Enzymatic de novo oligonucleotide synthesis: Emerging techniques and advancements

Oligonucleotide synthesis serves as a cornerstone of modern life sciences, enabling groundbreaking advancements across molecular diagnostics, therapeutic development, and transformative technologies including DNA data storage and programmable biological systems. While phosphoramidite-based chemical synthesis remains the industrial standard, its limitations in producing long-sequence constructs, cumulative error rates, and reliance on toxic solvents pose significant challenges for next-generation applications. Emerging enzymatic synthesis approaches offer a paradigm shift by harnessing the inherent precision and environmental sustainability of biological systems. This comprehensive review systematically examines the evolving landscape of oligonucleotide synthesis technologies. We first analyze the mechanistic foundations and persistent limitations of conventional chemical methods, followed by a critical evaluation of enzymatic strategies with particular emphasis on terminal deoxynucleotidyl transferase (TdT)-mediated template-independent polymerization. The work provides detailed insights into enzymatic reaction engineering, including substrate specificity profiling of nucleotide analogs and innovative solid-phase synthesis platforms enabling iterative nucleotide addition. Furthermore, we discuss emerging high-throughput synthesis architectures and commercial translation efforts. In summary, this review comprehensively encapsulates the advancements and commercialization status of enzymatic synthesis technologies, offering valuable guidance that can expedite the innovative development of enzymatic oligonucleotide manufacturing platforms.