Au satellite nanoparticles at controlled locations after DNA hybridization. Our approach enables selective engineering of oligo presentation at the single-particle level and opens prospects for sophisticated design of nanoscale assemblies that are important in a wide range of biological applications.

Graphical abstract

Core-satellite metal nanoparticle assemblies are emerging as nanoconstructs for sensing,^{1, 2} imaging,^{3, 4} and therapeutics.^{5, 6} Gold nanoparticles (AuNPs) are advantageous as building blocks for assembled structures because they can be synthesized in a wide variety of shapes and sizes^{7, 8} and functionalized with surface ligands containing a thiol group.^{9, 10} Thiolated oligonucleotides (oligos) are effective linkers for bio-programmable nanoparticle constructs because the sequences are tailorable, and hybridization between complementary strands is reversible.^{6, 11–13} Although spheres are the most common shape of oligo-functionalized AuNPs, control over the placement of different oligos at specific locations is challenging because of the uniform surface curvature and reactivity; also, the chemical and physical properties of different sequences of DNA are similar.^{14, 15} Methods have been developed to create AuNPs with regio-selective surface ligands by phase segregation, where oligos and polymers each cover different regions of the AuNP; however, this process introduces hydrophobic ligands that can cause NP aggregation.^16–19 Other approaches use blocking agents to prevent oligo functionalization in specific areas²⁰ or pre-assembled DNA templates to orient oligos with respect to each other,²¹ but they cannot control oligo location on a single NP or facilitate the attachment of different oligo sequences.

AuNPs with anisotropic shapes offer a complementary approach to construct building blocks for assemblies by functionalizing specific structural features with surface ligands.^{13, 22–24} Nanorods and nanoprisms terminate in different crystal facets with different binding energies for thiolated oligos that enable selective functionalization.^{12, 25–29} However, the synthesis of seed-mediated anisotropic AuNPs usually requires capping agents such as cetyl trimethyl ammonium bromide (CTAB), which have two key drawbacks for biological applications: (1) the surfactant is cytotoxic; and (2) the molecular bilayer formed binds tightly to the Au surface and inhibits functionalization of AuNPs with thiolated oligos.^{30, 31} Furthermore, the placement of oligos on the desired crystal facets depends sensitively on ligand concentration and incubation time, which could lead to oligos binding non-specifically.^{13, 29, 32–34}

Highly anisotropic AuNPs support multiple localized surface plasmon (LSP) resonances that can be individually excited to produce localized heat and hot electrons at specific wavelengths.^35–38 Excitation of LSPs has been exploited in the design of AuNPs for the delivery and release of therapeutic oligos.³⁹ Light irradiation at the LSP resonance can thermally dehybridize doublestranded DNA grafted to particle surfaces,^{38, 40} release single-stranded DNA by reshaping gold nanorods into spheres,^{33, 41} and excite hot electrons that can cleave Au-S bonds.^{36, 39} Gold nanostars (AuNS) are structures with multiple branches and sharp tips that show strong, concentrated near-field enhancement at their LSP wavelengths.^{39, 42–44} Previously, we demonstrated that therapeutic oligos (DNA aptamers) can be released from AuNS inside cancer cells near the cell nucleus under fs-pulses; notably, the AuNS maintained its shape both in vitro and after light irradiation.³⁹

Here we show a light-mediated approach to direct the placement of different DNA strands onto specific regions of AuNPs (Scheme 1). AuNS densely functionalized with single-stranded DNA are the starting nanoconstructs and subjected to fs-pulsed light at the LSP resonance of a branch to release DNA selectively from the tips. The extent of DNA cleavage is controlled by laser power and irradiation time. The ligand-free areas at the tips of the AuNS can then be functionalized with a second thiolated DNA sequence to generate nanoconstructs with two different oligo sequences at the body and tips of the AuNS. This site-selective functionalization of AuNS with different oligo sequences can facilitate the assembly of AuNPs into complex superstructures.

Scheme 1. DNA replacement on tips of AuNS branches.

DNA1 (grey) releases from the surface upon irradiation, leaving space at the tips for DNA2 (red) to conjugate.

AuNS were synthesized in a one-pot reaction with the biocompatible Good’s Buffer, HEPES, which acts as both the capping and reducing agent.⁴⁵ The heterogeneous mixture of AuNS shapes resulted in an average hydrodynamic diameter of ~40 nm and a peak LSP resonance near 800 nm (Figure 1a). Transmission electron microscopy (TEM) analysis on this AuNS synthesis procedure revealed that particles with three or more branches make up ~70% of the AuNS population.⁴⁶ Figures 1b–c show typical seven and six-branched particles, respectively. Finite-difference time-domain (FDTD) calculations of AuNS shapes under 820-nm light showed high electric field intensities at the tips of the branches aligned with the polarization of light (Figure 1d–e; Figure S1). Minimal enhancement occurred at the long branches not aligned with the light polarization or short branches that did not have LSP resonances at 820 nm.

Figure 1. Simulations of near-field enhancement of AuNS from 820-nm light irradiation.

(a) UV-Vis absorbance spectra of bulk AuNS solution (normalized to highest peak). (b-c) TEM images of AuNS with (b) one and (c) two long branches. (d-e) FDTD simulations show the near-field enhancement of plasmonic gold nanostars is unevenly distributed among branches when irradiated at the LSP of the bulk solution, which leads to selective tip DNA replacement.

AuNS were first fully functionalized with sequence 1 (DNA1; 5’ SH-R-TTT TTT TTT TTT TTT TTT TTT TTT 3’; Supporting Information, Methods). DNA1@AuNS (1900 ± 100 DNA1 strands/AuNS) in water were irradiated with a 1 kHz Ti:sapphire laser at 800 nm for either 4 or 10 s. Since AuNPs reshape in aqueous solution near 1.5 W/cm²,^41–47 which we also confirmed (Figure S2), we used lower powers (0.3, 0.5, 0.7, and 0.9 W/cm²) to break the Au-S bond and release the oligos from the AuNS tips.

Figure 2 shows how the amount of DNA1 released from AuNS is controlled by both irradiation time and laser power. At 0.3 W/cm² for 4 s, 200 ± 60 DNA1 strands were cleaved from the AuNS (10% of starting amount), and at 0.9 W/cm² for the same irradiation time, 1300 ± 90 DNA1 strands were cleaved (68%). At low laser powers and shorter irradiation times (≤0.9 W/cm², 4 s), the AuNS released DNA1 with unobserved or only minor reshaping of the NP cores (Figures S2-S3; Table S1). At higher laser powers and longer irradiation times (≥0.5 W/cm², 10 s), AuNS reshaped into spherical particles (Figures S2-S3; Table S1); therefore, DNA released only from the tips cannot be differentiated from the total amount of cleaved DNA at these higher irradiation powers because of contributions from reshaping (Figure S4).

Figure 2. Higher laser power and longer irradiation times increase DNA release from AuNS.

As laser power (0.0, 0.3, 0.5, 0.7, or 0.9 W/cm²) and exposure time (4 or 10 s) increases, more DNA releases from the AuNS. P-values were calculated using a T-test. All samples had p-values <0.05 in comparison to non-irradiated DNA1@AuNS (0% release).

Following irradiation of AuNS to release DNA1, AuNS were functionalized with a second thiolated DNA oligo sequence (DNA2; 5’ TCCATGACGTTCCTGACGTT-R-SH 3’) (Supporting Information, Methods). To quantify oligo attachment during this second round of conjugation, we added a Cy3 fluorophore to the 5’ end of DNA2 (Cy3-DNA2). We found that to avoid any additive adsorption of DNA2 oligos between DNA1 strands on the AuNS body and to ensure that DNA2 only adsorbed on the newly exposed bare regions, AuNS must be densely functionalization with DNA1 (>1900 strands/AuNS) prior to irradiation (Figure S5).

Figure 3 shows that the amount of DNA1 released during irradiation is similar to the amount of Cy3-DNA2 after 2 h of incubation in a salt solution (Supporting Information, Methods). The slightly higher than one-to-one replacement of Cy3-DNA2 with increased power and conjugation time (average 10% increase) may be attributed to a combination of the tips having a small, positive radius of curvature^{48, 49} and the oligo(ethylene glycol) spacer between the thiol and nucleotide bases in DNA2 having less electrostatic repulsion than the charged DNA1 backbone.⁵⁰ A 24-h incubation period increased the amount of DNA2 on the surface by an additional average of 30% for each power and time condition (Figure S6), which suggests that longer incubation times allow for increased density of DNA2 at the AuNS tips. In contrast, without high concentrations of salt (0.6 M Na⁺) during incubation, little DNA2 functionalization was observed (Figures S7-8). Moreover, Cy3-DNA2 was not detected on non-irradiated, AuNS fully functionalized with DNA1 (Figure 3); hence, there was no exchange of DNA1 for DNA2 during the second round of functionalization.

Figure 3. Irradiated DNA1@AuNS add DNA2 strands to the surface through a second round of functionalization.

DNA1@AuNS were irradiated at 0, 0.3, 0.5, 0.7 or 0.9 W/cm² to release DNA1 from the surface of AuNS. The particles then went through a second round of functionalization in a 0.6 M [Na⁺] solution for 2 h to add DNA2 to the vacant spaces. p-values were calculated using a t-test. All samples had p-values < 0.01 compared to non-irradiated DNA1@AuNS (0.0 W/cm²).

Because the NP sizes are below the diffraction limit at visible wavelengths, the Cy3-labeled DNA2 used for optical quantification cannot be used to characterize the location of DNA2 on the AuNS. Therefore, we attached 5-nm spherical AuNPs by DNA hybridization (DNA4@spheres) to DNA2 and then used TEM to visualize the assemblies.^{6, 51-54} To overcome NP-to-NP electrostatic repulsion from the negatively charged DNA shells and to increase hybridization within the assemblies,⁵³ we used a linker DNA (DNA3) complementary to both DNA2 on the AuNS and DNA4 on the 5-nm AuNPs (Figure 4a; Scheme S2).

Figure 4. DNA4@AuNP_5-nm hybridize to DNA2 on AuNS.

(a) Scheme of DNA2, DNA3, and DNA4 hybridization. (b-e) TEM images of DNA4@spheres hybridized to (b) DNA1@AuNS (DNA1@AuNS), (c) irradiated DNA1@AuNS (0.5 W/cm² for 4 s) followed by a second round of functionalization to place DNA2 at the tips and keep DNA1 at the body (DNA1_Body⁻DNA2_Tips@AuNS), (d) irradiated DNA2@AuNS (0.5 W/cm² for 4 s) followed by a second round of functionalization to place DNA1 at the tips and keep DNA2 at the body (DNA2_Body⁻DNA1_Tips@AuNS), and (e) DNA2@AuNS.

Figures 4b-d indicate the locations of DNA2 strands via hybridization with DNA4@spheres on three different AuNS nanoconstructs with different presentations of DNA1 and DNA2. Figure 4b shows a control experiment for the hybridization process; the association of DNA4@spheres occurs for all regions of AuNS functionalized with DNA2 (DNA2@AuNS) (Figures S9 and S10; Supporting Information, Methods). Figures 4c-d each show irradiated AuNS (0.5 W/cm² for 4 s) with selective placement of the second DNA oligo. In Figure 4c, DNA1@AuNS were first irradiated and then functionalized with DNA2 (DNA1_Body-DNA2_Tips@AuNS) similar to that in Figure 3 but without Cy3. These assemblies show spheres around the tips of the AuNS branches (Figure S11), locations that are consistent with the calculated near-field enhancement (Figure 1, Figures S1 and S12). In contrast, Figure 4d shows assemblies with the opposite configuration, where DNA2 surrounds the body (indicated by 5-nm spheres), and DNA1 is at the tips (DNA2_Body-DNA1_Tips@AuNS). These nanoconstructs demonstrate the specificity of the DNA2 placement and selective tip functionalization.

In summary, we developed an optical approach to direct the placement of two different DNA sequences on a single AuNP. Pulsed laser excitation at the LSP wavelength of the plasmonic AuNS can release DNA from the branch tips that can then be functionalized with a second thiolated DNA sequence. This biocompatible approach allows for regio-selective placement of multiple oligo sequences while preserving the properties of an oligo-only ligand shell that can enable unique assemblies of NP superstructures via DNA hybridization. We expect that the ability to position NPs relative to each with molecular precision will facilitate the design and synthesis of complex nanoconstruct architectures for sub-cellular sensing and imaging.