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
(Rs = 2.05 ± 0.3), peak efficiency
(N = 12,200 ± 230), and peak symmetry (As = 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.