Salting-Out of DNA Origami Nanostructures by Ammonium Sulfate.

Marcel Hanke,Ruiping Chen,Karim Fahmy,Niklas Hansen,Guido Grundmeier,Adrian Keller

doi:10.3390/ijms23052817

Abstract

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.

Highlights

Published: 4 March 2022DNA origami nanostructures, first introduced in 2006 [1], have developed into widely used, highly versatile tools for addressing important problems and challenges in biophysics [2,3], biomedicine [4,5], molecular [6,7], structural [8,9], and chemical biology [10,11], sensing [12,13], microscopy [14,15], and many other fields of fundamental and applied research
These applications have benefited from the numerous advantages that make DNA origami nanostructures superior to other, more conventional nanostructures, such as high biocompatibility [16,17], high stability in comparatively harsh environments [18,19], and the unprecedented possibility to arrange molecular species with sub-nanometer accuracy [11,20], there are still several challenges that need to be overcome for this technology to enter real-world applications
Since DNA origami assembly relies on the folding of a single-stranded scaffold upon hybridization with multiple oligonucleotide staples at a large molar excess, the total amount of assembled DNA origami nanostructures at a given assembly yield directly correlates with the amount of available scaffold

Summary

Introduction

DNA origami nanostructures, first introduced in 2006 [1], have developed into widely used, highly versatile tools for addressing important problems and challenges in biophysics [2,3], biomedicine [4,5], molecular [6,7], structural [8,9], and chemical biology [10,11], sensing [12,13], microscopy [14,15], and many other fields of fundamental and applied research These applications have benefited from the numerous advantages that make DNA origami nanostructures superior to other, more conventional nanostructures, such as high biocompatibility [16,17], high stability in comparatively harsh environments [18,19], and the unprecedented possibility to arrange molecular species with sub-nanometer accuracy [11,20], there are still several challenges that need to be overcome for this technology to enter real-world applications. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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