Comparison on binding interactions of quercetin and its metal complexes with calf thymus DNA by spectroscopic techniques and viscosity measurement

Article abstract of DOI:10.1002/jmr.2933

Quercetin (Qu) and its metal complexes have received great attention during the last years, due to their good antioxidant, antibacterial, and anticancer activities. In this contribution, binding interactions of Qu and Qu–metal complexes with calf thymus D

Full text of DOI:10.1002/jmr.2933

Received: 16 July 2021
DOI: 10.1002/jmr.2933
Revised: 10 August 2021
Accepted: 12 August 2021
R E S E A R C H A R T I C L E
Comparison on binding interactions of quercetin and its metal
complexes with calf thymus DNA by spectroscopic techniques
and viscosity measurement
Huajian Luo1 | Yu Liang1 | Huiying Zhang2 | Yi Liu1,3 | Qi Xiao1
Shan Huang1
|
1Guangxi Key Laboratory of Natural Polymer
Abstract
Chemistry and Physics, College of Chemistry
and Materials, Nanning Normal University,
Quercetin (Qu) and its metal complexes have received great attention during the last
years, due to their good antioxidant, antibacterial, and anticancer activities. In this
contribution, binding interactions of Qu and Qu–metal complexes with calf thymus
DNA (ctDNA) were investigated and compared systematically by using spectroscopic
techniques and viscosity measurement. UV-vis absorption spectra of ctDNA–
compound systems showed obvious hypochromic effect. Relative viscosity and melt-
ing temperature of ctDNA increased after the addition of Qu and Qu–metal com-
plexes, and the change tendency is Qu–Cr(III) > Qu–Mn(II) > Qu–Zn(II) > Qu–Cu(II)
> Qu. Fluorescence competition experiments show that hydrogen bonds and van der
Waals interaction play an important role in the intercalative binding of Qu and Qu–
metal complexes with ctDNA. Qu and Qu–metal complexes could unwind the right-
handed B-form helicity of ctDNA and further affect its base pair stacking. Space ste-
ric hindrance might be responsible for the differences in the intercalative binding
between ctDNA and different Qu–metal complexes. These results provide new infor-
mation for the molecular understanding of binding interactions of Qu–metal com-
plexes with DNA and the strategy for research of structural influences.
Nanning, China
2College of Chemistry and Biological
Engineering, Hechi University, Hechi, China
3State Key Laboratory of Separation
Membranes and Membrane Processes, School
of Chemistry and Chemical Engineering,
Tiangong University, Tianjin, China
Correspondence
Qi Xiao and Shan Huang, Guangxi Key
Laboratory of Natural Polymer Chemistry and
Physics, College of Chemistry and Materials,
Nanning Normal University, Nanning 530001,
China.
Email: qi.xiao@whu.edu.cn (Q. X.) and
huangs@nnnu.edu.cn (S. H.)
Funding information
National Natural Science Foundation of China,
Grant/Award Numbers: 21763005, 21563006,
21864006, 21563010; Natural Science
Foundation of Guangxi Province, Grant/Award
Numbers: 2017GXNSFDA198034,
2017GXNSFFA198005,
K E Y W O R D S
2021GXNSFAA075015; BAGUI Scholar
Program of Guangxi Province of China;
Thousands of Young Teachers Training
Program of Guangxi Province, Grant/Award
Number: guijiaoren[2018]18
calf thymus DNA, chiral conformation, intercalative binding, metal complexes, quercetin
1
|
INTRODUCTION
effects.4,5 Since Qu can scavenge reactive oxygen species (ROS)
including •OH, H2O2 and O2ꢀ efficiently, it can protect DNA from
Quercetin (Qu), which is one of the most abundant natural flavonoids
and polyphenolic compounds presented in various vegetables and
fruits, is one of the primary active components of plentiful natural
Chinese traditional medicines.1-3 Many researches have indicated that
Qu is equipped with a broad pharmacological activity, such as antican-
cer, antiviral, antibacterial, antioxidant, and anti-inflammatory
damage induced by ROS either as a free molecular species or at a site
where it binds to DNA.6,7 In recent years, various metal cations can
chelate with Qu to form stable Qu–metal complexes, which have
attracted great interest by biochemists and biomedical scientists due
to their better antioxidant, antibacterial, and anticancer activity.8,9 For
example, Qu–Cu(II) complex can prevent the production of ROS and
exhibits higher antioxidant activity in comparison with pure Qu.10
Qu–Cu(II) and Qu–Zn(II) complexes exhibited superior antitumor
Huajian Luo and Yu Liang contributed equally to this work.
J Mol Recognit. 2021;e2933.
wileyonlinelibrary.com/journal/jmr
© 2021 John Wiley & Sons Ltd.
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https://doi.org/10.1002/jmr.2933

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LUO ET AL.
activities to Qu alone.11 Qu–Ni(II) complex showed antitumor activity
which might be related to its intercalation into DNA structure and
DNA-binding selectivity.12 These results demonstrate that the combi-
nation of Qu and metal ions is a promising starting point for the devel-
opment of novel Qu-based antioxidant and anticancer drugs.
purchased from Sinopharm Chemical Reagent Factory Co. Ltd.
(Shanghai, China). The stock solution of Qu (3.0 ꢂ 10ꢀ3 mol Lꢀ1) was
prepared by dissolving its crystals in ethyl alcohol. The ctDNA was
dissolved in Tris-HCl buffer (pH 7.4) and stored in refrigerator. The
concentration of ctDNA was determined spectrophotometrically using
DNA, which plays a major role during the replication and tran-
scription of genetic information of life process, is one of the main
targeted biomacromolecules of several anticancer drugs.13 Investiga-
tion of binding interactions between DNA and important anticancer
drugs have become an active and important subject in many research
fields, since such researches can provide valuable information for the
development of effective target anticancer therapeutic agents.14-16
Binding interactions of Qu and its metal complexes with DNA have
been extensively studied, as their unusual binding properties, com-
bined with their general photochemical properties, make them appro-
priate probes for DNA secondary structure, conformation,
photocleavers, and antitumor drugs.17,18 Tan et al investigated the
DNA binding and oxidative DNA damage induced by Qu–Cu
(II) complex.19 Ni et al researched the interaction between Qu–Cu
(II) complex and DNA by using a neutral red dye fluorescent probe.20
Hu and coworkers have systematically studied the structure-activity
relationship of Qu and naringenin with DNA.21 Collectively, these
findings give an understanding of the structure-activity relationships
which may be helpful in the design of analogs of these flavonoids and
their application in drug and food industries.
an extinction coefficient at 260 nm of 6600 L molꢀ1 cmꢀ1 24
. The
purity of ctDNA was verified by monitoring the ratio of absorbance at
260 and 280 nm. All other chemical reagents were of analytical
reagent grade. Ultrapure water was used throughout the whole
experiment.
2.2
|
Apparatus
UV-vis absorption spectra were measured on Cary 100 UV-vis spec-
trophotometer (Agilent Technologies, Inc., Australia). Fluorescence
spectra were performed on RF-5301 PC luminescence spectrometer
(Shimadzu Co., Ltd., Tokyo, Japan). Circular dichroism (CD) spectra
were recorded on Chirascan CD spectrometer (Applied Photophysics,
England). Viscosity measurements were carried out using a viscosity
meter (Yinhua Flowmeter Co. Ltd., China). All pH measurements were
made with a basic pH meter PB-10 (Sartorius Scientific Instruments
Co., Ltd., China).
It is well-known that the binding modes between small molecules
and DNA are mainly electrostatic attraction, groove binding, and inter-
calative binding.7,22 Among three binding modes, intercalative binding
can lead to the unwinding and the lengthening of the DNA helix.23
Although some small molecules can intercalate with DNA bases,
detailed influencing factors are quite complicated and kinetic mecha-
nisms of their interactions are still unrevealed. Systematical investiga-
tion of binding modes and interaction mechanisms between small
molecules and DNA are very significant to the research of conforma-
tion variation of DNA and the directional design of efficient antican-
cer drugs against several diseases. Although the interactions between
some Qu–metal complexes and calf thymus DNA (ctDNA) have been
reported previously, the systematical investigation and comparison of
the interactions among these Qu–metal complexes with ctDNA are
still unrevealed clearly. Inspired by these facts, we investigated and
compared the binding interactions of Qu and Qu–metal complexes
with ctDNA by viscosity measurements, DNA melting, and spectro-
scopic techniques in this work. This study will be helpful to the devel-
opment of novel flavonoid based therapeutic agents that target DNA
for severe diseases and to the development of antioxidants that can
be used in drug and food industries.
2.3
|
Procedures
2.3.1
|
Preparation of Qu–metal complexes
Qu–metal complexes were prepared according to the literature
method.19 Solid Qu (3.0 ꢂ 10ꢀ3 mol Lꢀ1) was dissolved in 60 mL of
ethanol. The pH of solution was adjusted to around 7.0. After
5 minutes, different metal ion solution with same concentration
(1.5 ꢂ 10ꢀ3 mol Lꢀ1) was added to the mixture. The mixture was
stirred and heated to reflux for 5 hours at 50ꢃC, and then the mixture
was poured into H2O. The brown-yellow precipitate was set aside for
48 hours, filtered, and washed three times with ethanol/H2O solution
(volume ratio of 1:3). The solid product was dried under a vacuum for
48 hours at room temperature. The possible structure models of Qu–
metal complexes were shown in Figure 1.
2.3.2
|
Viscosity measurements
Viscosity of ctDNA with different concentration of ethidium bromide
(EB), acridine orange (AO), Qu, and Qu–metal complexes were mea-
sured in a viscometer that was kept at a constant temperature of 25
0.1ꢃC in a thermostatic water bath. Flow time measurements were
performed by a digital stopwatch with a resolution of 0.001 second.
At least five-time records reproducible to 0.02 second were obtained,
and the average value was used in the calculations. Data were pres-
ented as (η/η0)1/3 vs [compound]/[ctDNA] value,25 where η and η0
were the viscosity values of ctDNA in the presence and absence of
compound, respectively. Viscosity value was calculated from the
2
|
EXPERIMENTAL
| Reagents
2.1
Qu and ctDNA were purchased from Sigma-Aldrich (St. Louis, MO,
USA). ZnSO4, MnSO4ꢁH2O, CuCl2ꢁ2H2O, and CrCl3ꢁ6H2O were

LUO ET AL.
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FIGURE 1 Structure models and solution colors of Qu–metal complexes
observed flow time of ctDNA containing solutions (t > 100 s)
corrected for the flow time of buffer alone (t0), η = (tꢀt0)/t0.
3
|
RESULTS AND DISCUSSION
| UV-vis absorption spectrometry
3.1
2.3.3
|
DNA melting studies
UV-vis absorption spectra of ctDNA and compounds with different
concentration of ctDNA in Tris-HCl buffer (pH 7.4) were shown in
Figure 2. Due to the strong absorption of purine and pyrimidine bases,
ctDNA shows a characteristic and maximum absorption peak of
260 nm.26 Meanwhile, Qu, Qu–Cr(III), Qu–Mn(II), and Qu–Zn
(II) complexes exhibit two absorption peaks at 267 and 376 nm
(Figure 2A-D), respectively. However, Qu–Cu(II) complex shows an
obvious absorption peak at 330 nm (Figure 2E). As inserted in
Figure 2A-D, with the continuous addition of ctDNA, the absorbances
of Qu, Qu–Cr(III), Qu–Mn(II), and Qu–Zn(II) complexes at 376 nm are
decreased gradually, suggesting that ctDNA interacts with these com-
pounds and subsequently affects their absorption characteristics. Usu-
ally, hyperchromic and hypochromic effects are regarded as the
spectral characteristics of DNA-compound interactions, and hypo-
chromic effect usually originates from the intercalative binding of
small molecules with DNA double helical structure.27,28 As shown in
Figure 3A, obvious hypochromic effects exists at 376 nm after the
interactions of ctDNA with Qu, Qu–Cr(III), Qu–Mn(II), and Qu–Zn
(II) complexes. So Qu and these Qu–Cu(II) complex may interact with
ctDNA through electrostatic forces, indicating that the interaction
modes of ctDNA with these compounds may be the intercalative
binding. Further shown in Figures 2E and 3A, the absorbance of Qu–
Cu(II) complex at 330 nm increases slightly at lower concentration of
ctDNA but decreases weakly at higher concentration of ctDNA. These
results suggest that Qu–Cu(II) complex may interact with ctDNA at its
lower concentration through electrostatic forces but intercalate into
the double helical structure of ctDNA with higher concentration.19
In order to compare the interactions between ctDNA and these
compounds, the quantity of hypochromism value (H) was obtained by
the equation of H = (AFreeꢀABounded)/AFree ꢂ 100%.29 Herein, AFree
means the absorbances of Qu, Qu–Cr(III), Qu–Mn(II), and Qu–Zn
DNA melting experiments were carried out by monitoring the absor-
bance of ctDNA at 260 nm in the absence and presence of com-
pounds at different temperatures. The temperature was continuously
monitored with a thermostatic bath. The absorbance was then plotted
as a function of temperature ranging from 30ꢃC to 95ꢃC. The melting
temperature of ctDNA was determined as the transition midpoint.
2.3.4
|
Fluorescence spectra measurements
Fluorescence spectra of ctDNA–AO system at the presence of com-
pounds were recorded at 298, 304, and 310 K with the excitation/
emission slits of 3.0/3.0 nm. The excitation wavelength was 475 nm
and the fluorescence intensity at 533 nm was recorded. The appropri-
ate blanks corresponding to buffer were subtracted to correct the
background of fluorescence. Titrations were performed manually by
using trace syringes and each spectrum was the average of three
scans.
2.3.5
|
CD spectra measurements
CD spectra of ctDNA, compounds, and ctDNA-compound systems
with different molar ratio of [compound]/[ctDNA] in Tris-HCl buffer
(pH 7.4) were recorded from 220 to 320 nm at 25ꢃC under constant
nitrogen airflow. CD profiles were obtained using scan speed of
500 nm minꢀ1 and response time of 0.5 second. Each spectrum was
the average of three successive scans and was corrected by the buffer
solution. Appropriate baseline corrections in CD spectra were made.

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LUO ET AL.
FIGURE 2 UV-vis absorption spectra of ctDNA and Qu (A), Qu-Cr(III) complex (B), Qu-Mn(II) complex (C), Qu-Zn(II) complex (D), and Qu-Cu
(II) complex (E) with different concentration of ctDNA in Tris-HCl buffer (pH 7.4) at 25 oC. Inset: UV-vis absorption spectra of ctDNA with or
without compounds in the wavelength range of 345 to 410 nm
FIGURE 3 A, Plots of absorbance vs [ctDNA]. The absorbance at 376 nm for Qu, Qu-Cr(III), Qu-Mn(II), and Qu-Zn(II) complexes, while at
330 nm for Qu-Cu(II) complex. B, Curves of the quantity of hypochromism values vs [ctDNA]/[compound]. C, Plots of 1/(AꢀA0) vs 1/[ctDNA]
(II) complexes at 376 nm or the absorbance of Qu–Cu(II) complex at
330 nm, while ABounded is the absorbance of these compounds after
the addition of ctDNA. As shown in Figure 3B, the hypochromism
values of Qu, Qu–Cr(III), Qu–Mn(II), and Qu–Zn(II) complexes are all
decreased with the increase of the concentration of ctDNA. Upon the
addition of 1.0 ꢂ 10ꢀ4 mol Lꢀ1 ctDNA, the hypochromism values of
these compounds from higher to lower are: 17.6% of Qu–Cr(III) com-
plex, 16.4% of Qu–Mn(II) complex, 12.8% of Qu–Zn(II) complex, and
9.8% of Qu, respectively. Since the higher hypochromism value often
reflects the stronger intercalative binding ability between DNA and
small molecules, Qu–Cr(III), Qu–Mn(II), and Qu–Zn(II) complexes show
stronger binding ability with ctDNA than Qu alone. Therefore, the
addition of metal ions into Qu can enhance its binding ability with
ctDNA to different extent. In comparison, Qu–Cr(III) complex can bind
with ctDNA more tightly than the other two Qu–metal complexes.
Comparably, a very small hypochromic effect of 1.6% is occurred in
Qu–Cu(II) complex after the addition of 1.0 ꢂ 10ꢀ4 mol Lꢀ1 ctDNA,
so Qu–Cu(II) complex can only intercalate with the double helical
structure of ctDNA with high concentration, which is agreed well with
the reported results.9,19
To quantitatively compare the intercalative binding ability of
these compounds with ctDNA, the intrinsic binding constant (Kb)
of compounds with ctDNA can be determined by using the following
equation:29,30
1
1
1
1
¼
þ
ð1Þ
AꢀA0 A∞ ꢀA0 KbðA∞ ꢀA0Þ ½ctDNAꢄ
Herein, A0 and A are the absorbances of compound in the
absence and presence of ctDNA, and A∞ is the final absorbance of

LUO ET AL.
5 of 12
ctDNA-compound system, respectively. The absorption peaks of Qu,
Qu–Cr(III), Qu–Mn(II), and Qu–Zn(II) complexes are chosen at 376 nm,
but the absorption peak of Qu–Cu(II) complex is chosen at 330 nm. In
double reciprocal plots of 1/(AꢀA0) vs 1/[ctDNA], the Kb value can be
given by the ratio of the slope to the intercept. According to the plots
in Figure 3C, the Kb values are calculated to be 6.23 ꢂ 103 L molꢀ1
for ctDNA–Qu–Cr(III) system, 4.98 ꢂ 103 L molꢀ1 for ctDNA–Qu–
Mn(II) system, 4.64 ꢂ 103 L molꢀ1 for ctDNA–Qu–Zn(II) system,
4.18 ꢂ 103 L molꢀ1 for ctDNA–Qu–Cu(II) system and 4.01 ꢂ 103 L molꢀ1
for ctDNA–Qu system, respectively. The larger binding constant of
ctDNA–Qu–Cr(III) system indicates that Qu–Cr(III) complex binds
with the adjacent ctDNA double helical structure more tightly than
other compounds. On the other hand, the binding constant of
ctDNA–Qu system is the smallest, suggesting the weakest binding
capability of Qu with ctDNA among these compounds. It can be
speculated that the addition of metal ions can enhance the coplanar-
ity of Qu–metal complexes, therefore these complexes can interca-
late into the double helical structure of ctDNA mainly throng the π - π
stacking interactions, thus resulting in the stronger binding strength
with ctDNA.9 Since Cr(III) possesses a higher electronic charge and less
ionic radius, Qu–Cr(III) complex shows better stability and more copla-
narity. Thus, among these Qu–metal complexes, Qu–Cr(III) complex
exhibits the strongest intercalative binding interaction with ctDNA. This
speculation will be further supported from other experiments investi-
gated later, which gives convincing evidence of the possible inter-
calative binding between ctDNA and these compounds.
relative viscosity of ctDNA were shown in Figure 4A. It is obvious that
the relative viscosity of ctDNA is increased gradually upon the contin-
uous addition of these compounds, indicating that the interaction
modes between ctDNA and these compounds may be the classic
intercalative binding.32 The detailed viscosity can be measured accu-
rately by the slope value of the linear plot of (η/η0)1/3 vs [compound]/
[ctDNA] (from 0 to 0.12).22 As continuously shown in Figure 4B, the
slope values are 0.995 for ctDNA–EB system, 0.940 for ctDNA–AO
system, 0.924 for ctDNA–Qu–Cr(III) system, 0.889 for ctDNA–Qu–
Mn(II) system, 0.872 for ctDNA–Qu–Zn(II) system, 0.846 for
ctDNA–Qu–Cu(II) system and 0.777 for ctDNA–Qu system, respec-
tively. However, the increase in viscosity of ctDNA for these com-
pounds is lower than that for EB and AO. Usually, the slope value
near 1.0 means an ideal intercalative binding,33,34 so EB and AO are
widely used intercalative probes in DNA. Since the slope values of
these ctDNA–Qu–metal systems are relatively high, so Qu and Qu–
metal complexes indeed intercalate into the base pairs of ctDNA
and then increase the overall double helix length of ctDNA. The rel-
ative viscosity of ctDNA is increased steadily upon the addition of
increasing concentrations of these compounds in the following
order: Qu–Cr(III) > Qu–Mn(II) > Qu–Zn(II) > Qu–Cu(II) > Qu, show-
ing that Qu–Cr(III) complex can intercalate with ctDNA more
strongly and deeply than other compounds. These phenomena are
highly consistent with the results obtained by UV-vis absorption
spectrometry.
3.3
|
DNA melting analysis
3.2
|
Viscosity investigation
DNA melting analysis can be used for further elucidating the inter-
calative binding of small molecules and DNA. DNA melting tempera-
ture (Tm), which is the temperature at which half of duplex DNA
denatured into two single strands, is dependent on the strength and
the mode of its interaction with small molecules.35,36 Intercalative
binding of small molecules with DNA can stabilize the natural double
helical structure of DNA and thus increase its Tm value obviously,
Viscosity experiment is an effective strategy to clarify the binding
mode of DNA with small molecules. Usually, classical intercalative
binding of small molecules with DNA base pairs causes the elongation
of DNA and the increment of DNA viscosity significantly, while a par-
tial or non-classical intercalative binding often causes a tiny change in
the viscosity of DNA.31 The influences of these compounds on the
FIGURE 4 A, Curves of relative viscosity of ctDNA vs [compound]/[ctDNA]. c(ctDNA) = 5.0 ꢂ 10ꢀ5 mol Lꢀ1; c(AO) = c(EB) = c(compound)/
(10ꢀ6 mol Lꢀ1): 0, 1, 2, 3, 4, 5, 6, 8, 11, 15, and 20. B, Plots of relative viscosity of ctDNA vs [compound]/[ctDNA].
c(ctDNA) = 5.0 ꢂ 10ꢀ5 mol Lꢀ1; c(AO) = c(EB) = c(compound)/(10ꢀ6 mol Lꢀ1): 0, 1, 2, 3, 4, 5, and 6. C, Melting curves of ctDNA in the absence
and presence of these compounds. c(ctDNA) = 2.0 ꢂ 10ꢀ4 mol Lꢀ1; c(compound) = 3.0 ꢂ 10ꢀ5 mol Lꢀ1

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LUO ET AL.
while the electrostatic attraction and groove binding cause no distinct
variation in the Tm value of DNA.35,36 In order to clarify the interac-
tion mechanism of these compounds with ctDNA, we recorded the
absorbance of ctDNA at 260 nm in the absence and presence of com-
pounds at different temperature with the temperature increasing
gradually from 30ꢃC to 95ꢃC. Hence, the Tm value of ctDNA was
obtained from the transition midpoint of the melting curves based on
the relative absorbance (fss) vs temperature. Herein, fss = (A-A0)/(Af-
A0), where A0 is the initial absorbance of ctDNA-compound system,
A is the absorbance of ctDNA-compound system corresponding to
the temperature and Af is the final absorbance of ctDNA-compound
system, respectively.
3.4
|
Fluorescence spectrometry
3.4.1
|
Interaction modes
Interaction modes between DNA and small molecules can be eluci-
dated by using the competitive fluorescence approach. The endoge-
nous fluorescence of DNA is too weak, but some classic intercalating
molecules can emit intense fluorescence in the presence of duplex
DNA through strong stacking interactions.37 Fluorescence of DNA–
probe system can be efficiently quenched after the addition of other
molecules by replacing the probe, and the extent of the fluorescence
quenching of DNA–probe system can be used to clarify the detail
interaction mode between small molecules and DNA.22,37 Herein, the
classic DNA intercalating molecule AO was chosen as the fluorescent
probe. As indicated in Figure 5A-E, the fluorescence of AO is
increased after its intercalative binding with ctDNA. The fluorescence
of ctDNA(AO) system is decreased gradually after the addition of
these compounds with increasing concentration. In addition, the fluo-
rescence of ctDNA(AO) system is decreased linearly with the increas-
ing concentration of compounds (inserts in Figure 5A-E), suggesting
the intercalative binding of these compounds with ctDNA. Such
experiments imply that these compounds can intercalate into the dou-
ble helical structure of ctDNA and compete with AO for the inter-
calative site of ctDNA.
As represented in Figure 4C, the Tm value of ctDNA alone is
72.5ꢃC. After the addition of these compounds, the Tm value of
ctDNA is increased to 80.8ꢃC for ctDNA–Qu–Cr(III) system, 79.4ꢃC
for ctDNA–Qu–Mn(II) system, 78.5ꢃC for ctDNA–Qu–Zn(II) system,
77.7ꢃC for ctDNA–Qu–Cu(II) system and 76.9ꢃC for ctDNA–Qu sys-
tem, respectively. It is reported that the intercalative binding of natu-
ral or synthetic compounds into DNA often result in the increase of
T
m value of about 5ꢃC to 8ꢃC, but the non-intercalative binding gener-
ally causes no significant increase of the Tm value.36 These results fur-
ther reveal the intercalative binding of these compounds with ctDNA.
More interestingly, the Tm value of ctDNA is increased steadily upon
the addition of these compounds in the following order: Qu–Cr(III)
> Qu–Mn(II) > Qu–Zn(II) > Qu–Cu(II) > Qu, reconfirming the strongest
intercalative binding ability of Qu–Cr(III) complex with ctDNA among
these compounds.
In order to compare the intercalative binding ability between
ctDNA and these compounds, the fluorescence quenching effects
were obtained by the equation of Q = (I0ꢀI)/I0 ꢂ 100%. Herein, I0
FIGURE 5 A-E, Fluorescence spectra of ctDNA(AO) system with various concentration of compounds. Inserts correspond to the Stern–
Volmer plots. c(ctDNA) = 1.0 ꢂ 10ꢀ4 mol Lꢀ1; c(AO) = 1.2 ꢂ 10ꢀ6 mol Lꢀ1; c(Qu)/(10ꢀ6 mol Lꢀ1), 1-11: from 0 to 20.0 at increments of 2.0; c
(Qu metal complexes)/(10ꢀ6 mol Lꢀ1), 1-11: from 0 to 10.0 at increments of 1.0. F, Curves of the fluorescence quenching values vs [compound]

LUO ET AL.
7 of 12
and I are the fluorescence intensity of ctDNA(AO) system without or
with these compounds, respectively. As shown in Figure 5F, the fluo-
rescence quenching effects of ctDNA(AO) system are all increased
with the increase of the concentration of these compounds. The
results suggest that these compounds can substitute for some AO
molecules and partial AO molecules dissociates from ctDNA(AO) com-
plex into the solution. Upon the addition of 1.0 ꢂ 10ꢀ5 mol Lꢀ1
compounds, the fluorescence quenching effects of ctDNA(AO) system
are 43.3% for Qu–Cr(III) complex, 38.3% for Qu–Mn(II) complex,
33.5% for Qu–Zn(II) complex, 28.6% for Qu–Cu(II) complex, and
23.5% for Qu, respectively. Since the higher fluorescence quenching
effect often reflects the stronger intercalative binding ability between
DNA and small molecules,21 Qu–Cr(III) complex bind with ctDNA
more tightly and replace AO molecules more efficiently than other
compounds.
Herein, KSV is the Stern–Volmer quenching constant and [Q] is
the concentration of compound, respectively. So, the KSV value can be
determined by linear regression plot of I0/I vs [Q]. Since these com-
pounds can intercalate into the duplex ctDNA, the fluorescence
quenching mechanism of these systems should be the static fluores-
cence quenching mode. In order to confirm the fluorescence
quenching mechanism, the fluorescence spectra of ctDNA(AO) com-
plex with different concentration of compounds were measured at
three different temperatures (298, 304 and 310 K) and the fluores-
cence quenching data were all plotted by Stern–Volmer equation. As
shown in Figure 6, the results agree well with the Stern–Volmer equa-
tion, which indicates that only either static or dynamic fluorescence
quenching process is occurred. The calculated Stern–Volmer
quenching constants KSV values at three different temperatures were
listed in Table 1. For ctDNA(AO)–Qu system, the KSV values at three
different temperatures are 4.65 ꢂ 104 L molꢀ1 at 298 K,
3.05 ꢂ 104 L molꢀ1 at 304 K and 2.25 ꢂ 104 L molꢀ1 at 310 K,
respectively. For ctDNA(AO)–Qu–Cr(III) system, the KSV values at
three different temperatures are 5.19 ꢂ 104 L molꢀ1 at 298 K,
4.39 ꢂ 104 L molꢀ1 at 304 K and 3.64 ꢂ 104 L molꢀ1 at 310 K,
respectively. It is very clear that the increase of the temperature
results in the decrease of the KSV values in all these systems.
Quenching constant will be decreased with the increment of tempera-
ture for the static quenching process while the reverse is true for
dynamic quenching process,38 so the fluorescence quenching mecha-
nism between these compounds and ctDNA(AO) complex is static
fluorescence quenching.
3.4.2
|
Binding constants
Binding constants between these compounds and ctDNA can be cal-
culated according to the fluorescence quenching plots of ctDNA(AO)
complex after the addition of these compounds (inserts in Figure 5A-
E). The fluorescence quenching of ctDNA(AO) complex by these com-
pounds is in good agreement with the classical Stern–Volmer
equation:38
I0
¼ 1þKSV½Qꢄ
ð2Þ
I
FIGURE 6 Stern–Volmer plots of ctDNA(AO)–Qu system (A), ctDNA(AO)-Qu-Cr(III) system (B), ctDNA(AO)-Qu-Mn(II) system (C),
ctDNA(AO)-Qu-Zn(II) system (D), and ctDNA(AO)-Qu-Cu(II) system (E)

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LUO ET AL.
TABLE 1 Stern–Volmer quenching constants KSV, associative binding constants Ka and relative thermodynamic parameters for the
interactions between these compounds and ctDNA(AO) complex at three different temperatures
KSV
(104 L molꢀ1
Ka
ΔH
ΔG
ΔS
System
T (K)
298
304
310
298
304
310
298
304
310
298
304
310
298
304
310
)
R2a
(104 L molꢀ1
)
R2a
(kJ molꢀ1
)
(kJ molꢀ1
)
(J molꢀ1 Kꢀ1
)
R2a
ctDNA(AO)–Qu
4.65
3.05
2.25
7.08
6.23
5.57
6.29
5.71
4.64
5.19
4.39
3.64
3.88
2.83
2.15
0.999
0.997
0.996
0.995
0.999
0.999
0.999
0.998
0.998
0.997
0.995
0.995
0.999
0.999
0.999
2.70
1.21
0.59
2.92
1.97
1.04
3.67
1.22
0.46
5.03
2.73
1.69
2.85
1.66
0.81
0.998
0.999
0.999
0.998
0.997
0.998
0.998
0.998
0.997
0.997
0.995
0.996
0.999
0.998
0.998
ꢀ96.51
ꢀ66.20
ꢀ133.05
ꢀ69.83
ꢀ80.74
ꢀ25.25
ꢀ23.82
ꢀ22.38
ꢀ25.58
ꢀ24.77
ꢀ23.95
ꢀ26.01
ꢀ23.85
ꢀ21.69
ꢀ26.78
ꢀ25.91
ꢀ25.04
ꢀ25.50
ꢀ24.38
ꢀ23.27
ꢀ239.1
0.999
ctDNA(AO)–
Qu–Zn(II)
ꢀ136.28
ꢀ359.22
ꢀ144.47
ꢀ185.36
0.998
0.999
0.998
0.997
ctDNA(AO)–
Qu–Mn(II)
ctDNA(AO)–
Qu–Cr(III)
ctDNA(AO)–
Qu–Cu(II)
aR2 is the correlation coefficient.
FIGURE 7 A-E, Modified Stern–Volmer plots of ctDNA(AO)–compound systems. F, Van't Hoff plots of ctDNA(AO)–compound systems
For static fluorescence quenching process, the associative binding
Herein, fa is the mole fraction of solvent accessible fluorophore.
So, the associative binding constant Ka value can be calculated from
constants (Ka) can be calculated through the modified Stern–Volmer
equation38
:
the quotient of the ordinate fa and the slope (faKa)ꢀ1. The modified
ꢀ1
Stern–Volmer plots were shown in Figure 7A-E, and the
corresponding associative binding constants Ka were also listed in
Table 1. The decreasing trend of Ka values with increasing
I0
I0
1
1
¼
¼
þ
ð3Þ
I0 ꢀI ΔI faKa½Qꢄ fa

LUO ET AL.
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ΔH ΔS
temperature is in accord with KSV's dependence on temperature, which
reconfirms that the fluorescence quenching mechanism of ctDNA(AO)
complex by these compound has mainly arisen from static fluorescence
quenching mode. At the reaction temperature of 298 K, the Ka values
of ctDNA(AO)–compound systems are 5.03 ꢂ 104 L molꢀ1 for
ctDNA(AO)–Qu–Cr(III) system, 3.67 ꢂ 104 L molꢀ1 for ctDNA(AO)–
Qu–Mn(II) system, 2.92 ꢂ 104 L molꢀ1 for ctDNA(AO)–Qu–Zn
(II) system, 2.85 ꢂ 104 L molꢀ1 for ctDNA(AO)–Qu–Cu(II) system and
2.70 ꢂ 104 L molꢀ1 for ctDNA(AO)–Qu system, respectively. More
interestingly, the Ka value of ctDNA(AO)–compound systems is
increased steadily upon the addition of these compounds in the follow-
ing order: Qu–Cr(III) > Qu–Mn(II) > Qu–Zn(II) > Qu–Cu(II) > Qu. The
largest binding constant of ctDNA(AO)–Qu–Cr(III) system indicates that
Qu–Cr(III) complex can intercalative with the duplex ctDNA most
tightly, which is highly consistent with the above results.
ln Ka ¼ ꢀ
þ
ð4Þ
ð5Þ
RT
R
ΔG ¼ ΔHꢀTΔS
As shown in Figure 7F, good linear relationships are existed
between lnKa and 1/T in these ctDNA(AO)–compound systems. The
calculated ΔH, ΔS, and ΔG values were all incorporated in Table 1.
The negative values of ΔG suggest that the intercalative binding pro-
cesses of these compounds with ctDNA–AO complex have occurred
spontaneously.40 Moreover, the negative values of both ΔH and ΔS
imply that the hydrogen bonds and van der Waals interactions play
major roles during their intercalative binding reactions.40
3.5
|
CD spectrometry
3.4.3
|
Binding forces
CD spectrometry is an effective technique to investigate the chiral
conformation of DNA after its interaction with small molecules. In
order to reveal the influence of these compounds on the chiral con-
formation of ctDNA, CD spectra of compounds, and ctDNA without
or with different concentration of compounds were illustrated in
Figure 8. It is apparent that all compounds are not optically active and
exhibit almost no CD signals in the measuring UV regions. However,
ctDNA exhibits an obvious CD spectrum consisted of a negative band
at 246 nm due to the right-handed B-form helicity and a positive
Binding forces contributing to DNA interactions with small molecules
usually include hydrophobic forces, hydrogen bonds, electrostatic
interactions and van der Waals interactions.39 In general, binding
forces can be elucidated by the variation of some thermodynamic
parameters, such as enthalpy change (ΔH), entropy change (ΔS), and
free energy change (ΔG). The values of these thermodynamic parame-
ters can be calculated through the following equations:
FIGURE 8 A. CD spectra of Qu and ctDNA-Qu systems with different molar ratio of [Qu]/[ctDNA]. B. CD spectra of Qu-Cr(III) and ctDNA-
Qu-Cr(III) systems with different molar ratio of [Qu-Cr(III)]/[ctDNA]. C. CD spectra of Qu-Mn(II) and ctDNA-Qu-Mn(II) systems with different
molar ratio of [Qu-Mn(II)]/[ctDNA]. D. CD spectra of Qu-Zn(II) and ctDNA-Qu-Zn(II) systems with different molar ratio of [Qu-Zn(II)]/[ctDNA].
E. CD spectra of Qu-Cu(II) and ctDNA-Qu-Cu(II) systems with different molar ratio of [Qu-Cu(II)]/[ctDNA]

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LUO ET AL.
FIGURE 9 A, Curves of CD intensity at 246 nm vs [compound]/[ctDNA]. B, Curves of CD intensity at 276 nm vs [compound]/[ctDNA]
band at 276 nm ascribed to the base pair stacking.41 Various studies
suggest that both absorption bands are quite susceptible to the inter-
action with small molecules. The intercalative binding of small mole-
cules into the double helical structure of DNA can significantly affect
the positive and negative peaks, while DNA interaction with small
molecules at the electrostatic interaction and grooves binding only
induces less change on the native CD spectrum of DNA.42 As shown
in Figure 8A-D, with the increase of the molar ratio of [compound]/
[ctDNA], the intensity of the negative band at 246 nm of ctDNA is
decreased significantly but the intensity of the positive band at
276 nm of ctDNA is increased obviously without any remarkable shift
in the band position. These changes in CD spectra are attributed to
the decrease in the right-handed B-form helicity but the increase in
the base pair stacking.22 Therefore, Qu, Qu–Cr(III), Qu–Mn(II), and
Qu–Zn(II) can decrease the right-handed B-form helix structure of
ctDNA but increase the base pair stacking of ctDNA. However, with
the increase of the concentration of Qu–Cu(II) complex, the intensities
of both negative band at 246 nm and positive band at 276 nm are all
decreased obviously (Figure 8E). Consequently, Qu–Cu(II) complex
can decrease both the right-handed B-form helicity and the base pair
stacking of ctDNA. These significant changes of the right-handed B-
form helicity and base pair stacking of ctDNA structure are ascribed
to the intercalative binding of these compounds with ctDNA. Since
Qu–Cu(II) complex decreases the base pair stacking of ctDNA, the
double helical structure of ctDNA becomes much loosen, resulting in
the lower binding ability of Qu–Cu(II) complex with ctDNA than Qu
and other Qu–metal complexes.
ctDNA more obviously than other two Qu–metal complexes, while
Qu–Cr(III) complex shows the smallest influence on the chiral confor-
mation of ctDNA. Therefore, these Qu–metal complexes can interca-
late into the double helical structure of ctDNA and then induce its
unwinding but enhance the base pair stacking of ctDNA. However,
Qu–Cu(II) complex can not only unwind the double helical structure
of ctDNA but also reduce the base pair stacking, resulting in its lowest
binding constant among Qu–metal complexes.
4
|
CONCLUSIONS
In this work, binding interactions of Qu and Qu–metal complexes
with ctDNA were comparatively investigated by UV-vis absorption
spectrometry, fluorescence spectrometry, CD spectrometry, vis-
cosity measurement, and DNA melting techniques. These com-
pounds intercalated into the double helical structure of ctDNA and
affected the chiral conformation of ctDNA. The fluorescence of
ctDNA(AO) complex was quenched by these compounds via static
fluorescence quenching mode, and the binding interaction was
driven mainly by hydrogen bonds and van der Waals interactions.
Among these compounds, Qu–Cr(III) complex interacted with
ctDNA more strongly than other compounds, while Qu–Cu
(II) complex not only unwound the duplex ctDNA but also reduced
its base pair stacking. Therefore, Qu–Cr(III) complex exhibited the
strongest binding ability but Qu–Cu(II) complex showed the weak-
est binding capacity among these Qu–metal complexes. These
results make a better understanding of structural influences on the
interaction between Qu–metal complexes and ctDNA from molec-
ular biology level, which is very important for the biological applica-
tions of these compounds.
As exhibited in Figure 9A, the intensity of negative band at
246 nm is decreased gradually with the continuous addition of these
compounds. The decreasing tendency of the intensity of negative
band at 246 nm upon the addition of these compounds is in the fol-
lowing order: Qu > Qu–Zn(II) > Qu–Mn(II) > Qu–Cr(III) > Qu–Cu(II). In
addition, the increasing tendency of the intensity of positive band at
276 nm from high to low is Qu > Qu–Zn(II) > Qu–Mn(II) > Qu–Cr(III)
(Figure 9B). These results indicate that Qu can affect the chiral con-
formation of ctDNA more significantly than Qu–metal complexes,
mainly because of its less space steric hindrance. Among Qu–Zn(II),
Qu–Mn(II), and Qu–Cr(III) complexes, Qu–Zn(II) complex can decrease
the right-handed B-form helicity but increase the base pair stacking of
ACKNOWLEDGEMENTS
This work was financially supported by National Natural Science Foun-
dation of China (21763005, 21563006, 21864006, 21563010), Natural
Science Foundation of Guangxi Province (2017GXNSFDA198034,
2017GXNSFFA198005, 2021GXNSFAA075015), Thousands of Young
Teachers Training Program of Guangxi Province (guijiaoren[2018]18),
and BAGUI Scholar Program of Guangxi Province of China.

LUO ET AL.
11 of 12
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CONFLICT OF INTEREST
The authors declare that they have no known competing financial
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AUTHOR CONTRIBUTIONS
Huajian Luo and Yu Liang: Performed the research. Qi Xiao and Shan
Huang: Designed the research study. Huiying Zhang and Yi Liu: Con-
tributed essential reagents or tools. Shan Huang, Huajian Luo, and Yu
Liang: Analyzed the data. Shan Huang and Huajian Luo: Wrote the
paper.
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DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
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ORCID
Qi Xiao
https://orcid.org/0000-0002-6133-1853
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