ИСТИНА

Exonuclease III (exo III) hydrolyzes sequentially mononucleotides from the blunt 3'-end of double-stranded DNA. Enzymes like exo III play an important role in cellular and physiological processes that are necessary to maintain genome stability. Its excess or absence leads to the serious diseases. Also, exo III is often used in the development of amplification assays of nucleic acid. In this regard, it is necessary to have a simple, sensitive and cheap methods to detect the activity of this enzyme. In this work, a chemiluminescent assay was developed, where a hairpin oligonucleotide with a blunt end was used as a substrate of exo III. In the presence of exo III, one of stem chains of the hairpin is hydrolyzed, thereby producing the hemin aptamer EAD2. The obtained sequence has G-quadruplex structure, which, after its interaction with hemin, produces peroxidase-mimicking DNAzyme. The catalytic activity of DNAzyme was estimated towards luminol and hydrogen peroxide. The conditions of determining enzyme activity of exo III were optimized by varying the structure of oligonucleotide substrates, the temperature and time of the enzyme hydrolysis, the concentration of MgCl2, the substrate and hemin. Under favorable conditions the proposed chemiluminescent assay of exo III allows to determine up to 0.01 U/µl. The analytical parameters of the developed method are compared using colorimetric and chemiluminescent detection.