Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of <i>Pyrococcus furiosus</i> DNA Polymerase.

ABSTRACT: Engineering of a bifunctional enzyme that combines DNA-dependent DNA polymerase and reverse transcriptase (RT) activities is a highly promising biotechnological goal, as it would enable one-enzyme RT-PCR. For this purpose, we selected the high-fidelity Pyrococcus furiosus (Pfu) DNA polymerase as engineering scaffold. The selection of amino acid residues for replacement was carried out based on a multi-sequence alignment of diverse DNA polymerases and literature data, which allowed us to target amino acids, which presumably are triggers of the RT activity appearance. Six mutant variants of the Pfu enzyme were created and their activity was analyzed. Through enzymatic screening, we identified the Pfu-M6 variant, which exhibits dual DNA-dependent and RNA-dependent DNA polymerase activi

SUBMITTER: Kuznetsova AA 

PROVIDER: S-EPMC12650659 | biostudies-literature | 2025 Oct

REPOSITORIES: biostudies-literature