# 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  
It is still challenging to link proteins to DNA scaffolds through either covalent or supramolecular means, especially if rigid and multipoint attachment is desired. Here we highlight a few key aspects of this challenge as it pertains to the three applications covered in this perspective. First, most chemical linkages between proteins and DNA are accomplished through heterobifunctional linkers (Figure 6A), where a surface residue on a protein (e.g., cysteine) is linked to an oligonucleotide bearing a unique reactive group (e.g., an amine) at the 5\({}^{\prime}\) or 3\({}^{\prime}\) end. Most such linkers incorporate a flexible connector, such as an aliphatic chain or oligoethylene glycol, between the two reactive ends of the linker. In addition, commercially obtained, functionalized DNA often has an aliphatic linker (ranging from 3-12 carbons) between the 3\({}^{\prime}\) or 5\({}^{\prime}\) end and the terminal moiety (Figure 6B). Although this additional flexibility and length (on the order of 2-3 nm for a fully extended linker) facilitate the reaction yield between the protein and DNA, they preclude a rigid attachment of the protein to the DNA scaffold, and reduce the spatial accuracy of protein placement. Another common strategy for attaching a protein of interest (POI) to a DNAscaffolds is by fusing it to a protein that ligates itself to a specific functional group on the DNA oligonucleotide. These self-ligating proteins, such as the Halo, SNAP, or Clip tags, are highly efficient and site-specific, but introduce extra bulk to the POI and are often linked to it through flexible amino acid linkers [56]. Thus, a key opportunity for chemists and bioengineers is to design conjugation strategies that enable short, rigid linkers between the DNA and protein, as well as multisite attachment to more effective \"pin\" the protein on a DNA nanoscaffold. We have described the range of protein-DNA bioconjugation strategies in extensive detail elsewhere, [6] but here we highlight two approaches that will facilitate these goals: (1) custom synthesis of modified DNA, with use of efficient bioconjugation reactions, and (2) proximity-enhanced reactions for modifying proteins in a second location driven by hybridization of a protein-DNA conjugate to a DNA scaffold. To the first point, the main reason many protein-DNA studies use long and flexible bifunctional linkers is because they are commercially available and can target common residues on a protein surface (e.g., lysine), or uniquely reactive amino acids that can readily introduced through genetic methods (e.g., cysteine). However, solid-phase DNA synthesis can incorporate arbitrarily complex (and arbitrarily many) unique chemical moieties by introducing appropriately-functionalized phosphoramidites into the solid-phase synthesis protocol. It is not trivial, however, to make modified phosphoramidites, so this task will require collaboration between synthetic organic chemists and DNA nanotechnologists and engineers. As one illustration, we contrast two approaches that our lab has pursued [57, 58] to click chemistry with a protein or peptide modified with a noncanonical azide residue: (1) using a commercially available N-hydroxysuccinimide (NHS) ester linker with amine-DNA (Figure 6B), which is \(\sim\)2 nm in length, and (2) using a strand with a 5\({}^{\prime}\) custom-synthesized phosphoramidite (Figure 6C,D), which is around half that size. However, a range of additional, efficient and high-yielding bioconjugation reactions have been used to attach proteins to DNA in recent years (for example, the  
Figure 6: Chemical conjugation strategies. (**A**) Examples of two commercially available linkers for attaching thiols (e.g., a cysteine on a protein) to amines (e.g., a modified oligonucleotide). (**B**) Example of a protein-DNA conjugate from strain-promoted azide-alkyne cycloaddition (SPAAC) between an azide-containing protein and DNA modified with dibenzocycloTyne (DBCO)-N-hydroxysuccinimide (NHS) ester [57].