ISSN 0027-1314, Moscow University Chemistry Bulletin, 2018, Vol. 73, No. 1, pp. 7–12. © Allerton Press, Inc., 2018.
Original Russian Text © O.L. Bodulev, A.V. Gribas, M.M. Vdovenko, I.Yu. Sakharov, 2018, published in Vestnik Moskovskogo Universiteta, Seriya 2: Khimiya, 2018, No. 2, pp. 78–84.
Chemiluminescent Detection of HIV DNA Based on Allosteric
Activation of Peroxidase-Mimicking DNAzyme
O. L. Bodulev
, A. V. Gribas
, M. M. Vdovenko
, and I. Yu. Sakharov
Department of Chemistry, Lomonosov Moscow State University, Moscow, 119991 Russia
Received November 23, 2017
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 interac-
tion 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 mech-
anism of the DNA-dependent activation of PMDNAzyme containing probes was confirmed by CD spectros-
copy 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 (190 000 nM
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.
Keywords: Allosteric activation, Peroxidase-mimicking DNAzyme, Chemiluminescence, DNA, HIV
Detection of DNA sequences plays an important
role in the prognosis of cancer, pathogenic microor-
ganisms detection and forensic analysis [1, 2]. Sensi-
tive and specific assays for detection of nucleic acids
have applications in analytical practice [3–7]. The
determination of DNA target is usually performed by
amplifying trace amounts of analyte to detectable lev-
els. The most used nucleic acid amplification is the
polymerase chain reaction (PCR) [8, 9]. In recent
years, as an alternative to PCR some methods of iso-
thermal amplification such as rolling circle amplifica-
tion , loop-mediated amplification [11, 12], strand
displacement amplification [13, 14], helicase-depen-
dent amplification reaction [15, 16] and hybridization
chain reaction [17, 18] have been developed. Amplifi-
cation of nucleic acids at a constant temperature is
promising for the development of cheap methods of
In recent years, sensitive hybridization methods for
the detection of DNA have been developed [19–24].
Some of them such as DNA chips with different kinds
of detection of analytical signals are heterogeneous
methods . However, it is well known that hybrid-
ization of probes immobilized on solid surfaces pro-
ceeds slowly, hence, the heterogeneous methods are
The detection of DNA in homogeneous solution is
more rapid and simpler [26, 27]. Fluorescence has
wide application in homogeneous DNA assays. Some
fluorescent dyes and nanoparticles are used as labels of
DNA-probes . Sensitive DNA assays were con-
structed using molecular beacons that become fluo-
rescent upon their binding to a complementary
sequence of single-stranded nucleic acid [29, 30].
Drawbacks of this approach are that expensive instru-
mentation is requested to fluorescence measurement
and all fluorescent assays show high background.
Alternative strategy is based on use of chemilumi-
nescent methods which are as sensitive as fluorescent
ones, but have low background and cheaper instru-
mentation. Many of such DNA assays use peroxidase-
mimicking DNAzyme (PMDNAzyme) [21, 31–33].
It is well known that PMDNAzyme (noncovalent
complex of heme and its aptamer) shows a catalytic
activity towards horseradish peroxidase substrates
including luminol. Thus, both fragment probe respon-
sible for DNA sensing and the label-catalyst (PMD-
NAzyme) are oligonucleotides that allows a synthesis
of oligonucleotide probes for DNA detection.
The article was translated by the authors.