A novel thermostable and processive reverse transcriptase from a group II intron of Anoxybacillus flavithermus

Abstract

Reverse transcriptases (RTs) are a family of enzymes synthesizing DNA using an RNA template and participating in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology; they showed higher thermostability, fidelity and processivity comparing to commonly using M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still insufficiently studied. Here, we biochemically characterized a novel RT from a thermophilic bacterium Anoxybacillus flavithermus, isolated from a hot spring in New Zealand with an optimal temperature for growth around 60{degrees}C. The cloned RT, named Afl RT, retained around 40% of the specific activity after 45 min incubation at 50{degrees}C. Optimal pH was 8.5, temperature -- 45-50 {degrees}C and Mn2+ ions were an optimal cofactor. In a processivity analysis with MS2 phage gRNA (3569 b), Afl RT elongated fully up to 36% of template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed to detect up to 10 copies of MS2 phage genomic RNA per reaction. Thus, Afl RT is a promising enzyme for practical applications requiring thermostable processive RTs.