Oligonucleotide length- and probe number-dependent assembly of gold nanoparticle on triangular DNA origami

Abstract

Effects of oligonucleotide length and probe number on the assembly of gold nanoparticles (AuNPs) with DNA origami templates were investigated for the first time. The optimal length and probe number are proposed to efficiently anchor AuNPs for the construction of plasmonic chiral nanostructures.

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DNA is a biomolecule that carries most of the genetic information in living organisms. The inheritance of biological characteristics from one generation to the next greatly relies on the replication of DNA molecules via the complementary Watson–Crick base-pairing of the four bases: adenine (A), guanine (G), cytosine (C), and thymine (T).¹ Because the self-assembly of DNA strands strictly obeys the base-pairing rules, it is allowable to rationally design the base sequence of the polynucleotide chain for the construction of precisely controlled nanostructures.² The manufacture of DNA based nanostructures has attracted widespread interest since the concept was proposed by Nadrian Seeman in the early 1980s.²

DNA origami is a DNA-based 2-dimensional (2D)^3–5 or 3-dimensional (3D) nanoscale structure,^6–9 which is assembled by folding a long single-strand virus DNA with multiple short “staple” DNA strands. The complementary binding of the pre-designed “staple” strands on defined locations of the long DNA molecules leads to the fabrication of various nanostructures such as smiley faces, triangles, squares,³ maps of China,¹⁰ and even 3D boxes.^8,11 It has been demonstrated in a number of recent studies that DNA origami is very promising in drug delivery vessels,^12–15 enzyme immobilization,^16–18 programmable molecule-transporting nanorobots,^19,20 and super-resolution molecular rulers.²¹ Since the sequences and the ends of “staple” DNA strands can be easily modified at the nucleotide level, it is possible to rationally design binding sites with regular spaces or unique geometries on DNA origami to achieve the subsequent assembly of other components.²² The most attractive application is the use of DNA origami as a template to immobilize plasmonic nanoparticles for controllable optical properties.²³ Due to their capability to support surface plasmon resonance and easy surface modification using thiolated single-strand DNA (ssDNA), gold nanoparticles (AuNPs) have been accurately positioned on DNA origami to achieve unique plasmonic chirality for surface-enhanced Raman scattering applications.^24,25 Several joint ssDNA strands were allowed to stretch out from DNA origami templates as the probes at the defined positions. Subsequently, AuNPs functionalized with oligonucleotide strands, which are complimentary to the joint strands, were assembled at the specific locations via base pairing.²³ Undoubtedly, the effective anchoring of the AuNPs on the origami is of great importance for the construction of plasmonic chiral nanostructures.

It is well-known that the length of ssDNA determines its secondary structure, consequently affecting the efficiency and specificity of the hybridization. The probe number on a DNA-anchoring template could also influence the capture efficiency of a target oligonucleotide. Therefore, the fabrication of DNA-origami based plasmonic nanostructures may largely depend on the length and the number of the probe ssDNA stretched out from the origami templates, as well as the length of oligonucleotides immobilized on AuNPs. However, so far no specific study has been conducted to investigate the roles of oligonucleotide length and probe number in the assembly of AuNPs on DNA origami templates.

DNA origami has been used as a molecular scaffold and nanoscale ruler for the assembly of metallic NPs together with organic fluorophores to study the corresponding distance-dependent photonic interactions.²⁶ In the present work, we plan to investigate the influences of the lengths of AuNP surface-immobilized oligonucleotide and the origami-attached capture probe, as well as the number of the capture probes on the binding efficiency of AuNPs on the origami templates. Herein, triangular DNA origami was prepared using single-stranded M13mp18 DNA and staple strands, with a joint strand stretched out at the middle of one side (Fig. S1 in ESI†). Thiolated oligonucleotide strands, which are complementary to the probe ssDNA, were modified on the surface of AuNPs with an average diameter of 14 nm. Atomic force microscopy (AFM) image illustrates the triangular shape with the thickness of 1.6 nm and the side length of ∼110 nm (Fig. S2a and d in ESI†), matching well with those of M13mp18 DNA origami triangles reported in previous works.^5,22,26 The existence of 60-mer single joint strands does not obviously change the morphologies and the heights of the triangles (Fig. S2b and e in ESI†). After assembly of AuNPs a bright dot can be observed at one side of each triangle. The section curve of a typical triangle shows that a particle with the height of ∼13 nm is attached on the template (Fig. S2c and f in ESI†), strongly proving the successful binding of AuNPs on the DNA origami. To explore influences of the oligonucleotide length and probe strand number on the origami-templated assembly of AuNPs, three sets of experiments were designed (Scheme 1): (1) the AuNPs modified with 20/40/60-mer oligonucleotide chains were assembled on the triangular DNA origami containing a 60-mer complementary joint strand; (2) the 20-mer oligonucleotide-immobilized AuNPs were assembled on the triangular DNA origami with a 20-, 40- or 60-mer complementary joint strand; (3) the 20-mer oligonucleotide-immobilized AuNPs were assembled on the triangular DNA origami with three 20-mer complementary probe strands.

Scheme 1 Schematic representation of the binding of an oligonucleotide-AuNP on a triangular DNA origami template with complementary probes.

The surface functionalization of the AuNPs was characterized using UV-vis spectrometry, hydrodynamic size and zeta potentials (Fig. S3 in ESI†). The surface immobilization of oligonucleotides does not significantly change the UV-vis peak wavelength, suggesting the good dispersibility of the functionalized AuNPs. As the increase of the strand length, the hydrodynamic size of the functionalized AuNPs gradually enhances, accompanying with the reduction of their zeta potentials. The data indicate the successful attachment of the 20-, 40- or 60-mer ssDNA strands on the nanoparticle surface, respectively. In the first set of experiments, DNA origami templates, which possess one 60-mer probe strand at the side of each triangle, were mixed with the as-prepared ssDNA-modified AuNPs to investigate the length effects of AuNP-surface attached oligonucleotides on the assembly process. Surprisingly, the addition of origami templates into the 40- or 60-mer AuNPs solution induces an obvious colour change from pink to dark purple immediately. The UV-vis absorption peak of the AuNPs suspension becomes broader and exhibits a significant red shift (Fig. 1), showing the severe aggregation of the AuNPs.^27,28 It should be noted that long ssDNA molecules are more favourable to form secondary structures in the solution for the achievement of lowest energy state.²⁹ The 40- and 60-mer ssDNA may be possibly immobilized on AuNPs surface in a folded conformation. The density of surface anchoring ssDNA on the nanoparticles may be not quite high because of the great steric hindrance of neighbour ligands.²⁷ In the presence of complementary probe strands, the secondary structures of the molecules are open to expose the unoccupied sites on the AuNPs surface, consequently resulting in the nanoparticle agglomeration (Fig. S4 in ESI†). Thus, the length of AuNP-immobilized oligonucleotides is very critical to the construction of DNA origami-based metamaterials. According to the data, 20-mer ssDNA should be selected since it can balance both the nanoparticle stability and the assembling specificity.

Fig. 1 UV-vis spectra of triangular DNA origami with a ssDNA probe before and after incubation with 20-mer, 40-mer and 60-mer ssDNA-modified AuNPs. Inset: digital photo of the mixtures of the triangular DNA origami with a probe and AuNPs modified with 20-mer, 40-mer and 60-mer ssDNA after a 1 hour incubation at 43 °C.

In the second set of experiments, the length of DNA origami-anchored probe strand was adjusted from 20 to 60 mer, while maintaining the length of the AuNPs surface-attached oligonucleotides at 20 mer. AFM images were captured after assembly of AuNPs on the origami templates (Fig. 2). The AuNPs-attached DNA origami templates and the non-attached ones can be easily differentiated from the AFM images. Binding rate is calculated as the ratio between the number of AuNPs-attached origami and the total number of all origami (non-attached origami + AuNPs-attached origami) to evaluate the effect on the assembly process. The alteration of the probe length from 20 to 60 mer does not remarkably affect the binding rate. The binding of the AuNPs on DNA origami may rely on two aspects: (1) the contact probability of the AuNPs and the origami-anchored probe strands in the aqueous system, as well as (2) the hybridization efficiency of the AuNP-attached oligonucleotides with the probe. Long ssDNA with the linear conformation may possess better flexibility than the short one. However, long ssDNA molecules like to form secondary structures in solution,²⁹ which may limit their chance to meet with AuNPs. Therefore, the 40- and 60-mer strands may not have higher contact probability than the 20-mer one. Since the length and the sequence of the AuNPs-attached oligonucleotide are fixed in the system, there should be no significant difference on the hybridization efficiency either. Thus, it is found that the length of DNA origami-based probe has no influence on the assembly of AuNPs on the templates. Considering the cost of the oligomer synthesis, it is highly recommended to use 20-mer joint strand for the assembly of AuNPs on DNA origami templates.

Fig. 2 AFM images of the complexes of 20-mer ssDNA-BSPP-AuNP and DNA origami triangles with 20-mer (a), 40-mer (b) and 60-mer (c) joint strands. (d) Statistical analysis of the binding rates of (a–c). Data are presented as the mean ± standard deviation from four independent measurements. Scale bar = 1 μm.

From the first and the second sets of experiments, it can be seen that the control of the AuNPs-origami assembly could not be achieved by simply adjusting the lengths of the nanoparticle-attached oligonucleotide and the origami-positioned probe strand. Thus, in the third set of experiments, DNA origami with more joint strands were utilized to enhance the assembly efficiency of the AuNPs. As shown in Fig. 3, the binding rates for origami with 1, 2 and 3 probes are calculated based on the AFM images. As the probe strand number increases from 1 to 3, the binding rate gradually grows from 43% to 65%. In comparison to the 1-probe origami, the 2-probe and 3-probe ones exhibit significant enhancements of more than 10% and 20% on the binding rate, respectively. At a certain AuNPs concentration, more probes on origami templates definitely can increase the contact probability between the AuNPs and the joint strands, enabling the occurrence of the hybridization of the complementary ssDNA. That's why the 3-probe origami gives the highest binding rate in the present study. The results clearly reveal that DNA origami with more probes could be applied to efficiently capture oligonucleotides-modified AuNPs.

Fig. 3 AFM images of the complexes of 20-mer ssDNA-BSPP-AuNP and DNA origami triangles with 1 (a), 2 (b) and 3 (c) capture probes. (d) Statistical analysis of the binding rates of (a–c). Data are presented as the mean ± standard deviation from four independent measurements. **: p < 0.01, in comparison to (a); ##: p < 0.01, in comparison to (b). Scale bar = 1 μm.

Due to the different charge-to-mass ratios, the pure DNA origami triangles may be separated from the AuNPs-assembled ones using gel electrophoresis.^23,30,31 As shown in Fig. 4, two bands can be observed in the three lanes and the pure origami runs faster than the AuNP-assembled one. It seems that the anchoring of oligonucleotide-immobilized AuNPs reduces the charge-to-mass ratio of DNA origami. Although the loading amount of each sample may vary a little bit, the relative intensities of the upper band to the lower one keep constant for all lanes, indicating that the binding rates of DNA origami triangles with 20-mer, 40-mer and 60-mer probes has no obvious difference (Fig. 4a). As to the assembly of AuNPs on DNA origami with 1, 2 and 3 capture probes, the band intensity of the AuNPs-conjugated origami gradually enhances with the increase of the probe number, accompanying with the decrease of the band intensity of the pure origami (Fig. 4b). The data clearly show that more capture probes on the templates could give the higher probability for the binding of AuNPs. The gel electrophoresis results agree well with the findings in AFM studies. It should be noted that the sizes of AuNPs and origami templates must be matchable to reduce the steric hindrance during the assembly process. Since the size of AuNPs is of great importance in the plasmonic efficiency, the size effects on the AuNPs-DNA origami binding rate should be investigated in future studies.

Fig. 4 (a) Gel electrophoresis of the mixtures of 20-mer ssDNA-BSPP-AuNP and DNA origami triangles with 20-mer (lane 1), 40-mer (lane 2) and 60-mer (lane 3) probes after a 1 hour incubation at 43 °C. (b) Gel electrophoresis of the mixtures of 20-mer ssDNA-BSPP-AuNP and DNA origami triangles with 1 (lane 1), 2 (lane 2) and 3 (lane 3) capture probes after a 1 hour incubation at 43 °C.

In summary, we investigate the influences of the lengths of AuNP surface-immobilized oligonucleotide and the origami-attached capture probe, as well as the number of the capture probes on the assembly efficiency of AuNPs on the origami templates. It is found that AuNPs modified with 20-mer oligonucleotides have good mono-dispersibility for the construction of DNA origami-based metamaterials. In order to enhance the assembly rate, DNA origami with 3 capture probes could be selected as the templates for the highly efficient AuNPs anchoring. Although the length of DNA origami-anchored probe strand has no influence on the assembly of AuNPs on the templates, capture probes with 20-mer length is recommended to reduce the cost for the fabrication process. The finding of this work may provide useful information for the efficient construction of DNA origami-based plasmonic chiral nanostructures.

Acknowledgements

This work is financially supported by National Program on Key Basic Research Project of China (973 Program) under contract No. 2013CB127804 and Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies under cstc2011pt-sy90001. Z. S. Lu would like to thank the supports by the Specialized Research Fund for the Doctoral Program of Higher Education (RFDP) (Grant No. 20130182120025), Young Core Teacher Program of the Municipal Higher Educational Institution of Chongqing and Fundamental Research Funds for the Central Universities (XDJK2015B016).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03700c

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