TY - JOUR

T1 - Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of Pyrococcus furiosus DNA Polymerase

AU - Kuznetsova, Aleksandra A

AU - Grishina, Irina A

AU - Mikushina, Elena S

AU - Kuznetsov, Nikita A

N1 - This work was supported by a Russian-Government-funded project (No. 125041005003-5).

PY - 2025/10/24

Y1 - 2025/10/24

N2 - 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 activity. This thermostable enzyme retains its inherent DNA polymerase function and has acquired the ability to catalyze reverse transcription under standard PCR conditions, which allows the created mutant form to be used for efficient amplification of DNA starting from an RNA template.

AB - 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 activity. This thermostable enzyme retains its inherent DNA polymerase function and has acquired the ability to catalyze reverse transcription under standard PCR conditions, which allows the created mutant form to be used for efficient amplification of DNA starting from an RNA template.

KW - Pyrococcus furiosus/enzymology

KW - DNA-Directed DNA Polymerase/genetics

KW - RNA-Directed DNA Polymerase/genetics

KW - Protein Engineering

KW - Reverse Transcriptase Polymerase Chain Reaction/methods

KW - Enzyme Stability

KW - Mutation

KW - Temperature

KW - Amino Acid Sequence

U2 - 10.3390/biom15111507

DO - 10.3390/biom15111507

M3 - Article

C2 - 41301425

VL - 15

JO - Biomolecules

JF - Biomolecules

SN - 2218-273X

IS - 11

M1 - 1507

ER -