Programmable Assembly of Higher-Order DNA Nanostructures from Microbial ssDNA Staple Libraries.

Bottom-up manufacturing of structural DNA nanotechnology requires a long single-stranded DNA (ssDNA) scaffold and hundreds of short (∼30 nt) ssDNA staples. However, large-scale production is limited by the high cost and environmental impact of solid-phase chemical staple synthesis. To address these challenges, we developed a phage-free, biological nanomanufacturing platform engineered in Escherichia coli. Two intracellular strategies for producing programmable ssDNA were evaluated: retron-based multicopy ssDNA (msDNA) synthesis via the Ec67 system and plasmid-encoded rolling circle replication (RCR). Although sequence-design flexibility is constrained by structural requirements within the retron (msd) cassette, the RCR-based system decouples ssDNA replication from sequence structure, enabling synthesis of arbitrary staples. This RCR platform generated long circular ssDNA (cssDNA) precursors of at least 1.8 kb with >99% sequence fidelity. Integrating programmable BseGI cleavage sites allowed targeted strand-selective enzymatic processing to release stoichiometric pools of 32-nt, origami-grade staple strands. Atomic force microscopy (AFM) confirmed that these biologically produced staples directed high-fidelity self-assembly of complex DNA tiles and hollow tubules. Notably, structural folding was demonstrated directly in crude cellular lysates. Together, these findings establish a phage-free framework for programmable in vivo ssDNA production and lay the foundation for future biological DNA nanomanufacturing workflows.