Computer-Assisted Workflow for DNA Origami-Engineered Triangular Nanopores.

Synthetic nanopores engineered by DNA origami provide a highly biocompatible and structurally programmable platform for elucidating the transmembrane mechanisms of functional macromolecules. However, engineering large DNA nanopores exceeding 30 nm in width remains a significant challenge. Here, we introduce a computer-assisted workflow to significantly reduce the design time and characterization costs of 45 nm-wide triangular DNA nanopores compared to empirical approaches. By leveraging extensive computational simulations, the feasibility of the triangular nanopore design was first validated. Computer-assisted image processing and data analysis confirmed that the majority of nanopore monomers maintained consistent triangular configurations and were capable of single-molecule translocation of trypsin. Long-term fluorescence tracking revealed exponential uptake of dextran into the vesicle interior, suggesting that the triangular DNA nanopore holds promise as a gatekeeper of macromolecular transmembrane transport. We envision that this computer-assisted approach could enhance design efficiency and data-processing accuracy, establishing a robust foundation for the intelligent development of DNA nanopores.