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TitleCryo-Electron Microscopy and Mass Analysis of Oligolysine-Coated DNA Nanostructures.
Journal, issue, pagesACS Nano, Vol. 15, Issue 6, Page 9391-9403, Year 2021
Publish dateJun 22, 2021
AuthorsEva Bertosin / Pierre Stömmer / Elija Feigl / Maximilian Wenig / Maximilian N Honemann / Hendrik Dietz /
PubMed AbstractCationic 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), ...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.
External linksACS Nano / PubMed:33724780 / PubMed Central
MethodsEM (single particle)
Resolution10.38 - 21.28 Å
Structure data

EMDB-12467:
B-brick 0.2:1 N:P PEG-oligolysine coated in 5 mM Mg2+
Method: EM (single particle) / Resolution: 12.0 Å

EMDB-12468:
B-brick 100:1 N:P PEG-oligolysine coated in 5 mM Mg2+
Method: EM (single particle) / Resolution: 21.28 Å

EMDB-12469:
B-brick 100:1 N:P PEG-oligolysine coated in PBS
Method: EM (single particle) / Resolution: 16.95 Å

EMDB-12470:
B-brick 0.75:1 N:P PEG-oligolysine coated in 5 mM Mg2+
Method: EM (single particle) / Resolution: 14.95 Å

EMDB-12471:
B-brick 0.75:1 N:P PEG-oligolysine coated in PBS
Method: EM (single particle) / Resolution: 18.76 Å

EMDB-12472:
B-brick 0.5:1 N:P oligolysine coated in 5 mM Mg2+
Method: EM (single particle) / Resolution: 16.26 Å

EMDB-12473:
A-brick bare in 5 mM Mg2+
Method: EM (single particle) / Resolution: 11.88 Å

EMDB-12474:
A-brick 0.75:1 N:P PEG-oligolysine coated in 5 mM Mg2+
Method: EM (single particle) / Resolution: 12.42 Å

EMDB-12475:
A-brick 0.75:1 N:P PEG-oligolysine coated in PBS
Method: EM (single particle) / Resolution: 16.29 Å

EMDB-12476:
A-brick 0.5:1 N:P oligolysine coated in 5 mM Mg2+
Method: EM (single particle) / Resolution: 17.7 Å

12516

7npn

EMDB-12516, PDB-7npn:
B-brick bare in 5 mM Mg2+
Method: EM (single particle) / Resolution: 10.38 Å

Source
  • synthetic construct (others)
KeywordsDNA / DNA Origami

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