Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive but retain their structures such that the intein excises during a heat pulse delivered at 70–80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component ‘hot-start’ control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5–6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

Methods
Proteins were separated by 8% SDS-PAGE. The gels were stained by Coomassie Blue. The gel bands were quantified using ImageJ.

Fluorescence polarization was measured using SpectraMax M5e plate reader (Molecular Devices): 485 nm excitation (9 nm bandwidth), 525 nm emission (15 nm bandwidth).

Real-time fluorescence signals were recorded using CFX96 thermocycler (Bio-Rad).

Protein melting curves were recorded using CFX96 thermocycler (Bio-Rad).

All data were outputs of the software of the instruments or ImageJ without further processing. 

All images were untrimmed raw gel images captured by UVP Gel Studio.