Department of Physics, Technical University of Munich, Garching 85748, Germany.
ACS Nano. 2021 Jun 22;15(6):9391-9403. doi: 10.1021/acsnano.0c10137. Epub 2021 Mar 16.
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.
阳离子涂层可以增强合成 DNA 物体在低离子强度环境（如生理流体）中的稳定性。在这里，我们使用单颗粒冷冻电子显微镜（cryo-EM）、拟原子模型拟合和单分子质量光度法研究了寡聚赖氨酸和聚乙二醇（PEG）-寡聚赖氨酸涂层的多层 DNA 折纸物体。这些涂层在多个纳米的分辨率下很好地保留了粗结构特征，但也会引起扭曲和弯曲等变形。更高密度的涂层也会导致 DNA 折纸测试物体的内部结构变形，其中设计的蜂窝型螺旋晶格被变形为更类似于正方形晶格的图案。在生理离子强度下，未涂层的物体解体，而涂层的物体保持完整，但它们在螺旋方向上收缩，在垂直于螺旋轴的方向上扩展。在我们确定的寡聚赖氨酸和 PEG-寡聚赖氨酸涂层的 DNA 折纸中，无法在 cryo-EM 图谱中分辨出螺旋的细节，如主/小沟和交叉点的位置，而这些特征在未涂层的参考物体的 cryo-EM 图谱中是可见的。在涂层下，钝端双链的接口仍然可以访问，并且可能用于形成依赖于钝端螺旋之间堆积相互作用的多聚体 DNA 折纸组装体。从涂层 DNA 折纸形成多聚体所需的离子强度要求与未涂层物体不同。使用单分子质量光度法，我们发现涂层 DNA 折纸物体在低离子强度生理条件下孵育前后的质量保持不变。这一发现表明，该涂层有效地阻止了链的解离，但也表明该涂层本身在原位保持稳定。我们的结果验证了寡聚赖氨酸涂层作为 DNA 折纸的强大稳定方法，但也揭示了一些潜在的失效点，实验者应该注意避免使用错误的前提进行工作。
