Protein engineering study demonstrates efficient nucleotide addition using engineered avian terminal deoxynucleotidyl transferase, indicating a practical route for de novo enzymatic DNA synthesis.
Template-free enzymatic approaches are considered the most promising solution for next-generation artificial DNA synthesis. However, the development of these technologies has been hampered by the lack of efficient enzymes specialized for stepwise nucleotide addition. By combining evolutionary analysis, high-throughput mutagenesis scanning, and rational design, we identified a terminal deoxynucleotidyl transferase from Zonotrichia albicollis (ZaTdT) and reshaped its catalytic cavity to better accommodate 3′-ONH 2 -modified nucleotides. The catalytic activity of the engineered ZaTdT for 3′-ONH 2 -dNTPs is 3 orders of magnitude higher than that of the commonly used mammalian TdT. The engineered ZaTdT enables highly efficient single-nucleotide extension of the growing oligonucleotide chain with an average stepwise yield of 98.7%, which makes it practical for de novo enzymatic DNA synthesis.
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Lu et al. (2022) studied this question.
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