Dimensions and Global Twist of Single-Layer DNA Origami Measured by Small-Angle X-ray Scattering

Abstract

The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.

Design, optimization, and analysis of large DNA and RNA nanostructures through interactive visualization, editing, and molecular simulation

“…OxDNA was parameterized to correctly reproduce the global twist of large 3D DNA structures 48,62 , suggesting that this twist is likely significant while in solution. We note, however, that the global twist of 2D DNA nanostructures in the bulk remains a topic of active research 63 , and more experimental data is needed to establish a better comparison of oxDNA parametrization with experimentally determined structures. Mean structures are also the best method to compare simulation results to cryo-EM maps.…”

Design and Synthesis of Pleated DNA Origami Nanotubes with Adjustable Diameters

“…The search algorithm was used to design and synthesize a minimally-strained regular 20-helix nanotube with a defined inside and outside surface (Figure 2A Nanotubes were characterized with transmission electron microscopy (TEM), atomic force microscopy (AFM) and cryo-electron microscopy (cryo-EM) ( Figure 2C; Supplementary Figure S3). With all imaging methods, the nanotubes appeared mostly as uniform rectangular structures whose lengths (TEM = 118.1 ± 2.0 nm, cryo-EM = 118.4 ± 2.9 nm, AFM = 121.1 ± 7.6 nm) ( Figure 2E; Figure S3), which likely results from affinity to the surface and mechanical compression by the AFM tip (49,57). This interaction also likely contributed to the higher ratio of structures which appear to have split and unrolled in AFM (13.2%) as compared to those seen in TEM (5.3%) micrographs (49).…”

“…where X p is the crossover periodicity, θ i is the internal angle between any three sequential helices and D p is the periodicity of a DNA duplex, approximately 10.5 base-pairs (bp) per turn (4,56,57). For a regular DNA nanotube with n helices, θ i is the internal angle of a regular polygon with n sides:…”

“…The approach is applied to the design and synthesis of a regular rolled-rectangle nanotube, and for DNA origami nanotubes with a pleated wall structure similar to those conceptualized theoretically (58). The latter design allows for nanotubes with an arbitrary and continuous set of diameters and interhelical spacing, and multi-layer DNA origami walls providing additional rigidity over single-layer walls, which lack structural rigidity (57). Finally, we present a new staple routing that adds further rigidity, provides additional control over the dimensions of the pleated nanotube and which is generally applicable to other DNA nanostructures.…”

In‐Situ Configuration Studies on Segmented DNA Origami Nanotubes

“…The structural parameters obtained by SAXS are in good agreement with those determined by atomicf orce microscopy (AFM), transmission electron microscopy (TEM), andd ynamic light scattering (DLS). [20,21] Further,t he positioning of chiral gold nanoparticles (AuNPs) decorated on DNA nanotubes has been accurately determined by analyzing the pair-distance distribution function. Our resultsestablish SAXS as areliable structural analysism ethod for long DNA nanotubes and could assist in the rational design of these structures.…”