The attachment of a DNA‐binding Sso7d‐like protein improves processivity and resistance to inhibitors of M‐MuLV reverse transcriptase

Abstract

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT 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 results in more robust and efficient RTs.

Comparison of Reverse Transcriptase (RT) Activities of Various M-MuLV RTs for RT-LAMP Assays

“…The cloning of chimeric and mutant RTs was described in our previous work [ 20 ]. In this study, several enzymes were examined, namely: M-MuLV RT with Sto7d protein from Sulfolobus tokodaii at C-terminus, M-MuLV RT with previously characterized D200N, T330P, L139P mutations [ 16 ], M-MuLV RT with D200N, T330P, L139P, and Sto7d protein at the C-terminus.…”

“…The thermal stability of M-MuLV RT mutants is directly proportional to the enzyme’s affinity towards a primer–template complex [ 11 , 17 , 19 ]. Mutations in template-interacting regions or fusion with additional DNA-binding protein domains could result in tether binding to the template [ 16 , 17 , 18 , 20 , 21 ]. Fortunately, thermostable M-MuLV RT mutants also demonstrated a higher tolerance to common amplification inhibitors, including heparin, formamide, NaCl, and humic acids [ 20 , 22 ].…”

“…Mutations in template-interacting regions or fusion with additional DNA-binding protein domains could result in tether binding to the template [ 16 , 17 , 18 , 20 , 21 ]. Fortunately, thermostable M-MuLV RT mutants also demonstrated a higher tolerance to common amplification inhibitors, including heparin, formamide, NaCl, and humic acids [ 20 , 22 ]. The efficacy of RT-PCR with various improved M-MuLV RTs was also higher than with the native enzyme.…”

M-MuLV reverse transcriptase: Selected properties and improved mutants

“…The observed results are similar to what has been reported for fusions of DNA polymerases. They could be explained as consequences of strengthening binding to a template by the additional domain on the C-end of the enzyme, acting independently from the M-MuLV RT [88] .…”

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR

“…Here we engineer and characterize a new variant of the Neq polymerase, Neq2X7, that combines the two fidelity-increasing mutations (2X) previously reported with the addition of Sso7d 27 . The engineered polymerase shows improved processivity over Neq(A523R/N540R) (Neq2X) and PfuX7 and perform well in amplification of DNA up to 12000 bp, in high GC content DNA amplification, and in incorporation of dUTP.…”