Correlative Super-Resolution and Atomic Force Microscopy of DNA Nanostructures and Characterization of Addressable Site Defects

Abstract

To bring real-world applications of DNA nanostructures to fruition, advanced microscopy techniques are needed to shed light on factors limiting the availability of addressable sites. Correlative microscopy, where two or more microscopies are combined to characterize the same sample, is an approach to overcome the limitations of individual techniques, yet it has seen limited use for DNA nanotechnology. We have developed an accessible strategy for high resolution, correlative DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution and atomic force microscopy (AFM) of DNA nanostructures, enabled by a simple and robust method to selectively bind DNA origami to cover glass. Using this technique, we examined addressable “docking” sites on DNA origami to distinguish between two defect scenarios–structurally incorporated but inactive docking sites, and unincorporated docking sites. We found that over 75% of defective docking sites were incorporated but inactive, suggesting unincorporated strands played a minor role in limiting the availability of addressable sites. We further explored the effects of strand purification, UV irradiation, and photooxidation on availability, providing insight on potential sources of defects and pathways toward improving the fidelity of DNA nanostructures.

Understanding Förster Resonance Energy Transfer in the Sheet Regime with DNA Brick-Based Dye Networks

“…To characterize dye incorporation efficiency, we measured the absorbance of purified structures containing increasing ratios of a single dye species. We note that DNA strand incorporation efficiency has been studied for DNA origami structures, 51,52 but equivalent studies have yet to be realized for DNA brickbased assemblies. Comparing the dye absorbance normalized by the 260 nm DNA absorbance (to correct for the number of total DNA structures) with the predicted value for the estimated number of dyes that should be present, we find that for Cy3 and A647 the dye incorporation is near ideal (98 ± 9.0% incorporation, Figure 2d).…”

Rapid DNA origami nanostructure detection and classification using the YOLOv5 deep convolutional neural network

“…The clipped images can then be stacked and input into the secondary CNN which will analyze the structures on a per unit basis and evaluate which binding sites are occupied, what is the labeling efficiency, defect analysis, etc. For example the CNN utilized by Green et al 53 for defect analysis in DNA origami using DNA-PAINT would be an ideal candidate to use as a secondary CNN. Using this approach, we were able to cut the time required for AFM image annotation from hours to less than 30 s. Furthermore, the proposed method is not subject to operator dependent variability and thus is of high repeatability.…”

Suppressing high-dimensional crystallographic defects for ultra-scaled DNA arrays

“…In self-assembled DNA patterns, the intrinsic physical properties of DNA helix (i.e., chirality, base number per turn, and crossover density) may induce lattice twisting 27 , which can be corrected via corrugated designs 13 , 46 – 49 and optimized basepair number per helical turn 50 . Meanwhile, the incorporation errors of individual DNAs (i.e., 0D defects, such as missing, duplicating, and local swinging of individual DNA building block) 51 are occurring at a rate of 0.3–20% 33 , 52 – 54 , as results of the overall contributions from different designs 55 , 56 , synthetic quality 57 , concentrations of DNA motifs 33 , 38 , the assembly conditions 52 , 58 , and the measurement accuracy 52 – 54 , besides the impact of DNA sequence 55 , 58 . However, because 3D periodic DNA lines typically consist of thousands of DNA building blocks 33 , 59 , individual 0D incorporation errors of single DNA do not affect the approximate shape of the DNA lines and may not inevitably lead to the pattern failure.…”

“…However, because 3D periodic DNA lines typically consist of thousands of DNA building blocks 33 , 59 , individual 0D incorporation errors of single DNA do not affect the approximate shape of the DNA lines and may not inevitably lead to the pattern failure. Instead, pattern failures arise when the simultaneous accumulations of tens to hundreds of mis-assembled DNAs within nearby lattices extend into higher dimensions 56 , 59 , 60 , including 1D dislocated lines and 2D domains of distinct lattice orientations. Even though these high-dimensional defects correlate with the majority of pattern failures 33 , 38 , 56 , 59 , there still lack an effective strategy to suppress them in the periodic DNA patterns.…”

“…Instead, pattern failures arise when the simultaneous accumulations of tens to hundreds of mis-assembled DNAs within nearby lattices extend into higher dimensions 56 , 59 , 60 , including 1D dislocated lines and 2D domains of distinct lattice orientations. Even though these high-dimensional defects correlate with the majority of pattern failures 33 , 38 , 56 , 59 , there still lack an effective strategy to suppress them in the periodic DNA patterns. Furthermore, the defective DNA patterns exhibit similar molecular weights and dimensions to those of the correctly assembled ones, presenting challenges for effective purification via conventional separation methods 33 , 61 .…”