Manganese-independent Reverse Transcriptase Activity of Tth DNA Polymerase with Two Amino Acid Substitutions

Background: The DNA polymerase of Thermus thermophilus (Tth pol) presents reverse transcriptase activity with Mn²⁺, and can be used for one-step RT-qPCR. However, Mn²⁺ would reduce amplification fidelity and cause nonspecific products. Objective: Eliminating the Mn²⁺ dependence of the reverse transcriptase activity of Tth pol by point mutations. Methods: We constructed three variants I640F, I709K, and I640F/I709K, and measured their DNA polymerase and reverse transcriptase activities without Mn²⁺. Their enzymatic characteristics and PCR inhibitor resistance were also tested. Finally, these variants were applied in one-step RT-qPCR. Results: All three variants presented reverse transcriptase activity with Mg²⁺ only and increased DNA polymerase activity. The variants, except I709K, showed no significant difference in thermostability, optimal pH, optimal NaCl concentration, storage stability and PCR inhibitor resistance compared to the wild type. Variant I640F/I709K had good performance in one-step RT-qPCR with Mg²⁺ only, whereas both variants with single substitution exhibited nonspecific amplification. Conclusion: We successfully constructed three Tth pol variants possessing Mn²⁺ independent reverse transcriptase activity. The variant I640F/I709K was suitable for one-step RT-qPCR because of its good performance.