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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. 

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. 

In recent decades, nucleic acid self-assemblies have emerged as popular nanomaterials due to their programmable and robust assembly, prescribed geometry, and versatile functionality. However, it remains a challenge to purify large quantities of DNA nanostructures or DNA-templated nanocomplexes for various applications. Commonly used purification methods are either limited by a small scale or incompatible with functionalized structures. To address this unmet need, we present a robust and scalable method of purifying DNA nanostructures by Sepharose resin-based size exclusion. The resin column can be manually packed in-house with reusability. The separation is driven by a low-pressure gravity flow in which large DNA nanostructures are eluted first followed by smaller impurities of ssDNA and proteins. We demonstrated the efficiency of the method for purifying DNA origami assemblies and protein-immobilized DNA nanostructures. Compared to routine agarose gel electrophoresis that yields 1 μg or less of purified products, this method can purify ∼100–1000 μg of DNA nanostructures in less than 30 min, with the overall collection yield of 50–70% of crude preparation mixture. The purified nanocomplexes showed more precise activity in evaluating enzyme functions and antibody-triggered activation of complement protein reactions. 

Abstract Nucleic acid detection plays a crucial role in various applications, including disease diagnostics, research development, food safety, and environmental health monitoring. A rapid, point‐of‐care (POC) nucleic acid test can greatly benefit healthcare system by providing timely diagnosis for effective treatment and patient management, as well as supporting diseases surveillance for emerging pandemic diseases. Recent advancements in nucleic acids technology have led to rapid assays for single‐stranded nucleic acids that can be integrated into simple and miniaturized platforms for ease of use. In this review, the study focuses on the developments in isothermal amplification, nucleic acid hybridization circuits, various enzyme‐based signal reporting mechanisms, and detection platforms that show promise for POC testing. The study also evaluates critical technical breakthroughs to identify the advantages and disadvantages of these methods in various applications.