posted on 2021-03-16, 19:14authored byEva Bertosin, Pierre Stömmer, Elija Feigl, Maximilian Wenig, Maximilian N. Honemann, Hendrik Dietz
Cationic coatings
can enhance the stability of synthetic DNA objects
in low ionic strength environments such as physiological fluids. Here,
we used single-particle cryo-electron microscopy (cryo-EM), pseudoatomic
model fitting, and single-molecule mass photometry to study oligolysine
and polyethylene glycol (PEG)-oligolysine-coated multilayer DNA origami
objects. The coatings preserve coarse structural features well on
a resolution of multiple nanometers but can also induce deformations
such as twisting and bending. Higher-density coatings also led to
internal structural deformations in the DNA origami test objects,
in which a designed honeycomb-type helical lattice was deformed into
a more square-lattice-like pattern. Under physiological ionic strength,
where the uncoated objects disassembled, the coated objects remained
intact but they shrunk in the helical direction and expanded in the
direction perpendicular to the helical axis. Helical details like
major/minor grooves and crossover locations were not discernible in
cryo-EM maps that we determined of DNA origami coated with oligolysine
and PEG-oligolysine, whereas these features were visible in cryo-EM
maps determined from the uncoated reference objects. Blunt-ended double-helical
interfaces remained accessible underneath the coating and may be used
for the formation of multimeric DNA origami assemblies that rely on
stacking interactions between blunt-ended helices. The ionic strength
requirements for forming multimers from coated DNA origami differed
from those needed for uncoated objects. Using single-molecule mass
photometry, we found that the mass of coated DNA origami objects prior
to and after incubation in low ionic strength physiological conditions
remained unchanged. This finding indicated that the coating effectively
prevented strand dissociation but also that the coating itself remained
stable in place. Our results validate oligolysine coatings as a powerful
stabilization method for DNA origami but also reveal several potential
points of failure that experimenters should watch to avoid working
with false premises.