Overview

Initially, we folded and purified DNA origami and characterized its quality using transmission electron microscopy (TEM) and agarose gel electrophoresis.^[1]

DNA origami folding

We folded the DNA origami using a PCR amplifer annealing method and characterized the folding results using agarose gel electrophoresis. We set the annealing protocol for the DNA origami as follows:

And established a gradient of Mg²⁺ concentration to explore the optimal condition for DNA origami folding.^[2]

Fig. 1. DNA origami folding

The folding efficiency of DNA origami using agarose gel electrophoresis. The lanes from left to right are as follows: ladder, p7249 scaffold, DNA origami folded under 1-8 mM Mg²⁺ condition.

We observed the formation of DNA origami bands under all Mg²⁺ conditions, with the bands positioned slightly above the M13mp18 plasmid, consistent with the expected location of DNA origami bands, indicating successful folding of the DNA origami. We also noticed that as the concentration of Mg²⁺ increased, the DNA origami gradually formed dimers. This is attributed to the reduction of electrostatic repulsion between DNA origami structures by Mg²⁺, leading to the aggregation of some DNA origami into dimers. We found that with increasing Mg²⁺ concentration, the DNA origami bands shifted downward, likely due to the weakening of the repulsive forces between the upper and lower layers of the DNA origami by Mg²⁺, causing the DNA origami to trend towards a closed state with reduced steric hindrance, thus migrating faster in agarose gel electrophoresis. We ultimately selected 4 mM Mg²⁺ for the folding of DNA origami, as this condition resulted in the most intense bands with the fewest dimers formed.

DNA origami purification

We employed a PEG 8000 purification method to purify the DNA origami, removing excess staple strands from the folding process, and characterized the purification results using agarose gel electrophoresis.^[4]

Fig. 2. DNA origami purification

The purification efficiency of PEG 8000 was characterized using agarose gel electrophoresis, with lanes from left to right are as follows: the first two are purified DNA origami, ladder, p7249 scaffold.

Agarose gel electrophoresis results indicate that the excess staple strands were successfully removed after purification of the DNA origami, and the bright band corresponding to the DNA origami suggests that the purification process resulted in a high product yield.

DNA origami TEM characterization

We performed uranyl acetate negative staining and no-staining procedures on the purified DNA origami samples and characterized their morphology using TEM.^[5]

Fig. 3. DNA origami TEM charaterization

(A) DNA origami without negative staining, with a scale bar of 200 nm. (B) DNA origami after negative staining with uranyl acetate, with a scale bar of 200 nm.

TEM results indicate that the DNA origami was successfully synthesized into the desired morphology, with no aggregation observed, suggesting that the Mg²⁺ concentration used for folding was appropriate. Since the DNA origami was not conjugated with AuNR, It exhibits a V-shaped structure, indicating that it is in an open state.

Overview

Following this, we modified gold nanorods (AuNRs) with thiol-modified DNA, and confirmed the quality of the AuNRs and the efficiency of the thiol-DNA modification using a UV-Vis-NIR spectrophotometer, dynamic light scattering (DLS), zeta potential measurements, and TEM.

DNA ligation

Fig. 4. UV-Vis-NIR full-band scanning absorption curves

UV-Vis-NIR absorbance spectrum scan of AuNRs before and after the modification of sulfhydry-DNA.

To obtain the UV-Vis-NIR absorbance spectrum, a sample solution underwent an irradiation with monochromic light from 1100 nm to 400 nm with an interval of 2 nm subsequently constructed a continuous absorbance curve. Through this curve, we can precisely determine the absorbance peak positions and intensities^[6][7].
The concentration of AuNRs could be accurately calculated based on the absorbance intensity at 400 nm. The concentration of the raw material is 0.129 nM.
The concentration of the product AuNRs was calculated to be 1.04 nM Importantly, Tabsorption peak positions and peak shapes remained unchanged, indicates that the AuNRs underwent no polycondensation^[9][10].

Zeta potential

Zeta potential referred to the potential at the shear plane and was utilized to characterize the stability of the dispersion system of AuNRs in aqueous solution. The presence of a net negative charge on the surface of AuNRs influenced the ionic distribution in the vicinity surrounding the AuNRs, thereby generating a double electric layer around the particles. The potential at this boundary, known as the zeta potential, was altered when sulfhydryl-DNA was modified onto the surface of AuNRs. As a result, the modification of DNA could be qualitatively illustrated by the change in zeta potential.

Fig. 5. Zeta potential before and after the modification of DNA

After synthesizing the sulfhydryl-DNA-modified AuNRs, we conducted tests on their zeta potential and observed that the zeta potential changed from -12.5 mV to -21.7 mV.[11] This finding provided evidence for the successful surface modification with DNA.^[12]

Detection of Nanoparticle Hydration Radius via Dynamic Light Scattering (DLS)

Dynamic Light Scattering (DLS) was a technique used to measure the particle size of nanoparticles. Consequently, we chose to utilize dynamic light scattering to measure the size of gold nanorods before and after the modification of sulfhydry-DNA. This approach enabled us to qualitatively assess the successful modification of sulfhydry-DNA.

Fig. 6. Size before and after DNA modification

Based on the available data, it was observed that the particle size of the AuNRs changed from 51.7 nm to 114.8 nm which indicates that the modification of the sulfhydry-DNA was successful.

Transmission Electron Microscopy (TEM)

To gain a more intuitive visualization of the morphology of the gold nanorods, the raw material samples of the gold nanorods were subjected to transmission electron microscopy. The resultant images are depicted (Fig. 7).

Fig. 7. The TEM image of AuNRs

As observed in the transmission electron microscopy (TEM) image, the AuNRs exhibited high quality, uniformity in size, and their dimensions were found to meet the requirements.
In conclusion, following the modification of the sulfhydry-DNA to the AuNRs, the size of the AuNRs, as well as the successful modification of sulfhydry-DNA onto their surface, was demonstrated through the evaluation of UV-Vis-NIR full-band absorption, zeta potential measurements, dynamic light scattering (DLS) hydration radius analysis, and TEM.^[13]

Overview

Subsequently, we assembled the DNA origami with AuNRs and characterized the assembly using TEM and agarose gel electrophoresis.

The assembly of AuNR and DNA origami

We mixed AuNR and DNA origami at different molar ratios and elevated sample temperature to 37 ℃, followed by overnight incubation in a water bath for natural cooling, in order to ensure the proper conjugation of AuNR with DNA origami.^[14]

Fig. 8. The electrophoretic mobility shift assay of Doara

The conjugation of DNA origami and AuNR was characterized using electrophoretic mobility shift assay. With lanes from left to right, are as follows: DNA origami, DNA origami conjugated with AuNR at molar ratios of 3:2, DNA origami conjugated with AuNR at molar ratios of 1:1, DNA origami conjugated with AuNR at molar ratios of 2:3, p7249 scaffold, ladder.

We observed that no distinct bands were present in the lanes where AuNRs were connected to DNA origami. This is attributed to the high molecular weight of the AuNR we used, approximately 2.67 x 10⁸, which is nearly 50 times largger than DNA origami, about 4.2 x 10⁶. Consequently, the assembled complex of AuNR and DNA origami has a large molecular weight and becomes entangled in the sample wells or dispersed into the electrophoresis buffer, hence no bands are visible. The electrophoretic mobility shift assay result shows that the assembly of AuNR and DNA origami lacks bands, indicating that the DNA origami successfully assembled with the AuNR.

To prevent the assembly of multiple DNA origami on a single AuNR, which could lead to steric hindrance conflicts between the DNA origami resulting in incomplete closure, and the heterogeneous photothermal switching efficiency resulting from varying numbers of DNA origami assembled on a single AuNR, we controlled the molar ratio of AuNR : DNA origami to approximately 1:1 during assembly to achieve the optimal assembly result.

The TEM characterization of Doara

We performed negative staining of Doara with uranyl acetate and characterized its morphology using TEM.^[15]

Fig. 9. The TEM characterization of Doara

(A to C) A zoomed-in view of individual Doara, with a Scale bar of 50 nm. (D) A normal view of multiple Doaras, with a scale bar of 100 nm.

The TEM characterization results reveal that the DNA origami has assembled correctly with the AuNR in a closed configuration, with each AuNR adorned with a single DNA origami. This arrangement prevents the incomplete closure result from steric hindrance between the DNA origami and heterogeneous photothermal switching efficiency. Additionally, within the same TEM field of view, there is a high number of Doara structures that are uniformly distributed, indicating a high assembly efficiency for Doara.