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Open AccessArticle
Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of Pyrococcus furiosus DNA Polymerase
by
Aleksandra A. Kuznetsova
Aleksandra A. Kuznetsova
,
Irina A. Grishina
Irina A. Grishina
Elena S. Mikushina
Elena S. Mikushina
Nikita A. Kuznetsov
Nikita A. Kuznetsov
1
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia
2
Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia
*
Author to whom correspondence should be addressed.
Biomolecules 2025, 15(11), 1507; https://doi.org/10.3390/biom15111507 (registering DOI)
Submission received: 2 September 2025
/
Revised: 19 October 2025
/
Accepted: 22 October 2025
/
Published: 24 October 2025
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 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.
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MDPI and ACS Style
Kuznetsova, A.A.; Grishina, I.A.; Mikushina, E.S.; Kuznetsov, N.A.
Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of Pyrococcus furiosus DNA Polymerase. Biomolecules 2025, 15, 1507.
https://doi.org/10.3390/biom15111507
AMA Style
Kuznetsova AA, Grishina IA, Mikushina ES, Kuznetsov NA.
Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of Pyrococcus furiosus DNA Polymerase. Biomolecules. 2025; 15(11):1507.
https://doi.org/10.3390/biom15111507
Chicago/Turabian Style
Kuznetsova, Aleksandra A., Irina A. Grishina, Elena S. Mikushina, and Nikita A. Kuznetsov.
2025. "Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of Pyrococcus furiosus DNA Polymerase" Biomolecules 15, no. 11: 1507.
https://doi.org/10.3390/biom15111507
APA Style
Kuznetsova, A. A., Grishina, I. A., Mikushina, E. S., & Kuznetsov, N. A.
(2025). Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of Pyrococcus furiosus DNA Polymerase. Biomolecules, 15(11), 1507.
https://doi.org/10.3390/biom15111507
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