RESUMO
DNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 {degrees}C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.
RESUMO
Despite the fact that strand-displacing activity is of great utility for a variety of applications, including isothermal amplification assays, there are relatively few strand-displacing DNA polymerases. In particular, the thermotolerant DNA polymerase from Geobacillus stearothermophilus (previously Bacillus stearothermophilus), Bst DNA polymerase (Bst DNAP), is used in a variety of assays, including loop-mediated isothermal amplification. However, despite its wide use, its properties remain open to improvement, as has been demonstrated by a variety of engineering efforts, including the identification of point mutations that impact its robustness, strand-displacement capabilities, and nascent reverse transcriptase activity. Interestingly, a strategy that has been commonly used to alter the capabilities of DNA polymerases, the addition of additional DNA- or RNA-binding domains, has yet to be applied to Bst DNAP. To this end, we now show that by adding fusion domains the performance of Bst DNAP in isothermal amplification assays, including its nascent RT activity, can be greatly improved. The impact of these improvements on the development of LAMP assays for the detection of SARS-CoV-2 is fully explored.
