Visualization of unstained homo/heterogeneous DNA nanostructures by low-voltage scanning transmission electron microscopy

Three-dimensional (3D) homo/heterogeneous DNA nanostructures were studied with low-voltage scanning transmission electron microscopy (LV-STEM). Four types of 3D DNA nanostructures were designed and fabricated by the origami method including newly proposed protocols. The low-energy electron probe and optimized dark-field STEM detector enabled individual unstained DNA nanostructures to be clearly imaged by the single acquisition without the averaging process. For the vertically stacked double structures, assembled through modified single-stranded domains, and the structures containing a square opening (i.e., a hole) in the center, the LV-STEM successfully reveals the vertical information of these 3D structures as the contrast differences compared to the reference. For the heterogeneous structures, the LV-STEM visualized both regions of the functionalized gold nanoparticles and the DNA base structure with distinct contrasts. This study introduces a straightforward method to fabricate stackable DNA nanostructures or nanoparticles by replacing a relatively small number of incumbent DNA strands, which could realize the simple and sophisticated fabrication of higher-order 3D DNA homo/hetero nanostructures. Together with these design techniques of DNA nanostructures, this study has demonstrated that the LV-STEM is the swift and simple method for visualizing the 3D DNA nanostructures and certifying the fabricated products as the specified design, which is applicable to various research fields on soft materials including DNA nanotechnology.


[2] AFM imaging of the s-DNs and nsh-DNs
To obtain the AFM images, 2 μL of the samples were placed on freshly cleaved mica for 30 seconds, after which 48 μL of 1 × TAE/Mg 2+ buffer was pipetted onto the mica surface. AFM images were taken by a Digital Instruments Nanoscope 3 (Veeco Inc., New York, USA) in tapping mode under a buffer using DNP-S10 silicon nitride tips (maximum tip radius: 40 nm, Bruker, Massachusetts, USA).
The s-DNs were observed in Figure S1 under liquid conditions. The line profile shows that the length of the single DNA nanostructure is much longer than the designed value and has a blunt edge compared with the LV-DF-STEM image. Next, the nsh-DNs were observed in Figure S2 under liquid conditions. As shown in Figure S2, most of the structures for nsh-DNs seem to be separated into two smaller structures with unspecific reasons, while the nsh-DNs were clearly observed as holed structures with LV-STEM observation in Fig. 4A.  For the TEM observation, the DNA origami structures were prepared on a supporting 20 nm carbon film in a similar way to that described in the main text of the manuscript. TEM images were acquired by Tecnai G2 F30 (FEI company, OR, USA) with bright-field (BF) and high-angle annular dark-field (HAADF) modes at an acceleration voltage of 300 kV. The specimens including the DNA origami structures were observed under negatively stained conditions with uranyl acetate 1% solution in Figure S3 (a, b) and the same specimens were observed without staining in Figure   S3 (c). AuNPs, which are sphere-like particles in the TEM images, were not combined with DNAs in this experiment but deposited for the purpose of helping TEM operation such as focusing.
AuNPs of high Z element showed dark contrast with the BF mode in Figure S3 (a) but bright contrast with the HAADF mode in Figure S3 (b, c). The stained DNA origami structures were barely visible with bright and dark contrasts in Figure S3 (a) and (b), respectively. The DNA origami structures have a rectangular shape with the dimension identical with s-DN, but have a designed opening on the one side. For unstained specimens in Figure S3 (c), we tried to observe but failed to find DNA origami structures because the DNA regions under the unstained condition did not show any contrast difference to the background of the supporting film. This is mainly attributed to the high energy condition of TEM, which causes small scattering cross-sections of electron beams to light-element materials such as DNA origami structures.

[4] Theoretical calculation of the DF-STEM signals
We theoretically estimated the DF signals of the s-DN and d-DNs to support our experimental data based on Lenz's theory, 1 which quantified the electron signals transmitted through the specimen at the specified collection angle. This equation uses a mass thickness x that is defined as where ρ is the density and t is the thickness of a specimen. The transmitted signal T(n0) at the specific incident (scattering) angle α0 of the electron beam is defined as where the contrast thickness is given by Here, Z is the atomic number. The characteristic angle θ0 is calculated by where αH is the Bohr radius. The transmitted signal T(α0) is exponentially dependent on the value x.
Then, for estimation of the theoretical contrast of the DF images using these equations, the Based on Eq. (S9), the DF signal dependence on the thickness of the DNA origami structures was derived as shown in Figure S4. Here, the thickness of the DNA origami structures was varied from 0 to 30 nm and the thickness of the carbon supporting film was 3 nm. The two acceleration voltage conditions were 30 keV and 300 keV, which corresponded to the operational condition in this study and the conventional condition of TEM. 2 Figure S4. Dependence of the DF signal on the thickness of the DNA origami structure on the 3 nm carbon supporting film.
In Figure S4, the DF signal shows an approximately linear dependence on the thickness of the DNA origami. As designed, the s-DN and d-DN are 7.4 and 14.8 nm in thickness, respectively.
For the s-DN and d-DN, the DF signal at 30 keV is 11 times higher than that at 300 keV. Therefore, under the low-voltage conditions in this study, the DNA structure without staining can be visualized in the high contrast.
We calculated the signal ratio by dividing the signal of the target by that of the s-DN. Under this definition, the d-DN and the s-DN had the signal ratios of 1.94 and 1, respectively. The two values predicted by Lenz's theory are indicated in Figure 3D with red arrows. These theoretical DF ratios are consistent with the contrast ratios evaluated from the actual LV-DF-STEM image. specimens considering physical models in 3D. 3 We adopted the same simulation conditions as our experiment, where the collection angle was 15 to 55 mrad and the acceleration voltage was 30 keV. Figure S5 shows simulated STEM images of four kinds of AuNPs of the h-DN. Three regions including the DNA origami, the AuNP, and the supporting film are distinguishable. The simulated image in Figure S5 (b) corresponds to the experimental conditions of the image in Figure 4D. As the diameter of the AuNP increases, the DF image of the AuNP becomes darker in the center region. In the radial direction, the DF images of the AuNP become darker from the outside to the center because of the spherical shape of the AuNP, that is, the center region is thicker than the surrounding. This produces a weaker signal at the center for the STEM detector.

[6] LV-STEM images of the s-DNs for statistical analysis in Figure 3
The 110 sets of the s-DN structures have been observed to acquire the statistical data in Figure   3E and F. Figure S6 shows original LV-STEM images of the s-DNs without any image processing.
The unstained s-DNs clearly show bright rectangular structures with single image acquisition. The brighter regions with white color are thicker lacy structures to support ultra-thin carbon films in dark contrast.   Figure S7 shows a high magnification LV-STEM image on three DNA nanostructures, which were fabricated in a similar process with the s-DNs. Figure S7 clearly shows the rectangular structures with sufficient contrast and well-defined edges. In addition, on the DNA structure with a yellow arrow, a box-shaped defect is recognized as a lower contrast region compared to the normal region. This result shows that LV-STEM can image not only a simple box shape, but also a non-symmetric shape of DNA origami structures, which would be more important in the field of DNA nanotechnology. The result is another evidence showing the imaging capability of the LV-STEM as a process monitor.
[9] Purification of DNA origami nanostructures The structures were run under 1 × TAE/Mg 2+ running buffer and a 2% agarose gel at 60 V for 2 hours and then the gel was stained with SYBR Gold (Thermo Fisher Scientific, Massachusetts, USA) Each the bands were characterized by using a Molecular Imager Gel  Figure S10 (Lane 1, 2, 3) shows the agarose gel electrophoresis data, which is the origin of Figure 1B in the main text. Figure S11 shows the gel image on a High-Performance 2UV Transilluminator.