# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 2 Structural Characterization of Proteins on DNA Nanos scaffolds
Two years later, [PERSON] and coworkers reported the reconstitution of the membrane protein \(\alpha\)-hemolysin in a hydrophobic DNA origami cavity, and probed its structure by cryo-EM [22]. They were able to observe the structure of the protein at a resolution of 30 A, partly due to the absence of interactions that pinned it down in one conformation in the lipid cavity. In 2020, [PERSON] and coworkers extended the nanogoniometer approach to solve the structure of BurrH (another DNA-binding protein) to a resolution of 6.5 A (Figure 3D,E) [16]. The DNA origami served as a barcoded, asymmetric object that could directly report the relative conformation of the protein, which was again attached to DNA duplex spanning  
Figure 3: DNA nanostructure analysis by cryo-electron microscopy. (**A**) Structure of an asymmetric DNA origami “pointer” object at 9–14 Å resolution [21]. (**B**) DNA origami “frame” with a spanning helix for binding the transcription factor p. 53 [15]. (**C**) The orientation of the protein on the spanning duplex can be tuned by the relative positioning (and thus rotation) of the binding site on the helix. (**D**) Design of a 3D origami with a spanning helix, and several “bits” to determine the orientation, for binding the protein BurrH. Reprinted with permission from ref. [16], Copyright 2020 Springer Nature. (**E**) Cryo-EM image of the structure (**top**) with encoding bits boxed in blue; 3D reconstruction of BurrH (**bottom**). a cavity in the nanostructure. The nanostructure \"barcodes\" on the origami specified the rotation angle of the protein on the bound duplex, and the rotational tilt of the duplex between two orientations. Compared with [PERSON] and [PERSON]'s work, [PERSON] and coworkers were able to obtain higher resolution by increasing the yield of origami structures bearing proteins (resulting in more particles per image) and tuning the origami aspect ratio to ensure proper orientation upon adhesion to the transmission electron microscopy (TEM) grid. Three key innovations will likely enable researchers to push the resolution limit of DNA-scaffolded proteins down to <3 A, which would enable atomic resolution. The first is chemical conjugation to the scaffold in a rigid fashion, as outlined in Section 4 below. Interestingly, [PERSON] and coworkers proposed that highly rigid attachment of their protein was not strictly necessary and could even be detrimental, but chemical conjugation could also enable analysis of non-DNA binding proteins. The second innovation is increasing the resolution of the DNA scaffold itself to the atomic level. A third advancement would be to use computational simulation with atomic resolution to better fit cryo-EM maps, as also discussed in Section 4. In 2020, the [PERSON] and [PERSON] groups reported that by taking into account structural fluctuations in DNA origami, and interpreting the cryo-EM maps with molecular dynamics simulations, they could further improve the resolution of 3D origami nanostructures to ~4 A [23]. In this fashion, they could distinguish the fine features of the objects, including major and minor grooves, single-strand breaks and crossover positions. Importantly, this technique demonstrated the inherent flexibility and undesired or unintended deformations in DNA nanostructures, and allowed the authors to correct them through sequence design or covalent crosslinking. In this fashion, it should be possible to design a rigid and well-defined object with atomically-defined locations for protein attachment. By combining this advanced method with better chemical immobilization of arbitrary proteins on a DNA scaffold, as well as methods for covalently locking flexible points in the DNA structure itself [24], the potential for structural solution of individual proteins becomes ever more tantalizing.