A computational framework for designing micron-scale crisscross DNA megastructures

Crisscross polymerization enables the assembly of hundreds of unique DNA origami slats into micron-sized structures with nanoscale precision. To design these megastructures, thousands of handle sequences from a fixed library must be assigned to individual slats to encode the desired binding architecture. This complexity presents two major challenges: handles must be selected to minimize parasitic interactions that compete with desired assembly, and the fabrication of hundreds of unique slats creates a substantial logistical burden. Here, we develop a unified framework that standardizes the design and fabrication of crisscross megastructures. We use an evolutionary algorithm to optimize handle assignment and minimize parasitic binding between slats, paired with a graph-based algorithm that expands the handle library. Together, these algorithms enable the assembly of large, multi-layered megastructures that otherwise would be produced at negligible yields. We have released this framework as #-CAD, an open-source graphical application that integrates these algorithms, streamlines laboratory workflows, and makes crisscross DNA origami more broadly accessible.