Scaffolded DNA origami allows for the construction of custom-designed DNA nanostructures via molecular self-assembly. These structures have recently demonstrated exciting potential for a range of biological applications such as drug delivery, force measurement, and biomarker detection. However, translating these devices from research demonstrations to clinical or other biological applications require thorough evaluation of DNA nanostructure structural stability under harsh physiologic conditions that include the presence of nucleases. A handful of studies that have evaluated DNA origami stability physiological buffers have revealed that stability can vary from tens of minutes to several hours depending on the structure design and the specific buffer. Currently, how physiological stability relates to nanostructure design, and more importantly how to design devices with appropriate stability is poorly understood.
Therefore, the objective of the current study is to evaluate how different structural characteristics of DNA nanostructures affects stability under a range of physiological conditions. Here, we have assessed the stability and degradation rate of DNA nanostructures under physiologically relevant factors including varying levels of salinity and fetal bovine serum (FBS). Stability experiments were performed on a large panel of DNA nanostructures including structures that share similar designs, but varied in a single parameter such as surface area, crossover frequency, lattice cross section, scaffold routing, or number of overhangs. Structural stability of nanostructures was monitored across varying concentrations of magnesium chloride (0-20mM) and fetal bovine serum (0%-100%) during a 24-hour room temperature incubation period. Quantitative data was obtained using agarose gel electrophoresis coupled with a gel intensity analysis program to monitor the long-term stability of each structure and a spectrophotometer to collect degradation kinetics data. Results were compiled to create an algorithm to predict the structural stability of DNA nanostructures based on their characteristics and designing stable structures suitable for various physiologically relevant environment. An additional goal for this work is to make a publically accessible database that DNA origami nanostructure designers can use to create an optimal structure for any experimental conditions more efficiently and conveniently.