# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 3 DNA Nanoscaffolds for Interrogating Ligand Spacing, Valency, and Confinement  
A second area where DNA nanomechanical devices have played an important role in biology is in probing the required distance and multivalency of proteins for effectively binding to cell receptors or triggering a biological effect. DNA origami nanostructures are particularly effective at this role for several reasons. First, they are rigid objects (especially if multilayer, block-like nanostructures are used), so thermal fluctuations do not change the distance between attached ligands much [25]. Second, they typically span dimensions of tens to hundreds of nanometers, a size scale well matched to many biological receptor complexes on cells. Third, it is relatively straightforward to change the distance between ligands, or the number of ligands, by extending staple strands at arbitrary points on the structure (with a resolution of ~5-10 nm). Fourth, the shape of a DNA nanostructure can be tuned to match the application at hand, e.g., a linear rod to serve as a molecular caliper [26], a wireframe polyhedron to mimic a virus [27], or a ring to recapitulate the nuclear pore [28; 29]. Since the inception of DNA origami [1], one of the most attractive applications for these structures was as a \"molecular breadboard\" to attach other species, like nanoparticles or proteins, with nanoscale control. Indeed, a vast and rich literature exists on the attachment of proteins to DNA nanoscaffolds, with particular interest in enzymatic cascades as molecular assembly lines. One potential limitation of the original, two-dimensional designs reported by [PERSON], and used by many others since, is that they are quite flexible. With the advent of three-dimensional origami comprised of several layers of stacked helices linked by multiple crossovers [2], much more rigid objects can be obtained, which do not deform significantly and can thus enforce specific distances between bound ligands. Here we discuss several seminal examples of biological studies that would not be possible, or be much more difficult to carry out, without the unique properties of origami. Due to space limitations, we will not discuss the extensive work on enzymatic cascades, and instead direct the interested reader to several excellent reviews on this topic [30; 31; 32]. In 2014, the [PERSON] and Teixera labs used a 3D origami scaffold as a \"nanocaliper\" to position the ephrin-A5 protein ligand by attaching it to a single-stranded (ss) DNA handle complementary to extensions of staple strands from the nanostructure (Figure 4A) [26]. In this way, the authors were able to control not only the number of ligands, but also the distance separating them (either 40 or 100 nm). This technique demonstrated that not only were two ligands necessary for receptor activation (compared with a monovalent system), but the sample with 100 nm spacing worked just as well as the one with 40 nm distance between proteins. Furthermore, using eight ligands spaced 14 nm apart did not yield any further improvements in bioactivity compared with the two ligands. A key to this approach's success was the rigidity of the nanocaliper, which enforced the desired distances and allowed for a systematic study of both spacing and absolute number of receptors that would not be possible with other approaches (like antibody clustering of the receptors). In a follow-up study, [PERSON] and coworkers were able to use a similar nanocaliper to immobilize small molecule antigens with controlled distances to probe the spatial tolerance of antibody binding [33]. Once again, spacing the molecular species on a rigid and tunable scaffold allowed for a precise investigation of IgG bivalent binding not possible with traditional methods like surface plasmon resonance. The ability of DNA origami to control the nanoscale spacing and number of ligands can also be applied to study the proteins that must oligomerize to function, like the potassium channel Kir3 [34], or caspase 9 [35].