Recent advances in biocatalytic and chemoenzymatic synthesis of oligonucleotides

Access to synthetic oligonucleotides is crucial for applications in diagnostics, therapeutics, synthetic biology, and nanotechnology. Traditional solid phase synthesis is limited by sequence length and complexities, low yields, high costs and poor sustainability. Similarly, polymerase‐based approaches such as in vitro transcription and primer extension reactions do not permit any control on the positioning of modifications and display poor substrate tolerance. In response, biocatalytic and chemoenzymatic strategies have emerged as promising alternatives, offering selective and efficient pathways for oligonucleotide synthesis. These methods leverage the precision and efficiency of enzymes to construct oligonucleotides with high fidelity. Recent advancements have focused on optimized systems and/or engineered enzymes enabling the incorporation of chemically modified nucleotides. Biocatalytic approaches, particularly those using DNA/RNA polymerases provide advantages in milder reaction conditions and enhanced sustainability. Chemoenzymatic methods, combining chemical synthesis and enzymes, have proven to be effective in overcoming limitations of traditional solid phase synthesis. This review summarizes recent developments in biocatalytic and chemoenzymatic strategies to construct oligonucleotides, highlighting innovations in enzyme engineering, substrate and reaction condition optimization for various applications. We address crucial details of the methods, their advantages, and limitations as well as important insights for future research directions in oligonucleotide production.