Suppression of Aggregation Enhances Placement Fidelity of Asymmetrical Right Triangle DNA Origami

DNA origami placement (DOP) exploits shape complementarity between DNA nanostructures and lithographically patterned binding sites to achieve precise spatial positioning, offering a promising route for integrating bottom-up self-assembly with top-down nanomanufacturing. However, nonspecific aggregation of DNA origami on patterned binding sites limits both single-origami occupancy yield and orientational fidelity. Here, we investigated the aggregation mechanism in an asymmetrical right triangle DNA origami system and found that vertex-specific aggregation is primarily driven by unbalanced origami arm twisting. Atomic force microscopy (AFM) revealed that aggregation predominantly occurs at vertices associated with the hypotenuse arm C, where twisting exposes staple domains that promote interorigami binding. Selective removal of short staple strands at the C arm termini reduced rigidity at the vertex, thereby preventing exposure of the staple domain, suppressing interorigami aggregation, and increasing the monomer yield from 72 ± 1.1% to 87.3 ± 1.5%. In contrast, poly-(T) extension at the same sites yields only minor improvement, indicating that base stacking is not the dominant aggregation mechanism. When applied to directed placement on patterned substrates, the optimized design improved orientation matching yield from 33.1 ± 7.8% to 77.1 ± 4.8%, representing a significant improvement in placement fidelity. These results demonstrate that tuning local structural features provides an effective strategy to suppress aggregation and is essential for achieving high-fidelity placement of asymmetrical DNA origami.