Evolving a High-Performance Terminal Deoxynucleotidyl Transferase for Enzymatic DNA Synthesis

Abstract Enzymatic DNA synthesis, using stepwise nucleotide addition catalyzed by template-independent polymerases, promises higher efficiency, quality, and sustainability than today’s industry standard phosphoramidite-based processes. We report on the directed evolution of a terminal deoxynucleotidyl transferase that uses 3’-phosphate blocked dNTPs to control the polymerization reaction and demonstrates high activity for these modified substrates and improved template promiscuity and thermostability.