Computational Design of a Thermostable and Highly Active Terminal Deoxynucleotidyl Transferase for Synthesis of Long De Novo DNA Molecules
Yadan Niu, Binbin Chen, Hui‐Jun Zhang, Wenlong Zheng, Jianping Wu, Lirong Yang, Meng Yang, Haoran Yu
Abstract
It remains challenging for enzymatic synthesis of long DNA using a terminal deoxynucleotidyl transferase (TdT) due to its limited activity against intermediates containing a 3′ terminal hairpin structure that occurred during synthesis. Reverting DNA to a single strand at high temperature is a solution, while TdT exhibits limited thermostability. Here, we explored a computational design strategy to enhance the thermostability of TdT. Ten sequences designed by ProteinMPNN improved the T m value by up to 24.3 °C. Two rounds design using PROSS generated the most stable and active variant M7–8 with a half-life improved by 77-fold. The M7–8 variant was successfully used for highly efficient extension of a 52 nt DNA oligonucleotide containing a hairpin structure, which makes it promising for use in the de novo synthesis of long DNA.