# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 4 Measuring or Applying Forces with DNA Nanodevices  
The third application of DNA nanostructures that we discuss is in measuring or applying forces on proteins at the single-molecule level. Although other methods, like optical tweezers or attachment to an AFM tip, exist for pulling on proteins with forces in the piconewton (pN) regime, these require specialized equipment and are limited to analyzing one protein at a time. DNA nanodevices have the advantage that they are relatively inexpensive and allow analysis of many proteins in parallel in a single sample. Furthermore, the biophysical properties of DNA are fairly well understood, or can be readily probed using computational simulations, as we discuss in Section 4, and readily tunable with only minor changes to the DNA sequences that comprise the origami nanoobject. We also note that simple DNA duplexes are extremely promising as biophysical sensors, e.g., to probe the forces applied by cell integrins to the extracellular matrix [40,41], but since these do not involve DNA nanostructures we will not cover them here. The structural programmability of DNA origami structures led several groups to probe their use as nanoscale force-generating devices akin to calipers or mechanical jacks. An added advantage to these systems is that DNA duplexes could be used as \"cranks\" to apply a given force. Alternatively, purely nanoscale effects absent from the macroscale world (like electrostatic repulsion or entropic springs) can be employed as well. [PERSON] and [PERSON] reported a hinged DNA origami nanostructure where the distance between the rigid arms could be tuned with several DNA \"adjuster\" duplexes, with adjustments tunable down to a single base pair (Figure 5A) [42]. Remarkably, this device could achieve displacement steps of only 0.04 nanometers, slightly less than the Bohr radius, due to the lever-arm effect. [PERSON], [PERSON], and coworkers demonstrated a different nanomechanical device based on the entropic forces that oppose extension of a single strand of DNA [43]. By reducing the number of nucleotides in these strands, the force could be readily tuned. As an alternative trigger for applying forces at the nanoscale, [PERSON] and coworkers described a DNA tweezer with a hairpin loop that could be triggered to open with a displacement strand. By incorporating the displacement strand into the nanostructure, but photocaging it, the authors were able to generate tens of piconewtons of force with a brief pulse of UV light that removed the cages and allowed the strand to open the tweezer (Figure 5B) [44]. The [PERSON] lab reported a host of nanoscale analogues to macroscale mechanical devices, like crank-sliders, pistons, Bennett linkages and a waterbomb base [45,46], which can readily be extended to biophysical studies on proteins. [PERSON] and [PERSON] describedDNA \"nunchuck\" devices, comprised of two long DNA nanotubes linked by a flexible hinge that can mechanically magnify the angle of the hinge and measure single-molecule forces [47; 48]. All the above demonstrations relied intimately on both the rigidity of the DNA helices comprising them and the ability to use DNA hybridization or coiling to apply precise forces. In two reports from 2016, these caliper- and jack-like structures were applied to measure the forces between double stranded DNA and nucleosomes, the protein complexes that wrap the genomic material in the nucleus. Both works, one by the [PERSON] and [PERSON] groups [49] and the other by the [PERSON] lab [50], used a hinge-like structure to position double stranded DNA between the arms of the device and allow the nucleosomes to bind (Figure 4C,D). By measuring the angle between the arms of the caliper, which could be closed by the nucleosome binding to and winding the DNA, various molecular details of these protein complexes could be determined such as the effect of salt on binding strength, the number and orientation of neighboring nucleosomes, the length of DNA or transcription factor binding on the nucleosome.