The attachment of Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase

Abstract

ABSTRACTReverse transcriptases, RTs, are a standard tool in both fundamental studies and diagnostics used for transcriptome profiling, virus RNA testing and other tasks. RTs should possess elevated temperature optimum, high thermal stability, processivity, and tolerance to contaminants originating from the biological substances under analysis or the purification reagents. Here, we have constructed a set of chimeric RTs, based on the combination of MuLV-RT and DNA-binding domains: the DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein, Sso7d counterpart, from Sulfolobus tokodaii. Chimeric RTs showed the same optimal temperature and the efficacy of terminal transferase reaction as the original M-MuLV RT. Processivity and the efficiency in cDNA synthesis of the chimeric RT with Sto7d at C-end were increased several-fold. The attachment of Sto7d enhanced the M-MuLV RT tolerance to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood, and human blood plasma. Thus, fusing M-MuLV RT with an additional domain resulted in more robust and efficient RTs.