Enhancing thermostability of Moloney murine leukemia virus reverse transcriptase through greedy combination of multiple mutant residues

Reverse transcription is crucial in bioengineering and biomedical fields, particularly for genome sequencing and virus diagnosis. Enhancing the thermostability of reverse transcriptase can significantly improve its efficiency and accuracy by enabling it to function at higher temperatures, thereby reducing RNA secondary structures and minimizing interference from contaminating enzymes, particularly in clinical samples. Here, using a combinatorial strategy, a variant of Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT) with improved activity across a wide temperature range (30-50 °C) was identified and maintained 100% activity after incubation at 50 °C for 10 min. Eleven hot-spot residues were mutated in various combinations, and the mutant proteins were rapidly expressed in a cell-free system for reverse transcription activity testing. Variant M5, which carries five mutated residues (E47K/E280R/T284R/L413G/D631V), exhibited enhanced thermostability and activity compared to any variant with a single residue mutation. Using purified recombinant protein for precise characterization, the melting temperature (Tm) of M5 increased by 4.7 °C when assembled with a nucleotide template-primer (T/P). Consequently, the half-life of M5 at 50 °C extended to approximately 60 min, in contrast to less than 4 min for the wild type. These findings demonstrate that the epistasis of combining multiple mutant residues holds excellent potential for significantly enhancing enzyme activity, even with existing knowledge. This heat-stable MMLV RT variant M5 may potentially improve efficiency and accuracy in molecular biology research and clinical diagnostics.