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O. L. Bodulev, A. V. Gribas, M. M. Vdovenko, I. Yu. Sakharov
Chemiluminescent
detection of HIV
DNA using 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 contained PMDNAzyme
fragment and the oligonucleotide complementary to HIV DNA which interacted each
other resulting in changes in G-quadruplex structure of the PMDNAzyme
and decreasing peroxidase-like activity of the probe. In the presence of HIV
DNA such interaction was destroyed because the complementary fragment of the
probe formed stable duplex with DNA target. As a result of this, 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 and 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
sensitivity of the assay was very high (190 000 nM–1). The values of
coefficient of variation (CV) measured within the working range varied
less than 4%, which says about a high precision of the proposed assay.
Key words:
Allosteric activation; Peroxidase-mimicking DNAzyme; Chemiluminescence; HIV;
DNA.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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