Analysis of DNA Origami Nanostructures Using Capillary Electrophoresis

DNA origami nanostructures are engineered nanomaterials (ENMs) that possess significant customizability, biocompatibility, and tunable structural and functional properties, making them potentially useful materials in fields, such as medicine, biocomputing, biomedical engineering, and measurement science. Despite the potential of DNA origami as a functional nanomaterial, a major barrier to its applicability is the difficulty associated with obtaining pure, well-folded structures. Therefore, rapid methods of analysis to ensure purity are needed to support the rapid development of this class of nanomaterials. Here, we present the development of capillary electrophoresis (CE) as an analytical tool for DNA origami. CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ctITP separation mode demonstrated superior performance in terms of peak resolution (R_s = 2.05 ± 0.3), peak efficiency (N = 12,200 ± 230), and peak symmetry (A_s = 1.29 ± 0.032). The SYBR family dyes (Gold, Green I, and Green II) were investigated as highly efficient, noncovalent fluorophores for on-column labeling of DNA origami and detection using laser-induced fluorescence. Finally, ctITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.