# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 3 DNA Nanoscaffolds for Interrogating Ligand Spacing, Valency, and Confinement
In the caspase 9 example, the addressable pegboard also allowed the authors to determine that clustering of the proteins enhanced activity; even though dimerization alone did activate the enzymes, spatially tunable clusters of three or four enzymes worked even better, but only if the proteins were also within a given distance. Although the above example used linear templates to space proteins or ligands, a key advantage of DNA nanotechnology is the high degree of programmability in shape that it allows. In 2020, [PERSON] and [PERSON] and coworkers described a polyhedral DNA origami scaffold for presenting B-cell ligands in order to create a vaccine (Figure 4B) [27]. This approach allowed the researchers to investigate the effect of protein number and spacing, as well as compare the spherical particle to a linear scaffold (akin to the Hogberg caliper). The authors found that increasing the ligand spacing resulted in more potent B-cell receptor activation, and the rigid segregation of ligands on the origami scaffolds, enhanced activity compared with more flexible ssDNA or polyethylene glycol (PEG) linkers. As with the nanocaliper, the programmability of DNA nanostructures enabled a direct, apples-to-apples comparison of various ligand presentations on a readily tunable scaffold, which would have been impossible with other approaches. Very recently, DNA origami scaffolds were also used to study the clustering of T-cell receptors (TCRs) by attaching binding antibody fragments to the DNA scaffold [36]. Furthermore, the origami structures were modified with cholesterol and embedded in a supported lipid bilayer, facilitating the motion and clustering of the TCRs in a highly biomimetic fashion, allowing the authors to determine that T cell activation required at least two TCRs within a distance of 20 nm. The DNA origami thus enabled both nanoscale control of ligand spacing, but also served as a nanoscale \"adapter\" to mimic the biological arrangement by embedding the proteins in a lipid membrane. Another powerful application of DNA nanostructure templates in biology is to probe the cooperative actions of multiple proteins working in tandem. In 2012, [PERSON] and colleagues used a DNA origami 12-helix bundle to position multiple copies of the motor proteins dynein or kinesin-1 (Figure 4C) [37]. The rigid origami nanostructure thus served to place these proteins with controlled valence and spacing to probe their cooperativity, and found that increasing the number of motors did not increase the velocity of the origami on a microtubule-modified surface. However, the spatial addressability of the handles enabled a \"tug of war\" arrangement between the two types of proteins, which are opposite-polarity molecular motors. Furthermore, introducing photoleavable linkers in the DNA handles for one type of motor allowed their triggered \"release\" from the scaffold with light, resulting in the other protein dominating. In 2015, the [PERSON] lab used a DNA origami nanotube to mimic a myosin fiber, and used DNA handles to position myosin heads with the same pitch (14.3 nm) as in the natural filament (Figure 4D) [38]. Once again, the authors were able to probe the synergistic effects of multiple myosin heads on nanotube motion on an actin-modified surface, and found that neither the number of myosin heads, nor their density, affected the gliding speed. In this way, the researchers validated a key biochemical mechanism for these molecular motors. In both of these examples, the DNA origami nanostructures provide an unparalleled tool for positioning these motor proteins with not just controlled spacing and valency but, as in the [PERSON] paper, precise stoichiometry between multiple types of proteins. The rigidity of these multihelical objects is critical to prevent spatial fluctuations that would perturb the desired distances or allow for undesired interactions between the proteins. A third distinct area of biological exploration enabled by DNA nanostructures is probing the function of proteins in confined nanoscale volumes.