Co-engineering DNAzymes and Phagemid Systems for Efficient and Scalable Production of Functional Single-Stranded DNA Nanostructures

Abstract DNA nanostructures have been widely explored for diverse applications, yet their scalable and cost-efficient production remains a major limitation. Phagemid-based biosynthesis of single-stranded DNA (ssDNA) provides a promising approach, but its efficiency is constrained by limited deoxyribozyme (DNAzyme) performance and suboptimal system design. Here, we develop an optimized bioproduction platform for ssDNA nanostructures by simultaneously engineering DNAzymes and phagemid-based production systems. Sequence-guided DNAzyme design identifies determinants for catalytic efficiency and pH adaptability, enabling highly efficient and robust in cis cleavage. The optimized DNAzymes achieve 100% self-cleavage within 30 min and expand the operational pH range from a narrow window around 7.2 to 6.6–7.8, thus enabling more reliable production of target ssDNA for structural folding. By combining optimization of phagemid constructs and helper phage/plasmid combinations, the resulting platform supports the biotechnological production of different nanostructures, including square and aptamer-functionalized triangle assemblies. With the p3024 phagemid and VCSM13 helper phage, production of functionalized triangular ssDNA nanostructures was successfully scaled to a 5 L bioreactor, yielding 151.0 mg/L phagemid ssDNA. Doxorubicin-loaded, aptamer AS1411-functionalized triangular nanostructures (DOX@DN-AS1411) enabled targeted and rapid intracellular drug delivery in breast cancer cells. This work establishes a scalable platform to produce programmable DNA nanostructures, facilitating their translation toward biomedical and nanomanufacturing applications.