# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 2 Structural Characterization of Proteins on DNA Nanos scaffolds
Furthermore, because DNA origami nanostructures can be highly anisotropic, they can serve as \"nanoscale goniometers\" [15, 16] that define the absolute orientation of the attached molecules, further facilitating class-averaging and solution of protein structure. Unlike DNA crystals, which, for the time  
Figure 2: Self-assembled DNA crystal lattices. **(A)** Original concept of DNA nanotechnology as conceived by [PERSON]: using a self-assembled DNA lattice to scaffold proteins in well-defined orientations in 3D space [7].Reprinted with permission from ref. [8], Copyright 2011 Springer Nature. **(B)** Design (left) and 3D crystal structure (right) of [PERSON] and [PERSON]’s original tensegrity triangle crystal design. Reprinted with permission from ref. [9], Copyright 2009 Springer Nature. **(C)** Crystal design with a weaving strand (red) linking four duplexes via Holliday junctions. Reprinted with permission from ref. [11], Copyright 2016 American Chemical Society. **(D)** Two views of the 3D crystal structure using the weaving strand with stacked duplexes design. Reprinted with permission from ref. [11], Copyright 2017 American Chemical Society. **(E)** The asymmetric unit of a crystal design with 2.7 Å resolution, showing individual base stacks. Reprinted with permission from ref. [12], Copyright 2020 John Wiley and Sons. **(F)** This design enabled expanded cavities in the crystal, with void spaces approaching 10 nm in diameter. being, are restricted to rather small proteins (\(\sim\)30-50 kDa) that can fit in their cavities, DNA origami nanostructures can be designed with much larger void spaces for hosting proteins. Indeed, work by [PERSON] and coworkers demonstrated that simple 2D DNA lattices could host proteins like RuvA [17] or G-protein coupled receptors (GPCRs) [18] in their cavities to generate repeating arrays that could be visualized by cryo-EM. Although these approaches gave only low resolution (20-30 A) reconstructions of the protein, they demonstrated the potential for a DNA scaffold as a \"sample holder\" to image proteins, while preventing aggregation or undesired surface adhesion effects. Cryo-EM had been used to characterize the 3D structure of DNA cages and to demonstrate their three-dimensionality for many years [19; 20], but not until 2012 was the structure of a compact, rigid and block-like DNA nanostructure reported for the goal of protein structural characterization [21]. [PERSON], [PERSON], and coworkers designed a block-like \"pointer\" object with densely packed helices on a square lattice, and solved its structure to a maximum resolution of 9.7 A (for the more rigid core), and 14 A at the more flexible outer periphery (Figure 3A). This resolution, nonetheless, allowed clear discernment of the DNA strand routing in the object, including the crossover points, stacked Holliday junctions or deviations from the designed lattice geometry due to backbone repulsion. In 2016, these same groups applied this technique to solving the structure of a DNA-binding protein, the transcription factor p53 (Figure 3B) [15]. The researchers designed a DNA origami cage to specifically sit perpendicular to, and vertically span the vitreous ice sheet containing the sample. The center of the structure was spanned by a DNA duplex bearing the binding site for the p53 protein. Changing the relative location of this binding site (relative to the helical axis of the duplex, Figure 3C) resulted in a different viewing angle of the protein. In this way, the DNA origami served as a nanoscale goniometer, enabling different perspectives of the attached protein. The authors used the origami-enforced images to solve the structure of the protein to \(\sim\)15 A, and, thereby, elucidate new details about the symmetry of the oligomer. The ultimate resolution was limited, however, by the flexibility of the DNA origami construct, and specifically the single duplex spanning the cavity.