# DNA Nanodevices as Mechanical Probes of Protein Structure and Function
## 5 Future Enabling Technologies for Protein-DNA Nanodevices
### Rigid Attachment of Proteins to DNA Scaffolds
Both the DNA amine modification (red) and the DBCO-NHS (blue) introduce flexible alkyl linkers between the oligonucleotide and the protein of interest (POI). (**C**) Using a bicyclononyne (BCN) directly attached to the 5\({}^{\prime}\) end of DNA for SPAAC reduces the linker length by more than half compared with DBCO-NHS [58]. (**D**) Structure of a BCN phosphoramidite for introduction of the cyclootyc at the 5\({}^{\prime}\) end of DNA via solid-phase synthesis. (**E**) Attaching a POI to a DNA nanostructure with a first handle, and positioning a second reactive handle (purple) on the DNA structure in close proximity to the protein surface. (**F**) Using a coiled-coil, with one peptide attached to the DNA structure and the other genetically fused to the protein, avoids initial protein-DNA conjugation. (**G**) Positioning three binding peptides on a DNA nanostructure may allow for immobilization of an unmodified POI simply through multivalent binding interactions. _trans_-cyclooctene/tetrazine reaction) [59], and can, in turn, be incorporated into phosphoramidites in order to control the linker length, rigidity and point of attachment (e.g., off the DNA backbone or base, instead of the termini). The second key advantage that we propose for protein-DNA nanomechanical devices is multisite tethering of proteins on a DNA scaffold to enforce the spatial relationship between them. Although it is possible to attach two or more unique DNA strands to a protein, this task is quite challenging because it requires two selective and orthogonal bioconjugation reactions, and attachment of the second handle is often made sterically more challenging due to the first strand. As an alternative, we suggest that the first strand (introduced using site-specific chemistry) can be used to anchor the protein on a DNA nanostructure bearing a strand with a second reactive handle. The proximity of this handle to the protein surface can then drive the second reaction due enhanced local concentration. A seminal report by [PERSON] and coworkers demonstrated that one ssDNA handle attached to a protein (via the His\({}_{6}\)-Ni(NTA) interaction) could spatially direct the bioconjugation of a complementary strand bearing a reactive NHS ester moiety [60]. In a follow-up work, [PERSON] and coworkers demonstrated this approach for attaching IgG antibodies to a cavity in a two-dimensional origami [61]. We envision widespread use of this concept in the future to attach arbitrary proteins to a nanostructure of interest, as in Figure 6E. The chemistry for this second reaction could also be site-specific (with proximity simply serving to increase the yield), or nonselective, like the lysine-targeting acylation described by [PERSON]. In this fashion, proteins could be rigidly attached in a fixed orientation on 3D DNA crystals, or DNA origami nanogoniometers for cryo-EM experiments. Indeed, modifying the second site of attachment of the protein on the scaffold (e.g., by moving around a reactive amino acid through mutagenesis) could provide another way to direct protein orientation for optimal \"viewing\" in the cryo-EM image. This approach would also be powerful for biophysical studies of proteins, for example by probing the forces required to unfold a protein by tugging on different parts of its surface, or to determine allosteric effects. The ability to tether _any_ protein to a DNA nanoscaffold will circumvent the restriction to DNA-binding proteins, like transcription factors or histones, used in many of the examples discussed in Sections 2 and 4. Finally, the ability to control protein orientation on a DNA scaffold will facilitate more precise biological studies. In native biological settings, protein surfaces are tightly controlled, and orientation is critical to ligand-receptor binding. By more accurately recapitulating these orientations, studies using these systems would provide greater insight.