skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: A Robust and Efficient Method to Purify DNA-Scaffolded Nanostructures by Gravity-Driven Size Exclusion Chromatography
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.  more » « less
Award ID(s):
2141141
PAR ID:
10511543
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
Langmuir
Date Published:
Journal Name:
Langmuir
Volume:
40
Issue:
16
ISSN:
0743-7463
Page Range / eLocation ID:
8365 to 8372
Subject(s) / Keyword(s):
DNA nanotechnology, nucleic acids assembly, size-exclusion chromatography, protein immobilization, enzyme cascade
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; (, 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. 
    more » « less
  2. ; ; ; (, ELECTROPHORESIS)
    Abstract Phages used for phage therapy of multidrug resistant bacteria must be highly purified prior to use. There are limited purification approaches that are broadly applicable to many phage types. Electrokinetics has shown great potential to manipulate phages, but obstructions from the cell debris produced during phage propagation can severely diminish the capacity of an electrokinetic device to concentrate and purify phage samples. A multipart insulator‐based electrokinetic device is proposed here to remove the larger, undesirable components of mixtures from phage preparations while transferring the freshly purified and concentrated sample to a second stage for downstream analysis. By combining the large debris prescreen and analysis stages in a streamlined system, this approach simultaneously reduces the impact of clogging and minimizes the sample loss observed during manual transferring of purified samples. Polystyrene particles were used to demonstrate a diminished sample loss of approximately one order of magnitude when using the cascade device as opposed to a manual transfer scheme. The purification and concentration of three different phage samples were demonstrated using the first stage of the cascade device as a prescreen. This design provides a simple method of purifying and concentrating valuable samples from a complex mixture that might impede separation capacity in a single channel. 
    more » « less
  3. ; ; ; ; (, Nanoscale)
    null (Ed.)
    Crosslinked porous protein crystals are a new biomaterial that can be engineered to encapsulate, stabilize, and organize guest molecules, nanoparticles, and biological moieties. In this study, for the first time, the combined interactions of DNA strands with porous protein crystals are quantitatively measured by high-resolution atomic force microscopy (AFM) and chemical force microscopy. The surface structure of protein crystals with unusually large pores was observed in liquid via high-resolution AFM. Force–distance ( F – D ) curves were also obtained using AFM tips modified to present or capture DNA. The modification of AFM tips allowed the tips to covalently bind DNA that was pre-loaded in the protein crystal nanopores. The modified tips enabled the interactions of DNA molecules with protein crystals to be quantitatively studied while revealing the morphology of the buffer-immersed protein crystal surface in detail, thereby preserving the structure and properties of protein crystals that could be disrupted or destroyed by drying. The hexagonal space group was manifest at the crystal surface, as were the strong interactions between DNA and the porous protein crystals in question. In sum, this study furthered our understanding of how a new protein-based biomaterial can be used to bind guest DNA assemblies. 
    more » « less
  4. ; ; ; (, Advanced Science)
    Abstract DNA is not only a carrier of genetic information, but also a versatile structural tool for the engineering and self‐assembling of nanostructures. In this regard, the DNA template has dramatically enhanced the scalability, programmability, and functionality of the self‐assembled DNA nanostructures. These capabilities provide opportunities for a wide range of biomedical applications in biosensing, bioimaging, drug delivery, and disease therapy. In this review, the importance and advantages of DNA for programming and fabricating of DNA nanostructures are first highlighted. The recent progress in design and construction of DNA nanostructures are then summarized, including DNA conjugated nanoparticle systems, DNA‐based clusters and extended organizations, and DNA origami‐templated assemblies. An overview on biomedical applications of the self‐assembled DNA nanostructures is provided. Finally, the conclusion and perspectives on the self‐assembled DNA nanostructures are presented. 
    more » « less
  5. ; ; ; ; ; ; ; ; (, Journal of Materials Chemistry B)
    Over the past few decades, DNA has been recognized as a powerful self-assembling material capable of crafting supramolecular nanoarchitectures with quasi-angstrom precision, which promises various applications in the fields of materials science, nanoengineering, and biomedical science. Notable structural features include biocompatibility, biodegradability, high digital encodability by Watson–Crick base pairing, nanoscale dimension, and surface addressability. Bottom-up fabrication of complex DNA nanostructures relies on the design of fundamental DNA motifs, including parallel (PX) and antiparallel (AX) crossovers. However, paranemic or PX motifs have not been thoroughly explored for the construction of DNA-based nanostructures compared to AX motifs. In this review, we summarize the developments of PX-based DNA nanostructures, highlight the advantages as well as challenges of PX-based assemblies, and give an overview of the structural and chemical features that lend their utilization in a variety of applications. The works presented cover PX-based DNA nanostructures in biological systems, dynamic systems, and biomedical contexts. The possible future advances of PX structures and applications are also summarized, discussed, and postulated. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email