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Nucleic acids self-assembly has rapidly advanced to produce multi-dimensional nanostructures with precise sizes and shapes. DNA nanostructures hold great potential for a wide range of applications, including biocatalysis, smart materials, molecular diagnosis, and therapeutics. Here, we present a study of using dynamic light scattering (DLS) and nanoparticles tracking analysis (NTA) to analyze DNA origami nanostructures for their size distribution and particles concentrations. Compared to DLS, NTA demonstrated higher resolution of size measurement with a smaller FWHM and was well suited for characterizing multimerization of DNA nanostructures. We future used intercalation dye to enhance the fluorescence signals of DNA origami to increase the detection sensitivity. By optimizing intercalation dyes and the dye-to-DNA origami ratio, fluorescent NTA was able to accurately quantify the concentration of dye-intercalated DNA nanostructures, closely matching with values obtained by UV absorbance at 260 nm. This optimized fluorescent NTA method offers an alternative approach for determining the concentration of DNA nanostructures based on their size distribution, in addition to commonly used UV absorbance quantification. This detailed information of size and concentration is not only crucial for production and quality control but could also provide mechanistic insights in various applications of DNA nanomaterials.
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This content will become publicly available on June 19, 2026
Analyzing DNA Origami Nanostructure Assembly by Dynamic Light Scattering and Nanoparticle Tracking Analysis
Abstract The field of nucleic acid self‐assembly has advanced significantly, enabling the creation of multi‐dimensional nanostructures with precise sizes and shapes. These nanostructures hold great potential for various applications, including biocatalysis, smart materials, molecular diagnosis, and therapeutics. Here, dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are employed to investigate DNA origami nanostructures, focusing on size distribution and particle concentration. Compared to DLS, NTA provided higher resolution in size measurement with a smaller full‐width at half‐maximum (FWHM), making it particularly suitable for characterizing DNA nanostructure. To enhance sensitivity, a fluorescent NTA method is developed by incorporating an intercalation dye to amplify the fluorescence signals of DNA origami. This method is validated by analyzing various DNA origami structures, ranging from 1 and 2D flexible structures to 3D compact shapes, and evaluating structural assembly yields. Additionally, NTA is used to analyze dynamic DNA nanocages that undergo conformational switches among linear, square, and pyramid shapes in response to the addition of trigger strands. Quantitative size distribution data is crucial not only for production quality control but also for providing mechanistic insights into the various applications of DNA nanomaterials.
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- Award ID(s):
- 2141141
- PAR ID:
- 10613496
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Small Methods
- ISSN:
- 2366-9608
- Subject(s) / Keyword(s):
- DNA origami dynamic DNA self-assembly dynamic light scattering nanoparticle tracking analysis size distribution
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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