Position Accuracy of Gold Nanoparticles on DNA Origami Structures Studied with Small-Angle X-ray Scattering

DNA origami objects allow for accurate positioning of guest molecules in three dimensions. Validation and understanding of design strategies for particle attachment as well as analysis of specific particle arrangements are desirable. Small-angle X-ray scattering (SAXS) is suited to probe distances of nano-objects with subnanometer resolution at physiologically relevant conditions including pH and salt and at varying temperatures. Here, we show that the pair density distribution function (PDDF) obtained from an indirect Fourier transform of SAXS intensities in a model-free way allows to investigate prototypical DNA origami-mediated gold nanoparticle (AuNP) assemblies. We analyze the structure of three AuNP-dimers on a DNA origami block, an AuNP trimer constituted by those dimers, and a helical arrangement of nine AuNPs on a DNA origami cylinder. For the dimers, we compare the model-free PDDF and explicit modeling of the SAXS intensity data by superposition of scattering intensities of the scattering objects. The PDDF of the trimer is verified to be a superposition of its dimeric contributions, that is, here AuNP-DNA origami assemblies were used as test boards underlining the validity of the PDDF analysis beyond pairs of AuNPs. We obtain information about AuNP distances with an uncertainty margin of 1.2 nm. This readout accuracy in turn can be used for high precision placement of AuNP by careful design of the AuNP attachment sites on the DNA-structure and by fine-tuning of the connector types.