Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency.

ABSTRACT: Engineering improved B-family DNA polymerases to catalyze 3'-O-modified nucleotide reversible terminators is limited by an insufficient understanding of the structural determinants that define polymerization efficiency. To explore the key mechanism for unnatural nucleotide incorporation, we engineered a B-family DNA polymerase from Thermococcus Kodakaraenis (KOD pol) by using semi-rational design strategies. We first scanned the active pocket of KOD pol through site-directed saturation mutagenesis and combinatorial mutations and identified a variant Mut_C2 containing five mutation sites (D141A, E143A, L408I, Y409A, A485E) using a high-throughput microwell-based screening method. Mut_C2 demonstrated high catalytic efficiency in incorporating 3'-O-azidomethyl-dATP labeled with a Cy3 dye, whe

SUBMITTER: Zhai L 

PROVIDER: S-EPMC11828419 | biostudies-literature | 2025

REPOSITORIES: biostudies-literature