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, scaling production remains bottlenecked by the high economic cost and environmental footprint of solid-phase chemical staple synthesis. To address these limitations, a phage-free, biological nanomanufacturing platform engineered in Escherichia coli is developed here. Two intracellular strategies for producing programmable ssDNA were systematically evaluated: retron-based multicopy ssDNA (msDNA) synthesis via the Ec67 system and plasmid-encoded rolling circle replication (RCR). While native structural topology constraints within the retron (msd) cassette limit its sequence-design flexibility, the alternative RCR-driven engine successfully decouples ssDNA replication from sequence secondary structures, enabling the synthesis of arbitrary staples. This RCR platform reliably generates long circular ssDNA (cssDNA) precursors of at least 1.8 kb with exceptional sequence fidelity (>99%). Integrating programmable BseGI cleavage sites allows targeted strand-selective enzymatic processing to cleanly release stoichiometric, origami-grade pools of 32-nt staple strands. Atomic force microscopy (AFM) confirms that these biologically produced staples successfully drive the high-fidelity self-assembly of complex DNA tiles and hollow tubules. Crucially, robust structural folding is demonstrated directly within crude, unpurified cellular lysates, establishing a green, cost-effective framework for the one-pot fabrication of advanced DNA-based nanomaterials.