# 3D DNA Nanostructures: The Nanoscale Architect
## 4 Stability and Environmental Conditions  
DNA origami is typically synthesized in a standard molecular biology buffer solution of Tris and EDTA (pH 8) in the presence of divalent cations, typically Mg\({}^{2+}\), that offset repulsive electrostatic interactions due to the intrinsic negative charge in the DNA phosphate backbone which may otherwise interfere with the stability of designed DNA nanostructures. However, this environment often differs in targeted applications. For in vivo applications, physiological fluids typically have Mg\({}^{2+}\) concentrations that are too low to maintain DNA nanostructure stability and nucleases that may degrade DNA nanostructures. It has also been shown that DNA nanostructures are ultimately digested by lysosomes [73] rather than having penetrated their desired cell target. Applications related to nanofabrication may also involve organic solvents, which can make conditions too harsh for DNA nanostructures to survive. Notably, while surface-deposited DNA nanostructures have been shown to be stable in the presence of organic solvents, they rapidly degrade in solution [74]. Figure 4: Signal-responsive actuation of DNA nanomechanics. (**A**) A light-responsive spherical DNA capsule with a photo-cleavable site (reprinted with permission from The Royal Society of Chemistry [68]); (**B**) A DNA origami arm that can be controlled by an external electric field (reprinted with permission from AAAS [72]); (**C**) A hinged DNA nanostructure that places polymers with temperature-responsive hydrophobicity on opposite facing sides of the hinge. Above a specific temperature threshold, the polymers aggregate due to hydrophobic interactions and pull the DNA nanostructure into a closed conformation (reprinted with permission from Wiley-VCH [71]). The [PERSON] group has shown that it is still possible to synthesize DNA nanostructures and maintain their stability without the presence of Mg\({}^{2+}\) (whose presence in solution can be unfavorable to many biological agents), and instead can rely on Na\({}^{+}\), which is much more widely compatible [75]. Joint work by the [PERSON] and [PERSON] groups investigated the effect of lowering Mg\({}^{2+}\) concentration for a comparison between a few simple DNA origami nanostructures. It was noted that dense structures, such as a 24 HB, may require higher concentrations of Mg\({}^{2+}\) to remain stable due to higher density of internal negative charge. Additional work by the [PERSON], [PERSON], and [PERSON] groups has also thoroughly documented DNA nanostructure stability with respect to changes in temperature or harsh chemicals [74,76] and are good benchmark references for areas where DNA nanostructures may need to be improved. Attacking this problem from another perspective has been to bolster the stability of DNA nanostructures by design to withstand harsher conditions. Stricter design criteria for the placement of crossovers and nicks have been shown to increase stability and yields of DNA origami [24,25,77]. A design strategy by the [PERSON] group utilized a technique with strong covalent cyclobutane pyrimidine dimer (CPD) bonds which form between proximally close thymidines upon UV irradiation. By adding thymine bases at specific staple sequence locations within the DNA origami design, UV irradiation could induce a network of strong covalent bonds throughout the structure to strengthen it [78]. Joint work by the [PERSON] and [PERSON] groups have shown that shape and size of DNA nanostructures may have some effect on the degradation rate of simple, monolithic structures in an in vivo rat model, or be at least sufficient enough to achieve passive buildup of anti-cancer drugs at a tumor site [79]. However, it does not indicate that structure-dependent functions could survive. Several other strategies rely on coating the DNA nanostructure with other molecules that may either serve to stiffen, protect, or camouflage the structure.