Circular permutation engineering improves amplification efficiency of Bst DNA polymerase.

Previously, a chimeric Bst DNA polymerase (HpStBL) with high inhibitor tolerance and reverse transcriptase activity, was constructed by introducing Hp47-Sto7d domain. However, the complex primer design and the possibility of nonspecific amplification in loop-mediated isothermal amplification (LAMP) partly restricted the broader application of HpStBL in nucleic acid detection. In order to explore the polymerase with better performance for LAMP and reverse-transcription LAMP (RT-LAMP), four CP-Bst DNA polymerase mutants were generated by circular permutation of Hp47-Sto7d domain of HpStBL. The results showed that circular permutation had impacts on the hydrophobicity of the protein by altering the amino acid sequence thereby affecting the affinity of the polymerase for DNA, and the mutants exhibited both DNA synthesis and reverse transcriptase activity. Mutant CP-G23 especially showed highest amplification performance, which could successfully amplify 100 copies/μL Astroviruses plasmid DNA. Based on the CP-G23, a simple and rapid nucleic acid detection method was developed with the recombinase from Thermus thermophilus, in which the system can successfully amplify 100 copies/μL Astroviruses plasmid DNA and 0.01 pg/μL SARS CoV-2 RNA in 45 min with one pair of inner primers. This method simplified the primer design and eliminated the need for reverse transcriptase, can be used for quantitative or qualitative DNA/RNA detection, and holds significant potential for application in clinical diagnostics.