The rational design of complementary DNA sequences can be used to construct a variety of self-assembling DNA nanostructures with nanometre precision, enabling biodesign and synthetic biology. DNA nanostructures have been extensively verified with techniques such as atomic force microscopy and electron microscopy, but these methods require edge definition and spatial calibration respectively, and require the adsorption of ‘soft’ biomolecules onto a surface, which can often deform their structure, especially in the case of single-layered DNA origami structures.
Here we demonstrate that SAXS can distinguish between different, modified single layered DNA origami tiles that look identical when immobilised on a mica surface and imaged with AFM. We use SAXS to explore the effect of individual staple location on the global twist of single layered DNA origami structures. We show that a substantial global twist, similar in magnitude to those previously observed in multi-layered DNA origami structures, can be induced with far more subtle deviations of staple crossover periodicities in single-layered DNA origami structures.
We then address the challenge of making direct measurements of the overall dimensions of DNA origami structures by immobilising pairs of gold nanoparticles at fixed positions. These result in discernable diffraction peaks in SAXS profiles that allow for the precise measurements of distances of at least ~900 Å, far beyond point-to-point distance resolution currently attainable using single molecule FRET.
Finally, we demonstrate that SAXS and Fourier analysis can measure internal geometries of a DNA origami nanotube, including interhelical spacing and the diameter of an individual double helix at concentrations as low as 0.1 mg/ml. Together, these results establish SAXS as a technique that can accurately measure large distances between specific regions of a molecule and also provide high-resolution measurements of overall geometry.