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Homogeneous method for Hg2+ detection based on allosteric activation of peroxidasemimicking DNAzyme (PMDNAzyme) was developed. In this method, two oligonucleotides probes are used. First probe (A) contains a noncovalent complex of hemin and its aptamer EAD2 (PMDNAzyme) associated with DNA oligonucleotide sequence, which is complementary to another oligonucleotide probe (B). Both probes can anneal to each other with a formation of a DNA duplex, resulting in the activation of PMDNAzyme by probe B [1]. If T–T mismatches are introduced into this DNA duplex, the binding energy of DNA probes will decrease. Previously it was demonstrated that Hg2+ can stabilize T–T mismatches by forming a T–Hg2+–T bonds [2]. Therefore, in Hg2+ presence the probes with T–T mismatches can anneal each other to produce a stable DNA duplex (Scheme 1). The formation of such duplex should increase the activity of PMDNAzyme. To test this hypothesis, we studied the interaction of three different pairs of the probes A and B in the absence and presence of Hg2+, where the catalytic activity of PMDNAzyme was measured towards luminol and hydrogen peroxide as substrates. The obtained results confirmed that the activity of probes A increases at addition of mercury cations to the reaction mixture containing probes A and B, which were used by us as calibration curves. The calculations showed that the best analytical parameters were obtained for probe A and probe B with sequences of 5’-TCG-TTT-CTC-TCT-CCA-ACT-GGG-AGG-GAG-GGA-GGG-A-3’ and 5’-TTG-GTT-TGA-GAA-ACG-A-3’, respectively. In this case after the optimization of concentrations of the probes and hemin in the reaction solution the detection limit and a linear range are 12 nM and 12-600 nM, respectively. Coefficient of variation (CV) measured within the working range varied from 0.7 to 3.0%.