# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 2 Structural Characterization of Proteins on DNA Nanos scaffolds  
The original goal of DNA nanotechnology, as conceived by [PERSON] in 1982 [7], was to generate a three-dimensional crystal from the self-assembly of individual DNA strands linked by immobile Holliday junctions. Such a crystal could, in turn, be used to scaffold proteins in a repeating lattice in space and solve their structure using X-ray crystallography without having to crystalize the proteins (Figure 2A) [8]. Key to this endeavor is the rigid attachment of the proteins to a mechanically robust, 3D DNA latticework, since even slight variations in their orientation would result in poor diffraction and preclude high-resolution characterization. The first rationally designed, self-assembled DNA crystal was reported by [PERSON], [PERSON] and coworkers in 2009, based on a tensegrip triangle motif--a nanoscale analogue of a mechanically rigid macroscopic object--comprised of three DNA strands (Figure 2B) [9]. This crystal was characterized to 4-14 A resolution (depending on the design), with the largest cavities surpassing 1100 nm\({}^{3}\) in volume. In 2016 and 2017, [PERSON], [PERSON] and coworkers reported a second DNA crystal design, once again employing three strands but relying on four stacked duplexes linked by Holliday junctions as a key motif (Figure 2C,D) [10; 11]. These motifs diffracted to \(\sim\)3 A, and a subsequent report that optimized the Holliday junction sequence yielded crystals that diffracted to 2.6 A, which allowed for visualization of individual purine-pyrimidine base stacks (Figure 2E,F) [12]. Critically, this latter design allowed for crystal cavities with \(\sim\)50% larger edges (albeit at a reduced resolution) and volumes of 1250 nm\({}^{3}\), paving the way for incorporation of larger proteins. Figure 1: Scope of this Perspective. (**A**) Controlling protein orientation by attachment to rigid DNA nanostructures that enables visualization by another technique (e.g., cryo-EM). (**B**) Controlling protein spacing (at the nanometer scale) and the number or type of protein attached. (**C**) Using a DNA nanostructure to either apply a force to, or measure the force exerted by, an attached protein. The above examples demonstrated that mechanically rigid 3D lattices of DNA duplexes could form crystals with defined void spaces that could host proteins. A key next step to this work will be to demonstrate attachment of proteins, either by using DNA binding proteins, or through chemical conjugation (see Section 4), in a rigid and predictable fashion, in order to solve their structure using the crystal with a known structure as a host. In 2006 [PERSON] demonstrated a different DNA crystal design could serve as a \"molecular sieve\", allowing the incorporation of a small protein (GFP, 28 kDa) but not a larger one (MBP-RFP, 280 kDa) [13]. This report was followed in 2014 with a demonstration that enzymes immobilized inside crystals were still catalytically active [14]. Although these reports did not attempt to immobilize the proteins for structural solution, the fact that they could permeate the 3D volume of the crystal and retain their structure and function (as evidenced by the GFP fluorescence and enzyme activity) bode well for achieving structural solution in the future. In recent years, rigid DNA nanostructures have also been applied to protein structural determination using cryoelectron microscopy (cryo-EM). Although cryo-EM methods are constantly improving, it is still difficult to characterize the structure of small proteins (<100 kDa) due to the lack of image contrast with the background. To circumvent this limitation, proteins can be complexed with larger species like antibodies to increase their effective size and make them easier to discern in the image. Inspired by these studies, several groups have explored using DNA nanostructures, which are readily visualized by cryo-EM due to their large size (-tens of nanometers) and high contrast relative to the background, as \"markers\" to find the protein.