Computational Design of a Thermostable and Highly Active Terminal Deoxynucleotidyl Transferase for Synthesis of Long De Novo DNA Molecules

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 Tm 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.