Lead-start isothermal polymerase amplification controlled by DNAzymatic switches

Abstract

As DNA polymerases are even active at ambient temperature, there is inevitable non-specific amplification; to avoid the undesired amplification of analytes, a heat activation-based polymerase chain reaction (PCR), called hot-start PCR, is widely used to be highly precise and quantitative in detection. Unlike thermocycling amplification, isothermal amplification, compatible for point-of-care (PoC) tests, cannot be benefited by the heat-activation technique, making the method qualitative rather than quantitative. In this work, we newly developed a lead ion (Pb²⁺) activation technique, called lead-start isothermal amplification, allowing on-demand activation or deactivation of DNA polymerases at room temperature. We systematically correlated the DNA polymerase inhibition by the TQ30 aptamer with Pb²⁺-responsive strand cleavage by the GR5 DNAzyme, and relying on the type of interconnectors, Pb²⁺ successfully served as an initiator or a terminator of isothermal DNA amplification. Our lead-start isothermal amplification was exceptionally Pb²⁺-specific, dramatically increasing the enzymatic activity of DNA polymerase (>25 times) only by Pb²⁺ introduction. Despite one-by-one sample preparation, a number of reactions can begin and end at the same time, sharing the identical amplification conditions, and thereby allowing their quantitative analysis and comparison. Using a portable UV lamp and a smartphone camera, we also succeeded in quantifying the amounts of clinically important and human papillomavirus type 16 genes in human serum and SARS-CoV-2's nucleocapsid genes in human serum and saliva, and the limit of detection was as low as 0.1 nM, highly applicable for actual PoC tests in the field with no purification process.