A new self-passivating template with the phosphorothioate strategy to effectively improve the detection limit and applicability of exponential amplification reaction

Exponential amplification reaction (EXPAR) has attracted much attention due to its simple primers and high amplification efficiency, but its applications are hindered by severe non-specificity amplification. Convenient exogenous chemical modification methods modified the entire template while inhibiting both non-specific and specific amplification. In this paper, we proposed a new self-passivating template with the phosphorothioate strategy to effectively improve the detection limit and applicability of EXPAR. We phosphorothioated several bases where the sequence was prone to form transient intermolecular 3'-end hybridization, thereby inhibiting the non-specific interactions and preventing the extension of templates by DNA polymerase. The melting temperature (Tm) curve and density functional theory (DFT) proved that the stability of hydrogen bonds between phosphorothioated bases did decrease. Benefitting from this strategy, the detection limit had been improved by 3 orders of magnitude. Moreover, due to the antioxidation property of phosphorothioate, this strategy showed good stability in serum, reflecting its excellent prospects in clinical sampling and detection.