Scalable purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures: Exploring anion-exchange and multimodal chromatography

Abstract

DNA-origami biomanufacturing relies in many cases on the use of asymmetric PCR (aPCR) to generate 500–3500 base, object-specific, single-stranded DNA (ssDNA) scaffolds. Each scaffold is usually purified by agarose gel extraction, a technique that is laborious, limited, not scalable, presents low recovery yields and a low-quality product. Alternatively, we present a chromatography-based method to purify ssDNA scaffolds from aPCR mixtures, which can be used in the context of DNA-origami techniques.aPCR was performed to generate single and double-stranded DNA (dsDNA) from the M13mp18 genome. To isolate the target ssDNA from dsDNA and other PCR impurities, anion-exchange (Q-ligand) and multimodal chromatography (CaptoTM adhere ImpRes) were explored using stepwise gradients with increasing NaCl concentrations. In anion exchange chromatography, the less-charged ssDNA eluted before the dsDNA. In multimodal chromatography, however, the elution pattern was reversed, highlighting the importance played by hydrophobicity. In either case, collected ssDNA-containing fractions were homogeneous and impurity free.Finally, 8.4 μg of a 1000-nt ssDNA fragment was purified and used alongside with site-specific short oligonucleotides (staples) to assemble 63-bp edge length tetrahedrons. Gel electrophoresis showed high assembly yield and purity, whereas fluorescence correlation spectroscopy confirmed that the tetrahedrons had a diffusion coefficient (26.7 μm2 s−1) consistent with the expected size (20 nm).