Scalable and sustainable manufacturing of functional DNA nanoassemblies via self-folding circular single-stranded DNA.

Functional DNA nanoassemblies (fDns) hold promise for biomedical and translational applications, but broader deployment is limited by manufacturing barriers that constrain scalability, reproducibility, and stability. Current biosynthetic approaches rely heavily on downstream enzymatic processing and purification, which dominate economic, operational, and environmental costs at scale. Here we report an integrated manufacturing strategy that combines programmed circular single-stranded DNA with bacteriophage-driven biosynthesis to enable direct production of high-quality fDns with minimal post-assembly purification. By reducing reliance on purification as a major bottleneck, this approach delivers topology-enhanced stability, high assembly efficiency, and batch reproducibility, achieving sub-gram laboratory production at ~$6 per milligram while suggesting a pathway toward scalable manufacturing. We demonstrate versatility through 11 distinct fDns, including an aptamer-functionalized construct that neutralizes SARS-CoV-2 pseudovirus and authentic Omicron variants in cellular and animal models. This work provides a cost-effective, scalable manufacturing framework for fDns, advancing their sustainable and reproducible deployment across life-science applications.