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1. Analyzing DNA Origami Nanostructure Assembly by Dynamic Light Scattering and Nanoparticle Tracking Analysis

   https://doi.org/10.1002/smtd.202500295  

   Zhang, Qiaochu; Chang, Xu; Ebrahimimojarad, Alireza; Shah, Akshay; Zhang, Fei; Fu, Jinglin (June 2025, Small Methods)

   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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2. Binding Site Programmable Self-Assembly of 3D Hierarchical DNA Origami Nanostructures

   https://doi.org/10.1021/acs.jpca.4c02603  

   Wei, Xingfei; Chen, Chi; Popov, Alexander V; Bathe, Mark; Hernandez, Rigoberto (June 2024, The Journal of Physical Chemistry A)

   DNA nanotechnology has broad applications in biomedical drug delivery and pro- grammable materials. Characterization of the self-assembly of DNA origami and quan- tum dots (QDs) is necessary for the development of new DNA-based nanostructures. We use computation and experiment to show that the self-assembly of 3D hierarchi- cal nanostructures can be controlled by programming the binding site number and their positions on DNA origami. Using biotinylated pentagonal pyramid wireframe DNA origamis and streptavidin capped QDs, we demonstrate that DNA origami with 1 binding site at the outer vertex can assemble multi-meric origamis with up to 6 DNA origamis on 1 QD, and DNA origami with 1 binding site at the inner center can only assemble monomeric and dimeric origamis. Meanwhile, the yield percentages of differ- ent multi-meric origamis are controlled by the QD:DNA-origami stoichiometric mixing ratio. DNA origamis with 2 binding sites at the αγ positions (of the pentagon) make larger nanostructures than those with binding sites at the αβ positions. In general, increasing the number of binding sites leads to increases in the nanostructure size. At high DNA origami concentration, the QD number in each cluster becomes the limiting factor for the growth of nanostructures. We find that reducing the QD size can also affect the self-assembly because of the reduced access to the binding sites from more densely packed origamis. 

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3. Conformational Control of DNA Origami by DNA Oligomers, Intercalators and UV Light

   https://doi.org/10.3390/mps4020038  

   Li, Ruixin; Chen, Haorong; Lee, Hyeongwoon; Choi, Jong Hyun (June 2021, Methods and Protocols)

   null (Ed.)

   DNA origami has garnered great attention due to its excellent programmability and precision. It offers a powerful means to create complex nanostructures which may not be possible by other methods. The macromolecular structures may be used as static templates for arranging proteins and other molecules. They are also capable of undergoing structural transformation in response to external signals, which may be exploited for sensing and actuation at the nanoscale. Such on-demand reconfigurations are executed mostly by DNA oligomers through base-pairing and/or strand displacement, demonstrating drastic shape changes between two different states, for example, open and close. Recent studies have developed new mechanisms to modulate the origami conformation in a controllable, progressive manner. Here we present several methods for conformational control of DNA origami nanostructures including chemical adducts and UV light as well as widely applied DNA oligomers. The detailed methods should be useful for beginners in the field of DNA nanotechnology. 

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4. Gradient-mixing LEGO robots for purifying DNA origami nanostructures of multiple components by rate-zonal centrifugation

   https://doi.org/10.1371/journal.pone.0283134  

   Sentosa, Jason; Djutanta, Franky; Horne, Brian; Showkeir, Dominic; Rezvani, Robert; Leff, Chloe; Pradhan, Swechchha; Hariadi, Rizal F. (July 2023, PLOS ONE)

   Zhang, Yuliang (Ed.)

   DNA origami purification is essential for many fields, including biophysics, molecular engineering, and therapeutics. The increasing interest in DNA origami has led to the development of rate-zonal centrifugation (RZC) as a scalable, high yield, and contamination-free method for purifying DNA origami nanostructures. RZC purification uses a linear density gradient of viscous media, such as glycerol or sucrose, to separate molecules according to their mass and shape. However, many methods for creating density gradients are time-consuming because they rely on slow passive diffusion. To expedite the preparation time, we used a LEGO gradient mixer to generate rotational motion and rapidly create a quasi-continuous density gradient with a minimal layering of the viscous media. Rotating two layers of differing concentrations at an angle decreases the time needed to form the density gradient from a few hours to minutes. In this study, the density gradients created by the LEGO gradient mixer were used to purify 3 DNA origami shapes that have different aspect ratios and numbers of components, with an aspect ratio ranging from 1:1 to 1:100 and the number of components up to 2. The gradient created by our LEGO gradient mixer is sufficient to purify folded DNA origami nanostructures from excess staple strands, regardless of their aspect ratios. Moreover, the gradient was able to separate DNA origami dimers from DNA origami monomers. In light of recent advances in large-scale DNA origami production, our method provides an alternative for purifying DNA origami nanostructures in large (gram) quantities in resource-limited settings. 

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5. CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

   https://doi.org/10.1093/nar/gkac049  

   Lin-Shiao, Enrique; Pfeifer, Wolfgang G.; Shy, Brian R.; Saffari Doost, Mohammad; Chen, Evelyn; Vykunta, Vivasvan S.; Hamilton, Jennifer R.; Stahl, Elizabeth C.; Lopez, Diana M.; Sandoval Espinoza, Cindy R.; et al (February 2022, Nucleic Acids Research)

   Abstract DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei. 

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