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.