The sequence of events during folding of a DNA origami.

We provide a comprehensive reference dataset of the kinetics of a multilayer DNA origami folding. To this end, we measured the folding kinetics of every staple strand and its two terminal segments during constant-temperature assembly of a multilayer DNA origami object. Our data illuminate the processes occurring during folding of the DNA origami in fine detail, starting with the first nucleating double-helical domains and ending with the fully folded DNA origami object. We found a complex sequence of folding events that cannot be explained with simplistic local design analysis. Our real-time data, although derived from one specific DNA origami object, through its sheer massive detail, could provide the crucial input needed to construct and test a quantitatively predictive, general model of DNA origami assembly.