John Jensen and Dr. Adam T. Woolley, Department of Chemistry and Biochemistry
Introduction
DNA origami is the method of folding a large single stranded DNA (ssDNA) with multiple smaller ssDNA “staple” strands into a predesigned shape, making it an attractive option in the bottom-up construction of nanoelectronic components, such as nanowires. These structures can be used as templates for placement of conductive species1, such as gold nanoparticles (Au NPs). In order to attach Au NPs to the structure, polyadenine (polyA) tails are added to ends of certain staple strands, allowing attachment of Au NP “seeds” that are functionalized with a thiolated polythymine (polyT) sequence. After attachment, the Au NP seeds are connected using an electroless plating process to form nanowires. The desired line width of these nanowires is ~5 nm. In the past, 5 nm gold nanospheres (Au NSs) have been used as seeds2, yet incomplete seeding led to gaps between the seeds and necessitated greater amounts of plating, thus increasing the line width of the nanowire. Two types of Au NPs were studied, 2 nm Au NSs, which theoretically should bind in higher densities leading to smaller gaps, as well as gold nanorods (Au NRs) with a diameter of 3 nm and a length of 10 nm.
Methods
The process previously used to functionalize the 5 nm Au NSs with the thiolated polyT sequence was unusable with the Au NSs and Au NRs, requiring novel methods to attach the polyT ligands to the Au NPs to be developed. In the case of the 2 nm Au NSs, concentration via ultracentrifugation (3 hrs, 35,000 rpm, 10°C) was required. The stock 2nm Au NSs were coated in citrate as a stabilizing agent. An excess of polyT (at a ratio of 1:200) was then added to the solution and incubated for ~24 hours. The Au NSs were purified using 30 kDa Amicon filters then rinsed and suspended in 0.5xTBE buffer. Variations to this method to functionalize the 2 nm Au NSs were studied, such as the use of bis(p-sulfonatophenyl)-phenylphosphine dihydrate dipotassium salt (BSPP) and NaCl to displace the citrate molecules before ultracentrifugation, and various incubation times for the polyT sequence (24, 48, 72 hours. The functionalization of the Au NRs proved to be somewhat more difficult due to the cetyltrimethylammonium bromide (CTAB) required for their synthesis. The CTAB directionalizes the growth of Au NRs during synthesis and forms a bilayer around the Au NRs. The covalent interaction between the thiol and the gold should allow replacement of this bilayer with the polyT sequence, but aggregation was an omnipresent threat once the CTAB bilayer was disrupted. Various methods were used in order to replace the CTAB bilayer, including the method used to functionalize the 5 nm Au NPs, phase transfer via ligand attachment3, and long incubation times with the polyT ssDNA (~48 hrs). To assess the effectiveness of the different methods for both the Au NSs and NRs, the concentrations of both Au NPs and ssDNA was measured using a NanoDrop ND-1000.
To test the application of these methods, Au NSs are hybridized onto DNA origami. This process includes first absorbing 3 μL of 2 nM DNA origami in 1xTAE buffer with Magnesium ions (Mg2+) onto a silicon dioxide (SiO2) surface for ~40 min in a humidified chamber to prevent evaporation. The excess liquid is then rinsed off and 15 μL of a certain concentration of 40 nM, 80 nM, or 160 nM Au NS solution is added to the surface and allowed to react for ~40 min. The excess liquid is again rinsed off and atomic force microscopy (AFM) can be utilized to analyze the seeding of the sample.
Results and Discussion
Experiments found the NaCl concentration and incubation period had no significant effect upon the functionalizing of Au NSs. The addition of BSPP prior to ultracentrifugation did show an increase in polyT addition to the Au NSs, giving an average of 17.3 ssDNA per Au NSs instead of the 14.3 obtained with out the BSPP. AFM images taken from the first seeding revealed promising signs, showing possible rises along the long arm of a T-shaped DNA origami, approximately matching the height of a 2 nm Au NS (see figure 1). Unfortunately, upon analyzing the sample with a scanning electron microscope (SEM), no Au NPs were found to have bound to the origami structures. Iterative trials confirmed this, as any SiO2 surface upon which the 2 nm Au NSs had been placed as seeds was cleared of DNA origami after seeding. This recurrence suggested a problem in the purification method of the 2 nm Au NSs.
Purification of the Au NRs consistently resulted in the aggregation of the Au NRs, which failed to return to solution once aggregated. The phase transfer method in which CTAB was to be replaced with dodecanethiol (DDT), transferring the Au NRs into an organic phase, proved ineffective as the Au NRs remained in the aqueous phase. Mixing produced a cloudy aqueous layer, which upon centrifugation produced Au NRs pelleted at the bottom of the centrifuge tube and a mucous-like substance between the aqueous and organic layer. The pelleted Au NRs were collected and were found to contain a distinct peak at 230 nm, indicative of DDT attachment and lacking the broad character of a CTAB peak. Upon functionalizing the Au NRs via incubation with the polyT sequence, the Au NRs aggregated and failed to dissolve in solution. This aggregation was consistent across all methods examined.
Conclusion
The recurring loss of DNA origami from the SiO2 surface prevented its further analysis and suggests a problem in the attachment of DNA origami to the SiO2 surface. As Mg2+ plays a vital role in the attachment of DNA origami to this surface, it is possible residual citrate is complexing with Mg2+ ions, thereby preventing the origami’s attachment. Functionalization of Au NRs still remains an attractive endeavor as it allows relatively large spaces to be covered using a thin particle, and will be studied further.
1 Elisabeth Pound , Jeffrey R. Ashton , Héctor A. Becerril, and Adam T. Woolley, Polymerase Chain Reaction Based Scaffold Preparation for the
Production of Thin, Branched DNA Origami Nanostructures of Arbitrary Sizes, Nano Lett., Vol. 9, No. 12, 2009
2 Anthony C. Pearson, Jianfei Liu, Elisabeth Pound, Bibek Uprety, Adam T. Woolley, Robert C. Davis, and John N. Harb, DNA Origami
Metallized Site Specifically to Form Electrically Conductive Nanowires, J. Phys. Chem. B, 116 (35), pp 10551–10560
3 Wijaya, A. & Hamad-Schifferli, K. Ligand customization and DNA functionalization of gold nanorods via round-trip phase transfer ligand
exchange. Langmuir 24,9966–9 (2008).