Affinity-Based Magnetic Particles for the Purification of Single-Stranded DNA Scaffolds for Biomanufacturing DNA-Origami Nanostructures

DNA nanotechnology encompasses the self-assembly of nucleic acids into complex nanostructures via Watson–Crick base pairing. Asymmetric PCR (aPCR) is often used to generate 500–3500 nucleotide (nt) long, object-specific, single-stranded DNA (ssDNA) scaffolds from DNA templates, which can then be assembled into nanoobjects by the DNA-origami technique. One crucial step in ssDNA scaffold preparation is purification. Scaffolds are usually purified by agarose gel extraction, a laborious, time consuming, limited, and nonscalable technique that presents low recovery yields, delivers low-quality products, and requires specific equipment. To overcome such pitfalls, we present a simple, fast, and potentially scalable affinity-based method comprising magnetic particles and a simple magnet. The system was used to purify ssDNA scaffolds from aPCR mixtures. Scaffolds with 449 and 1000 nt were synthesized by aPCR along with double-stranded DNA (dsDNA) using the genome of the M13mp18 phage as the template. Magnetic particles were functionalized with a 20 nt oligonucleotide complementary to the 3′ end of ssDNA scaffolds. Hybridization between the ssDNA scaffolds in the aPCR mixture and the affinity beads was promoted, which allowed (i) the removal of the dsDNA and (ii) subsequent recovery of ssDNA upon melting to denaturing temperatures. The purified scaffolds were used to assemble 31 and 63 bp edge length tetrahedra using site-specific short oligonucleotides, thermal annealing, and high magnesium concentrations. The resulting DNA-origami structures showed high assembly yield and purity, as observed using agarose gel electrophoresis. In conclusion, the method enabled the purification of 550 ng of 449 nt and 880 ng of 1000 nt ssDNA fragments per aPCR reaction (50 μL), demonstrating its potential as a helpful and versatile tool in the production of DNA-origami nanostructures.