Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR
Year: 2019
Type: article
Abstract: Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5'→3' nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.
Source: Nucleic Acids Research
DOI: 10.1093/nar/gkz104
Authors: Ryan C Heller, Suhman Chung, Katarzyna Crissy, Kyle Dumas, David Schuster, Thomas W Schoenfeld
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,992
OpenAlex ID: W2913824730