Unraveling the Folding Dynamics of DNA Origami Structures

Achieving high folding yield remains a major challenge in DNA origami, particularly as structures increase in complexity and scale. Here, we investigate how DNA origami design influences folding yield and kinetics using a combination of real-time fluorometry, gel electrophoresis, electron microscopy, and theoretical analysis. Results reveal a balance of the free energy changes from loop formation and hybridization that govern nucleation of nanostructure assembly, while the extent of cooperativity determines the overall assembly behavior. We measure the effect of structural complexity, staple design, and scaffold design on each energetic parameter, folding yield, and kinetics. We show that the scaffold crossover pattern determines the extent of cooperativity and subsequent folding kinetics, where fewer scaffold crossovers result in more cooperative folding. We also demonstrate that limiting the number of crossovers per staple should be prioritized over extending staple binding domains. The entropic penalty dominates the lower energy binding, disrupting folding. Finally, we demonstrate a 1-2 hour focused annealing ramp strategy that can increase yield up to 17% relative to traditional multi-day ramps. Optimizing energy changes and the contribution of cooperativity through design can significantly enhance folding yield and assembly time, particularly for complex structures, aiding the design and assembly of large-scale materials.