Research progress in the production, purification, and applications of long single-stranded DNA

Long single-stranded DNA (long ssDNA) plays important roles in cutting-edge fields such as DNA origami, gene therapy, and synthetic biology. However, the production, preparation, and purification of long ssDNA remain technically challenging. This review systematically summarizes current strategies for long ssDNA production and preparation, including phage-E. coli-based biological production, template-dependent enzymatic amplification, and viral-vector-based generation of ssDNA genomes. CRISPR-Cas-assisted approaches are discussed separately as complementary tools for post-synthetic processing, sequence-selective cleavage, enrichment, and quality control, whereas other enzymatic approaches are considered for post-synthetic strand conversion. We compare the characteristics and limitations of these production and preparation strategies, discuss purification techniques for long ssDNA, and summarize its applications in DNA origami nanotechnology and gene therapy. This review comprehensively synthesizes the bottlenecks in long ssDNA production, preparation, and purification, and outlines further development trends. We highlight template-independent de novo enzymatic DNA synthesis, automated DNA manufacturing, synthetic biology-based production systems, and AI-assisted sequence optimization. These developments are expected to accelerate the transition of long ssDNA technologies from laboratory research toward scalable manufacturing and practical applications.