Isothermal Disorder-to-Order Transitions of DNA Origami Structures Induced by Alternative Component Subsets

Abstract

DNA origami technology has shown potential across various applications, including the construction of molecular machines. Among these, mimicking the complex structural transitions of natural biomolecules in physiological environments remains a long-standing pursuit. Here, inspired by intrinsically disordered proteins, we propose a strategy for inducing disorder-to-order transitions in DNA origami structures at room temperature using alternative component subsets. In a triangular DNA origami model, we define three subsets of its constitutional DNA staples based on their spatial distributions along the scaffold. Atomic force microscopy and molecular dynamics simulations show that the individual subsets result in metastable assemblies with disordered morphologies and elevated free-energy fluctuations compared with those generated by the complete set of staples. Notably, after the addition of the remaining staples, the irregular structures transform into ordered triangular architectures within 2 h at room temperature, achieving yields of up to ∼60%. These findings suggest that these controlled folding pathways in DNA origami can robustly converge on the global energy minimum at room temperature, thereby providing a promising alternative strategy for engineering biomimetic DNA molecular machines.

Figure 1

Scheme of the self-assembly of DNA origami with stepwise introduced subsets. (a) Flow diagram of the two-stage assembly. (b) Schematic illustration of the layouts of the staple subsets in triangular DNA origami. Blue lines, staples involved in the subsets.

Figure 2

Partially folded DNA origami structures resulting from different subsets. (a) Schematic of the layouts of different staple subsets in DNA origami structures in Stage 1; (b) Snapshots of coarse-grained molecular dynamics simulations of different staple subset products from Stage 1. (c) Representative AFM images of different subset products from Stage 1, and the area distribution statistics of the monolithic structure (N ≈ 1000 structures). Red solid lines are GaussAmp fitting. (d) Histogram of the area distribution of monomer structures of different subset products from Stage 1 (N ≈ 1000 structures). (e) Yield statistics for uncompleted structures of different subsets from Stage 1. Data are presented as mean ± s.d. (N ≈ 1000 structures). ***, p < 0.001, analyzed with one-way ANOVA.

Figure 3

Complete DNA origami structures resulting from different pathways. (a) Schematic of the different subset types in Stage 2, the assembled AFM results, and the statistical analysis of the area of monolithic structures (N ≈ 1000 structures). White dashed circles, incomplete shapes. (b) Histogram of the statistical analysis of the area of monolithic structures produced by the different Staple subsets in Stage 2 (N ≈ 1000 structures). (c) Yield statistics for complete structures of each subset in Stage 2. Red solid lines, GaussAmp fitting. Data are presented as mean ± s.d. (N ≈ 1000 structures). **, p < 0.01; ***, p < 0.001, analyzed with one-way ANOVA.

Figure 4

Simulated free energy landscapes resulting from different subsets. (a) Energy equilibrium oscillation curves of the free energies of the structures of different subsets; (b) Energy frequency distributions of the free energies of the structures of different subsets; (c) Energy histograms of the structures at different stages of the folding process for each set type; gray denotes the initial unfolded state; blue denotes the different structures generated by the folding of the different subset staples; and green denotes the completed shapes. Scale bar: 50 nm. The free energies of the structures are in units of k_BT, where k_B is the Boltzmann constant, and T is the absolute temperature. Data are presented as mean ± s.d. (N ≈ 20,000 samplings in a simulation). ***, p < 0.001, analyzed with one-way ANOVA.