Direct, live imaging of protein-DNA interactions under physiological conditions is invaluable for understanding the mechanism and kinetics of binding and understanding the topological changes of the DNA strand. The DNA origami technology allows for precise placement of target molecules in a designed nanostructure. Here, we describe a protocol for the self-assembly of DNA origami frames with 2 stretched DNA sequences containing the binding site of a transcription factor, i.e., the Protein FadR, which is a TetR-family tanscription factor regulator for fatty acid metabolism in the archaeal organism Sulfolobus acidocaldarius. These frames can be used to study the dynamics of transcription factor binding using high-speed AFM and obtain mechanistic insights into the mechanism of action of transcription factors.