Chemiluminescent Detection of HIV DNA Based on Allosteric Activation of Peroxidase-Mimicking DNAzyme

Abstract

Homogeneous chemiluminescent method for HIV DNA detection based on allosteric activation of peroxidase-mimicking DNAzyme (PMDNAzyme) was developed. The probes used in the assay contain PMDNAzyme fragment and the additional oligonucleotide sequence complementary to HIV DNA. The interaction of PMDNAzyme fragment and the additional oligonucleotide sequence results in changes in G-quadruplex structure of the PMDNAzyme and decreases peroxidase-like activity of the probe. In the presence of HIV DNA such interaction was destroyed due to the formation of stable duplex between the additional fragment of the probe and DNA-analyte. Consequently, some reorganizations in G-quadruplex structure of the probe are observed, which are accompanied by enhancement of catalytic activity of the PMDNAzyme. The mechanism of the DNA-dependent activation of PMDNAzyme containing probes was confirmed by CD spectroscopy as well as modeling of the probes and their complexes with DNA target. The calibration curves for HIV DNA determination allowed estimating the analytical parameters of the assay. The detection limit value and the linear range were shown to be 0.3 nM and 0.3–15 nM, respectively. The assay sensitivity was high (190000 nM^–1). The values of coefficient of variation (CV) measured within the working range varied less than 4%, which indicates the high accuracy of the proposed assay.