Department of Chemistry and Biochemistry, Swarthmore College, Swarthmore, Pennsylvania 19081, United States.
Physical Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, Maryland 20899, United States.
Anal Chem. 2023 Dec 26;95(51):18783-18792. doi: 10.1021/acs.analchem.3c03641. Epub 2023 Dec 13.
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 (ITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ITP separation mode demonstrated superior performance in terms of peak resolution ( = 2.05 ± 0.3), peak efficiency ( = 12,200 ± 230), and peak symmetry ( = 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, ITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.
DNA 折纸纳米结构是一种工程纳米材料 (ENM)，具有显著的可定制性、生物相容性和可调的结构和功能特性，使其在医学、生物计算、生物医学工程和测量科学等领域具有潜在的应用价值。尽管 DNA 折纸作为一种功能纳米材料具有很大的潜力，但它的应用面临的一个主要障碍是难以获得纯的、折叠良好的结构。因此，需要快速的分析方法来确保纯度，以支持这一类纳米材料的快速发展。在这里，我们提出了毛细管电泳 (CE) 作为 DNA 折纸的分析工具。在毛细管区带电泳 (CZE) 和毛细管瞬态等速电泳 (ITP) 两种模式下对 CE 进行了研究。对两种系统的优化都得到了折叠 DNA 折纸纳米结构与过量的订书钉链的基线分离。ITP 分离模式在峰分辨率 (=2.05±0.3)、峰效率 (=12,200±230)和峰对称性 (=1.29±0.032)方面表现出优越的性能。SYBR 家族染料（金、绿 I 和绿 II）被研究作为高效的非共价荧光染料，用于 DNA 折纸的柱上标记，并通过激光诱导荧光进行检测。最后，ITP 分析条件也适用于不同形状的 DNA 折纸纳米结构，并可用于区分 DNA 折纸聚集体。
