ИСТИНА

Peroxidase-mimicking DNAzyme (PMDNAzyme) is a noncovalent complex of hemin and its aptamer in G-quadruplex conformation. In the present work, aptamer EAD2 (CTGGGAGGGAGGGAGGGA) was used. This peroxidase mimetic, PMDNAzyme is able to catalyze luminol oxidation with a formation of light. An extension of the aptamer by attaching additional DNA sequence prevents a formation of G-quadruplex of EAD2 or strongly deformed it, if the additional sequence is able to specifically react with the aptamer sequence. In turn, these structural changes diminish the catalytic activity of PMDNAzyme. In the same time, in the presence of targets PMDNAzyme reduces its activity. Based on this fact, we modeled and synthesized some probes with additional sequences which were complementary both to targets (fragment of HBV DNA and miRNA-141) and to EAD2. Such probes were used in the development of chemiluminescent assays for detection of HIV DNA and miRNA-141. The detection limit for both targets was 100 pM. The linear range values for HIV DNA and miRNA-141were 0.1-50 nM and 0.1–10 nM, respectively. The values of coefficient of variation measured within the working range varied within 2-4%, which indicates a high precision of the proposed assays. The effect of allosteric activation of PMDNAzyme was also applied in the development of a method to measure exonuclease III activity. For this, we synthesized similar probes (hairpins) with blunt 3’-ends. The catalytic activity of such probes was low. In the presence of exonuclease III (exo III) one of stands of a stem was digested with a production of aptamer EAD2, which after reaction with hemin shows the high activity. This effect allowed developing simple and versatile assay for estimation of exo III activity with a detection limit of 0.01 U/μL.