A General Enzymatic Strategy for Site‐Specific Incorporation of Modified Genetic Building Blocks Into DNA

Methods for site-specific incorporation of modified DNA building blocks remain limited. Phosphoramidite chemistry, the most common approach, suffers from low coupling efficiency, error-prone synthesis, and toxic waste generation. Enzymatic approaches on the other hand operate under mild aqueous conditions but are highly context-dependent, cannot incorporate multiple modifications, or lack generalizability. Here, we present a proof-of-concept for a general enzymatic strategy that uses template-dependent polymerases to incorporate modified nucleotides at defined positions with high fidelity, providing highly pure products through intrinsic error correction. There is scope for this approach to be automated on DNA sequencers using Sequencing-by-Synthesis (SBS) technology, offering a pathway for a routine, eco-friendly synthesis of long, site-specifically modified DNA sequences, including promoters and gene fragments.