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

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

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 origam...

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Bibliographic Details
Published in:PloS one Vol. 18; no. 7; p. e0283134
Main Authors: Sentosa, Jason, Djutanta, Franky, Horne, Brian, Showkeir, Dominic, Rezvani, Robert, Leff, Chloe, Pradhan, Swechchha, Hariadi, Rizal F
Format: Journal Article
Language:English
Published: United States Public Library of Science 19-07-2023
Subjects:
Analysis
Aspect ratio
Biology and life sciences
Biophysics
Centrifugation
Centrifugation, Zonal
Centrifuges
Contamination
Density gradients
Deoxyribonucleic acid
Diffusion rate
DNA
DNA - chemistry
Engineering and Technology
Glycerin
Glycerol
Methods
Microscopy
Monomers
Nanostructure
Nanostructures - chemistry
Nanotechnology - methods
Nucleic Acid Conformation
Oligonucleotides
Physical Sciences
Properties
Purification
Research and analysis methods
Robotics
Robots
Sucrose
United States
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Summary: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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Competing Interests: The authors have declared that no competing interests exist.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0283134