posted on 2021-11-24, 20:34authored byA. Rita Silva-Santos, Rui Oliveira-Silva, Sara Sousa Rosa, Pedro M. R. Paulo, Duarte Miguel F. Prazeres
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.