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However, large\u2010scale production is limited by the high cost and environmental impact of solid\u2010phase chemical staple synthesis. To address these challenges, we developed a phage\u2010free, biological nanomanufacturing platform engineered in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    . Two intracellular strategies for producing programmable ssDNA were evaluated: retron\u2010based multicopy ssDNA (msDNA) synthesis via the Ec67 system and plasmid\u2010encoded rolling circle replication (RCR). Although sequence\u2010design flexibility is constrained by structural requirements within the retron (\n                    <jats:italic>msd<\/jats:italic>\n                    ) cassette, the RCR\u2010based 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\u00a0kb with &gt;99% sequence fidelity. Integrating programmable BseGI cleavage sites allowed targeted strand\u2010selective enzymatic processing to release stoichiometric pools of 32\u2010nt, origami\u2010grade staple strands. Atomic force microscopy (AFM) confirmed that these biologically produced staples directed high\u2010fidelity self\u2010assembly of complex DNA tiles and hollow tubules. Notably, structural folding was demonstrated directly in crude cellular lysates. Together, these findings establish a phage\u2010free framework for programmable in vivo ssDNA production and lay the foundation for future biological DNA nanomanufacturing workflows.\n                  <\/jats:p>","DOI":"10.1002\/smll.75880","type":"journal-article","created":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T07:34:03Z","timestamp":1790408043000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Programmable Assembly of Higher\u2010Order DNA Nanostructures from Microbial ssDNA Staple Libraries"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2594-0408","authenticated-orcid":false,"given":"Ming Hung","family":"Yen","sequence":"first","affiliation":[{"name":"Chemical &amp; Biomolecular Engineering University of Delaware  Newark Delaware USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1708-5605","authenticated-orcid":false,"given":"Mallikarjuna Reddy","family":"Kesama","sequence":"additional","affiliation":[{"name":"School of Mechanical Engineering Purdue University  West Lafayette Indiana USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-7943-4342","authenticated-orcid":false,"given":"Yancheng","family":"Du","sequence":"additional","affiliation":[{"name":"School of Mechanical Engineering Purdue University  West Lafayette Indiana USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0507-3052","authenticated-orcid":false,"given":"Jong Hyun","family":"Choi","sequence":"additional","affiliation":[{"name":"School of Mechanical Engineering Purdue University  West Lafayette Indiana USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2904-9118","authenticated-orcid":false,"given":"Kevin V.","family":"Solomon","sequence":"additional","affiliation":[{"name":"Chemical &amp; Biomolecular Engineering University of Delaware  Newark Delaware 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