Noise-reduced nicking enzyme-based isothermal amplification via blocking the 3'-end of the amplicon using a novel fluorophore-immobilized binder

Nicking enzyme amplification reaction (NEAR) is a promising isothermal DNA amplification method that has been applied in various sensing platforms as a promising signal amplifier. Although NEAR offers useful features for isothermal amplification, the risk of non-specific amplification imposes constraints on its potential usability. Here, we suggest a novel method to reduce the noise of NEAR, enabled by providing a specific binder to the 3'-end of amplicons as a reaction element, named Stabilizer. Stabilizer was designed to prevent undesirable interactions among the accumulated amplicons, thereby eliminating non-specific amplification caused by mis-annealing. Normal sequence Stabilizer without any modification (STB.N) was not effective in reducing the noise of NEAR. We employed a thermodynamic property of fluorophore-labeling that strengthens its annealing toward a complementary strand. We found that Stabilizer with a fluorophore at the 5'-end (F-STB) significantly reduces the noise level compared to cases of STB.N and Stabilizer-free. The NEAR with enhanced specificity would increase performance and enable applications in need of accuracy.