Abstract
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.
Highlights
DNA nanotechnology, a branch of structural nanotechnology, enables the fabrication of nanostructures with various shapes and structures in all dimensions based on the designability and structural stability of the DNA duplex[1]
A doubly stacking DNA origami structure (d-DN) was fabricated by combining two single DNA nanostructure (s-DN) vertically. Another s-DN was redesigned to a nano-square holed DNA nanostructure, which had an opening at the center in www.nature.com/scientificreports the normal direction to the DNA helix
Four types of 3D DNA nanostructures were studied with the visualizing strategy of the low-voltage scanning transmission electron microscopy (LV-scanning transmission electron microscopy (STEM))
Summary
DNA nanotechnology, a branch of structural nanotechnology, enables the fabrication of nanostructures with various shapes and structures in all dimensions based on the designability and structural stability of the DNA duplex[1]. AFM measurement struggles to measure the exact inner morphologies of 3D DNA origami structures, which originates from the inherent principle of AFM via tip-sample interactions[19,20] At this point, electron microscopy (EM) measurement rather stands out as a minimally invasive tool that can see the inner features of 3D DNA structures. Through the TEM observation, the specimen can be damaged by several effects such as surface charge interaction, dehydration, and electron beam exposure, and staining process[21]. Among these damages, the staining process is an unavoidable step to observe the DNA origami structures for the TEM imaging with sufficient quality. Staining material such as uranyl acetate is toxic and environmentally hazardous and causes additional side effects, for example, undesirable interaction with the specimen and structural artifacts in images[25,26,27]
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