Scaffolded DNA origami nanostructures enable the self-assembly of arbitrarily shaped objects with unprecedented accuracy. Yet, varying physiological conditions are prone to induce slight structural changes in the nanoscale architecture. Here, we report on high precision measurements of overall shape and interhelical distance of three prototypic DNA origami structures in solution using synchrotron small-angle X-ray scattering. Sheet-, brick-, and cylinder-shaped DNA constructs were assembled and the shape factors determined with angstrom resolution from fits to the scattering profiles. With decreasing MgCl₂ concentration electrostatic swelling of both shape cross section and interhelical DNA spacing of the DNA origami structures is observed. The structures tolerate up to 10% interhelical expansion before they disintegrate. In contrast, with increasing temperature, the cylinder-shaped structures show no thermal expansion in a wide temperature window before they abruptly melt above 50 °C. Details on molecular structure of DNA origami can also be obtained using in-house X-ray scattering equipment and, hence, allow for routine folding and stability testing of DNA-based agents that are designed to operate under varying salt conditions.

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