Abstract
Self-assembled DNA nanostructures feature an unprecedented addressability with sub-nanometer precision and accuracy. This addressability relies on the ability to attach functional entities to single DNA strands in these structures. The efficiency of this attachment depends on two factors: incorporation of the strand of interest and accessibility of this strand for downstream modification. Here we use DNA-PAINT super-resolution microscopy to quantify both incorporation and accessibility of all individual strands in DNA origami with molecular resolution. We find that strand incorporation strongly correlates with the position in the structure, ranging from a minimum of 48% on the edges to a maximum of 95% in the center. Our method offers a direct feedback for the rational refinement of the design and assembly process of DNA nanostructures and provides a long sought-after quantitative explanation for efficiencies of DNA-based nanomachines.
Highlights
Self-assembled DNA nanostructures feature an unprecedented addressability with subnanometer precision and accuracy
To translate accessibility to absolute staple incorporation efficiency, we added an offset of +7% when imaging the accessibility of a 3′-end site
The apparent blinking is used to reconstruct super-resolution images that visualize the designed pattern of docking sites, e.g., a 20-nm-grid structure (based on the twodimensional (2D) rectangular DNA origami, details about the design are shown in Supplementary Figs. 1 and 2)
Summary
Self-assembled DNA nanostructures feature an unprecedented addressability with subnanometer precision and accuracy. DNA nanostructures because it can achieve the far unprecedented spatial resolution of ~5 nm, enabling the quantification of the accessibility and absolute incorporation efficiency of every single staple in a DNA origami structure[22]. We concluded that in the 9.3% of the cases (in which no site was detected) the staple was not incorporated This allowed us to assess the accessibility of docking sites for downstream studies, as well as to quantify the actual incorporation efficiency of single staples. As the vision of structural DNA nanotechnology[24] is to arrange matter in a prescribed manner by site-specific attachment of molecular entities, we believe the ultimate measure of quality for DNA origami should be the accessibility of docking sites. We focus on detection efficiencies (i.e., accessibility) for the rest of this study
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