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

PLOS ONE RESEARCH ARTICLE Gradient-mixing LEGO robots for purifying DNA origami nanostructures of multiple components by rate-zonal centrifugation Jason Sentosa1,2☯, Franky Djutanta1,3☯*, Brian Horne4☯, Dominic Showkeir4☯, Robert Rezvani1,3, Chloe Leff1,5, Swechchha Pradhan1,3, Rizal F. Hariadi ID1,5* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Biodesign Center for Molecular Design and Biomimetics (at the Biodesign Institute) at Arizona State University, Tempe, AZ, United States of America, 2 Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, United States of America, 3 School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, United States of America, 4 Department of Physics, Arizona State University, Tempe, AZ, United States of America, 5 School of Molecular Sciences, Arizona State University, Tempe, AZ, United States of America ☯ These authors contributed equally to this work. * (FD); (RFH) Abstract OPEN ACCESS Citation: Sentosa J, Djutanta F, Horne B, Showkeir D, Rezvani R, Leff C, et al. (2023) Gradient-mixing LEGO robots for purifying DNA origami nanostructures of multiple components by ratezonal centrifugation. PLoS ONE 18(7): e0283134. https://doi.org/10.1371/journal.pone.0283134 Editor: Yuliang Zhang, Lawrence Livermore National Laboratory, UNITED STATES Received: January 23, 2022 Accepted: March 2, 2023 Published: July 19, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0283134 Copyright: © 2023 Sentosa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All the data supporting this study are provided within the paper and in https://zenodo.org/badge/latestdoi/ 446256259. 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. Introduction DNA origami, a method of self-assembling complex nanostructures from long, single-stranded DNA (scaffold strands) and large excess of shorter oligonucleotides (staple strands) [1–8], has PLOS ONE | https://doi.org/10.1371/journal.pone.0283134 July 19, 2023 1 / 14 PLOS ONE Funding: The research in Hariadi lab was supported by the National Institutes of Health Director’s New Innovator Award (1DP2AI144247), National Science Foundation SemiSynBio II (2027215), and Arizona Biomedical Research Consortium (ADHS17-00007401). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Gradient-mixing LEGO robots for purifying DNA origami nanostructures by ultracentrifugation proven to be a robust and efficient method for generating nanostructures with arbitrary shapes at *5 nm resolution. Furthermore, the exquisite positional control of DNA origami enables precise patterning of biomolecules and inorganic molecules [8–11]. The programmable DNA origami has been used in fields such as medicine [12–16], super-resolution microscopy [17– 20] and electronics [21–23]. Although efforts have been made to optimize DNA origami assembly [24, 25], the yield of well-folded origami for complex 3D structures is far less than 100% [26]. Most applications of DNA origami, however, require uncontaminated samples that are free of staple strands and misfolded structures [26–28]. Thus, for downstream applications, a purification step is added after the folding step to isolate the well-folded structures. Rate-Zonal Centrifugation (RZC) is a high-yield, contamination-free method for purifying DNA origami [27]. The technique subjects a linear density gradient to high centrifugal force in order to separate heterogeneous molecules by their distinct mass and shape [29]. Within the context of DNA origami, RZC can separate well-folded structures from other undesired species (misfolded structures, staple strands, and aggregates) as a purification step. RZC has several advantages compared to other purification methods, such as agarose gel electrophoresis (AGE). RZC keeps the samples in an aqueous solution throughout the process, is free from contaminants such as agarose gel residues, and is scalable to accommodate a large amount of sample [27]. In most cases, RZC purification requires fewer steps than multi-round spin filter purification or agarose gel extraction [30]. RZC purification starts with the traditional preparation of the density gradient, which can be time consuming [27, 31]. There are several methods to prepare a density gradient of glycerol: (1) layering two solutions of different glycerol concentrations in a tube and resting the tube horizontally for 1–2 hours to allow the glycerol to passively diffuse, (2) layering several solutions of different glycerol concentrations and resting the tube upright so that the layers can passively diffuse, and (3) mixing the glycerol gradient using a commercially available gradient mixer. Since the first two methods employ passive (...truncated)