Enzymatic Strategies for Next-Generation DNA Synthesis: Boosting Efficiency and Overcoming Secondary Structures

Chemical DNA synthesis is the gold standard for producing synthetic oligonucleotides. Despite four decades of success, fragment length and secondary DNA structure formation remain limitations, constraining access to long, complex sequences. Enzymatic de novo DNA synthesis has emerged as a promising next-generation alternative, offering a catalytic nature, higher coupling efficiencies, and uninterrupted strand growth. Recent advances, such as Codexis’ evolved terminal deoxynucleotidyl transferase (TdT) achieving near-chemical coupling efficiencies with 3′-phosphate-protected nucleotides, show that enzyme engineering is a viable path forward. The mitigation of secondary DNA structure formation is equally crucial to avoid synthesis stalls and complicated DNA assembly yet it remains surprisingly underexplored. This Perspective highlights current strategies to address both bottlenecks: improving TdT catalysis and controlling DNA folding. Together, these efforts could enable routine, cost-effective synthesis of kilobase-long, structure-rich sequences, transforming research and industrial applications alike.