Engineering thermostable friend mouse leukemia virus reverse transcriptase through mutational combination.

Reverse transcriptase (RTs) is an essential tool in molecular biology and medical research; however, its typical lack of thermostability poses significant limitations. In this study, we engineered thermostable RTs derived from Friend mouse leukemia virus reverse transcriptase (FrMLV RT) through a mutational combination. The thermostable FrM5 variant (D178C/E280R/T284R/W291F/L581W) is obtained through iterative rounds of mutational combination and rapid cell-free RTs activity assays. The FrM5 variant exhibited robust RTs activity across a broad temperature range (35-50 °C) with the template-primer (T/P). Notably, the half-life of the FrM5 variant at 50 °C was approximately 20 min, in contrast to less than 2 min for the wild-type (FrWT) in the presence of T/P. Furthermore, the melting temperature difference between the FrWT and FrM5 variants was less than 2 °C, regardless of the presence or absence of T/P. Finally, we demonstrated that FrM5 exhibits tighter binding to T/P, which likely protects against heat inactivation. This advancement could substantially improve the efficiency and accuracy of molecular biology and medical research applications.