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

ABSTRACT

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.