<i>De novo</i> binders overcome the MMLV RT stability-activity trade-off.

ABSTRACT: Moloney murine leukemia virus reverse transcriptase (MMLV RT) is an RNA-dependent DNA polymerase widely used in molecular diagnostics and gene editing. Its application is limited by poor thermal stability and inactivation induced by long-term storage. We combined multi-site mutagenesis with de novo-designed negatively charged protein binders targeting the nucleic acid-binding pocket to enhance stability without compromising catalysis. The engineered MMLV RT-SV variant showed improved reaction thermostability, and the addition of electrostatically complementary binders further increased intrinsic and storage stability. The resulting enzyme-binder complexes maintained full polymerase activity, exhibited a 9°C higher melting temperature, and showed minimal activity loss after accelerat

SUBMITTER: Zhu Y 

PROVIDER: S-EPMC12719197 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature