SYNTHETIC ENZYMOLOGY FOR NEXT-GENERATION-OMICS APPLICATIONS

Abstract

Terminal deoxynucleotidyl transferase (TdT) is a Family X DNA polymerase with dominant template-independent activity. One alluring possibility is the use of TdT for de novo DNA synthesis by iterative incorporation of reversibly 3’ blocked nucleotides followed by deblocking. However, WT TdT is incapable of incorporating 3’ modified nucleotides, and TdT engineering is hindered by its marginal stability. We desired to evolve a TdT variant of enhanced thermostability, which act as a scaffold for directed evolution towards efficient incorporation of 3’ blocked nucleotides. Thermostable TdT variant would also have potential applications in current library preparation. Here, we developed a FRET-based assay and screened about 10,000 mutants to identify a thermostable TdT, TdT3-2. Subsequent verification confirmed that TdT3-2 is about 10 °C more thermostable than WT TdT. This study highlights the successful directed evolution of thermostable TdT which serves as an excellent primer for further engineering towards enzymatic de novo DNA synthesis.