# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 4 Measuring or Applying Forces with DNA Nanodevices
In a related study that same year, the [PERSON] group used their caliper device to measure the forces between two different nucleosomes (as opposed to between nucleosomes and DNA), which is key for understanding the higher-order organization of chromatin into structures in the nucleus (Figure 4E) [51]. Both the relative force applied on the nucleosomes, as well as their distance, orientation and chemical modification (e.g., acetylation) could be probed to yield an energy landscape as a function of internucleosome distance. These examples all demonstrate the great potential in DNA nanodevices for studying DNA-binding proteins with single-molecule resolution, with an emphasis on controlling their displacement and energy landscape, or other molecular properties of the proteins and their substrates. As we discuss in Section 4 below, using site-specific bioconjugation methods would extend these studies to arbitrary proteins, and enhancing the rigidity of attachment would enable greater control over protein orientation for measuring protein-protein interaction energies. Figure 5: Applying forces using DNA nanodevices. (**A**) DNA nanocaliper with “adjuster” helices for Bohr radius resolution. Reprinted with permission from ref. [42], Copyright 2015 Springer Nature. (**B**) DNA nanotweezr with a photocaged trigger strand, allowing for rapid opening with a short pulse of UV light. Reprinted with permission from ref. [44], Copyright 2018 John Wiley and Sons. (**C,D**) Application of hinged DNA nanostructures to measure the biophysical properties of nucleosomes binding to DNA. Reprinted with permission from ref. [49; 50], Copyright 2016 American Chemical Society. (**E**) DNA nanostructure for measuring the forces between two nucleosomes. Reprinted with permission from ref. [51], Copyright 2016 American Association for the Advancement of Science. (**F**) DNA origami nanospring for measuring the stall force of myosin motors. Reprinted with permission from ref. [52], Copyright 2016 Springer Nature. An alternative design for applying and measuring forces on proteins was reported by [PERSON] and coworkers in 2016, and relied on a DNA nanostructure \"spring\" to tug on the motor protein myosin VI (Figure 4F) [52]. Unlike the caliper-like designs mentioned above, this spring consisted of a two-helix DNA bundle with negative superhelical strain (following design rules for curved and twisted origami) [53] to give a structure similar to a macroscopic spring. The authors first characterized the force-extension curve of this spring using an optical trap and demonstrated that the DNA nanostructure had a roughly 10-fold lower spring constant than double-stranded (ds) DNA. As a result, their design matched the stall force of the myosin motors (\(\sim\)2 pN) at a much shorter length than dsDNA, which in turn allowed for shorter run lengths (\(\sim\)700-800 nm). The nanostructure could thus precisely \"match\" the intrinsic biophysical properties of the motor, whereas simpler force-applying devices like dsDNA were unsuited to this particular protein. Using this device, the authors were able to demonstrate that at higher forces the mechanism of myosin VI motion switched (from hand-over-hand to inchworm motion), as well as set up a tug of war between myosin VI and myosin V by attaching them to opposite ends of the spring. The programmable mechanical properties of DNA nanostructures played a key role in the ultimate application, and although optical tweezers were used to calibrate the device it could be used thereafter to probe protein function without a complicated experimental setup. The application of DNA origami to single molecule biophysics also presents challenges in the correct interpretation of the measured force-extension properties, as one needs to correctly interpret measured values of a statistical ensemble of forces and extensions [54], as well as take into account the mechanical properties and extensions of double-stranded and single-stranded segments under tension in the DNA origami constructs that are used to apply force [55].