Monitoring the Instant Creation of a New Fluorescent Signal for Evaluation of DNA Conformation Based on Intercalation Complex

Article abstract of DOI:10.1007/s10895-018-2294-4

Here we report the monitoring the instant creation of a new fluorescent signal (FS) aroused from a positively charged water-soluble fluorogenic probe, ethidium bromide (EtBr) in the presence of a radical initiator, ammonium persulfate (APS) and an acceler

Full text of DOI:10.1007/s10895-018-2294-4

Journal of Fluorescence
https://doi.org/10.1007/s10895-018-2294-4
ORIGINAL ARTICLE
Monitoring the Instant Creation of a New Fluorescent Signal
for Evaluation of DNA Conformation Based on Intercalation Complex
Ahmet T. Uzumcu1 & Orhan Guney1 & Oguz Okay1
Received: 17 July 2018 /Accepted: 3 September 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Here we report the monitoring the instant creation of a new fluorescent signal (FS) aroused from a positively charged water-
soluble fluorogenic probe, ethidium bromide (EtBr) in the presence of a radical initiator, ammonium persulfate (APS) and an
accelerator, tetraethylmetilendiamine (TEMED) for evaluation of deoxyribonucleic acid (DNA) conformation. The results re-
vealed that the occurred FS (λex = 430 nm; λmax = 525 nm) is a reduced form of EtBr (λex = 480 nm; λmax = 617 nm) and it is
completely distinct from hydroethidine (λex = 350 nm; λmax = 430 nm), which is two-electron reduced form of EtBr. It was
noticed that EtBr was reduced to a new FS during the polymerization of N, N dimethyacrylamide (DMAA) too, at 25 °C in the
presence of APS and TEMED or at 55 °C with only APS, and the rate of formation of FS was increased upon treatment time. The
effect of nanoclays such as Laponite XLG® and Laponite XLS®, which provide a protective environment for DNA in nature,
were also investigated through the reduction process of EtBr in the absence and presence of a water soluble monomer DMAA.
We demonstrated that DNA conformation might be evaluated by monitoring FS effectuated during the reduction of EtBr in the
presence of nanoclays having positively and negatively charged surfaces. Protective property of DNA against the formation of
reduced product was elucidated by carrying out the polymerization at 55 °C. The results revealed that the monitoring of formation
of FS in the presence of radical initiator could lead to elucidate the conformation of DNA upon formation of intercalator complex.
.
.
.
Keywords EtBr DNA conformation Intercalation complex Nanoclay
Introduction
intercalates between the adjacent base pairs within the strands
of DNA thanks to the prevention of the consisted hydrophobic
micro-environment from the fluorescent quenchers such as
oxygen or water, upon complexation [8–11]. By intercalation,
fluorescence quantum yield of EtBr drastically increases and
forms a complex with DNA by displaying an optical activity
in the visible region [12, 13].
Fluorescent probes, which are of great importance for the
improvement of biological imaging systems and assays, are
requisite tools for biological chemistry and being used almost
everywhere as tags for biomolecules, enzyme substrates, en-
vironmental markers, and cellular stains [1–3]. Ethidium bro-
mide (EtBr) is widely utilized as a fluorescent probe for the
determination of DNA conformation or interaction of DNA
with physiologically important molecules [4–7]. Fluorescence
intensity of free EtBr is weak but enhanced when it
Increasing interest has recently been dedicated to synthetic
organic molecules, especially anti-viral and anti-cancer drugs
which are able to interact with DNA, arising from electrostatic
interaction, intercalation between base pairs of DNA, or adher-
ing to the groove of DNA [14–17]. Dihydroethidium also
known as hydroethidine (HE) is the two-electron reduced form
of EtBr and is converted (dehydrogenated) into EtBr inside the
living cell, which then intercalates with DNA [18–20] and its
fluorescence emission at 617 nm greatly enhances when excit-
ed at 530 nm. To investigate the oxidative stress, reactive oxy-
gen species in cellular systems are determined by using HE as a
fluorescent probe since oxidation product of HE strongly fluo-
resces upon interaction with DNA, making them easily detect-
able in cells [18, 19, 21]. The reduction of EtBr to HE was
directly demonstrated by disappearance of the absorbance of
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10895-018-2294-4) contains supplementary
material, which is available to authorized users.
* Orhan Guney
oguney@itu.edu.tr
* Oguz Okay
okayo@itu.edu.tr
1
Departments of Chemistry and Polymer Science & Technology,
Istanbul Technical University, Maslak, 34469 Istanbul, Turkey

J Fluoresc
Scheme 1 Synthesis of HE by
using EtBr
EtBr at 480 nm, where HE does not exhibit any absorption at
the same wavelength of excitation [22–25].
Synthesis of Hydroethidium
In this study, UV-vis and fluorescence spectroscopy techniques
were used to gain insight into the reduction product of EtBr during
the free radical polymerization of DMAA and to enlighten the
conformational changes of DNA upon interaction with an
intercalator molecule in the absence and presence of nanoclay.
Hydroethidium (HE) was prepared as follows: 54 mg of
NaBH4 was added into 100 mg of EtBr, which was previously
dissolved in 2 ml absolute methanol at 0 °C under nitrogen
atmosphere in dark condition. After 20 h stirring, methanol
was evaporated under vacuum and 3 ml of 3 v/v% NaOH
solution was added. The precipitate was extracted several
times with ether and washed with water. The precipitate was
dried over anhydrous sodium sulfate and then recrystallized in
ether to yield a light brown powder (Scheme 1).
Materials and Methods
Materials
Fluorescence and UV-Vis Measurements
Ethidium bromide (EtBr), Deoxyribonucleic acid sodium salt
(DNA) of salmon testes (∼2000 base pairs), N,N,N,N′-
tetramethylethylenediamine (TEMED), Dimethyl sulfoxide
(DMSO) and N-N Dimethylacrylamide (DMAA) were pur-
chased from Sigma and used as received. Ammonium persulfate
(APS) was obtained from Fluka. Sodium borohydride (NaBH4)
was purchased from Sigma-Aldrich. The synthetic hectorite clays,
Laponite XLG (Mg5.34Li0.66- Si8O20(OH)4Na0.66) and Laponite
XLS (92.32 wt% of Mg5.34Li0.66- Si8O20(OH)4Na0.66 and
7.68 wt% of Na4P2O7) were kindly provided by Rockwood Ltd.
The fluorescence spectra of ethidium bromide (EtBr) were re-
corded on a Carry eclipse spectrofluorometer equipped with a
thermostated cell holder. The excitation and emission slit widths
were adjusted to 5 nm. The measurements were performed at
various excitation wavelengths; 430, 475 and 530 nm and fluo-
rescence spectra were recorded as a function of time. The UV-
Vis spectra were obtained from a VWR UV-1600 PC spectro-
photometer and the data were recorded via PC software.
Concentration and Functions of Materials
Results and Discussion
The concentrations of fluorogenic probe, EtBr (10 μM), the
monomer, DMAA (1.0 M), the initiator, APS (3.51 mM) and
the accelerator, TEMED (0.08 v/v%) were fixed for all of the
prepared samples. The concentration of ds-DNA was set to
2 w/v% while Laponite content was fixed to 3 w/v% through-
out all measurements.
Absorption Spectra of EtBr upon Addition of Diverse
Reagents
Fig. 1a shows UV absorption spectra of 10 μM aqueous
EtBr solution without and with the additives APS,
Fig. 1 UV absorption spectra of
10 μM aqueous EtBr solution with
various additives. (a): EtBr alone
(dashed curve) and after addition
of APS, TEMED and APS/
TEMED (solid curves). (b): EtBr+
DMAA alone (dashed curve) and
after addition of APS or APS/
TEMED (solid curves). (c): EtBr+
DMAA+DNA before (dashed
curve) and after addition of APS or
APS/TEMED (solid curves)

J Fluoresc
Scheme 2 Proposed scheme for
the reaction of radicals with EtBr
TEMED, or both. EtBr alone exhibits an absorption
maximum at 478 nm whereas a structureless spectrum
appears upon addition of APS, which we attribute to the
electrostatic interaction between anionic APS and cat-
ionic EtBr causing the alteration of conjugation on the
structure [8]. Moreover, the addition of TEMED alone
does not induce a spectral change but the addition of
both APS and TEMED to the EtBr solution produces a
large hypochromic shift to 430 nm in absorption maxi-
mum. Because TEMED is an accelerator for the decom-
position of APS and able to generate free radical at or
below the room temperature, this finding reveals that
the radicals generated using APS + TEMED redox initi-
ator system leads to an electron transfer, resulting in a
reduction of EtBr (Scheme 2). Considering the fact that
the formation of hydroethidine requires a two- electron
transfer reaction that exhibits an absorption maximum at
350 nm [26], it was deduced that the observed product
is novel and unfamiliar to our knowledge.
10 μM EtBr solution containing DMAA monomer at a con-
centration of 1 M. The dashed curve in Fig. 1b presents the
spectrum of aqueous EtBr + DMAA solution whereas the
solid curves are the spectra after addition of APS and APS+
TEMED. Because APS and TEMED initiate the polymeri-
zation of DMA via free-radical mechanism, the appearance
of the absorbance maximum at the same wavelength (430
2 nm) reveals that the electron transfer mechanism from
radical to EtBr was not affected due to the polymerization
of DMAA. Interestingly, including ds-DNA in the aqueous
solution of EtBr and DMAA, no hypechromic shift was
observed, as seen in Fig. 1c. Instead, a bathochromic shift
of about 50 nm in peak maximum of EtBr was observed
when DNA was included to the EtBr- DMAA solution con-
taining APS with or without TEMED. Noticeably, the peak
maximum of EtBr at 530 nm unchanged in the presence of
DNA, indicating that reduction of EtBr could not take place
during the polymerization process due to intercalation be-
tween the strands of DNA. Hence, DNA-EtBr intercalation
mechanism prevents the electron transfer to EtBr from free
radicals in the system.
A similar hypochromic shift to 430 nm was also ob-
served when APS or APS + TEMED were added to
Fig. 2 Excitation spectra of
10 μM EtBr in different
environments. (a) In water (1),
upon addition of APS (2), APS +
TEMED (3) and APS + TEMED
+ DNA (4). (b) In solution of 1 M
DMAA (1), upon addition of APS
(2), APS + TEMED (3) and
APS + TEMED + DNA (4).
λem = 615 nm

J Fluoresc
Fig. 3 Time-dependent emission
spectra of FS formed during the
reduction of 10 μM EtBr in pre-
polymer solution containing
DMAA+APS + TEMED
depending on excitation
wavelengths; (a) λex = 430 nm,
(b) λex = 475 nm, (c) λex
=
530 nm. Time periods are
indicated
Excitation Spectra of EtBr in the Presence of Different
Species
the time dependent emission spectra. On the other side, the
peak maximum of EtBr apparently red-shifted to 530 nm, fol-
lowing the addition of DNA to the pre-polymer solution with
or without DMAA containing APS and TEMED (curve 4 in
Fig. 2a and b). It is noticeable that the intercalation of EtBr
between the strands of DNA is much stronger than the other
interactions and intercalation was achieved for both systems
with or without DMAA.
Excitation spectrum of 10 μM aqueous EtBr solution is sim-
ilar to absorption spectrum with displaying maximum at
475 nm (Fig. 2a). As seen from Fig. 2a, the wavelength of
maximum intensity shifts to 450 nm upon addition of APS
(curve 2) and there is no spectral change in the presence of
only TEMED (data not shown) but shifts to 430 nm by addi-
tion of both APS and TEMED (curve 3). However, EtBr dis-
plays peak maximum at 500 nm in the presence of DMAA
(Fig. 2b, curve 1), showing the hydrophobic environment
formed around EtBr due to interaction with DMAA. After
addition of APS into pre-polymer solution, there was a blue
shift (70 nm) in maximum of wavelength (Fig. 2b, curve 2).
Nevertheless, EtBr exhibits excitation maximum at both
440 nm with a shoulder at around 500 nm when both APS
and TEMED were added (Fig. 2b, curve 3). The shoulder
appears due to formation of a new product during the course
of polymerization and vanishes after exhibiting a blue shift at
the end of the reaction. Detailed explanation is given in the
latter section and in the supporting information by presenting
Fluorescence Spectra of EtBr during Reduction
and Formation of FS
The emission spectra of EtBr during the reduction in 1 M
DMAA solution containing APS and TEMED at various ex-
citation wavelengths were monitored at different time periods
prior to addition of all reagents (Fig. 3). Strikingly, at earlier
times of polymerization, the peak maximum at 617 2 nm
diminishes and the peak appeared at 525 nm (λex = 430 nm)
starts to rise as the polymerization proceeds (Fig. 3a). The
intensity of the peak at 525 nm depends on excitation wave-
length and almost vanished when excited at 530 nm (Fig. 3a
and b) showing that besides EtBr, another product also existed
Fig. 4 Emission spectra of 10 μM aqueous EtBr solution (1), upon
addition of APS (2), APS + TEMED (3), DMAA+APS (4), DMAA+
APS + TEMED (5), DNA + APS + TEMED (6) DNA + DMAA+
APS + TEMED (7). λex = 430 nm)
Fig. 5 Emission intensities at 525 nm as a function of time of 10 μM EtBr
solution upon addition of APS (1), APS + TEMED (2), APS + TEMED+
DMAA (3), APS + TEMED+DNA (4) and APS + TEMED+DMAA+
DNA (5). λex = 430 nm

J Fluoresc
Fig. 8 Variation of emission intensities at 500 nm of solutions (DMAA+
APS + TEMED) containing nanoclays; Laponite XLG (1) and Laponite
XLS (2). (λex = 430 nm). Inset figure: Intensities at 617 nm for same
solutions upon excitation at 530 nm
Fig. 6 Fluorescence spectra of 10 μM aqueous EtBr solution (1), upon
addition of APS (2), APS + TEMED (3), APS + TEMED+DMAA(4),
APS + TEMED+DNA(5) and APS + TEMED+DMAA+ DNA (6).
λex = 530 nm
Meanwhile, the spectrum obtained upon exciting at 475 nm
exhibits the emissions at 600 and 530 nm, indicating that both
EtBr and the formed product exist in solution (Fig.S4).
Fluorescence spectra of EtBr upon excitation at 430 nm ex-
hibit maximum peak at 518 nm during the polymerization of
DMAA initiated by APS with or without TEMED (Fig. 4,
curve 4 and 5), whereas there was no emission peak around
530 nm (Fig. 4, curves 6 and 7) in the presence of DNA with
or without DMAA upon excitation wavelengths (Fig.S7 and
Fig. S8). This indicates that either the interaction of DNAwith
EtBr creates the hydrophobic environment or EtBr intercalates
into DNA and therefore prevents the process of reduction.
Figure 5 shows the emission intensities at 525 2 nm of all
samples as a function of time. In the presence of only APS,
there seems to be a small increment in emission intensity,
whereas there is an apparent rise of intensity in the presence
of APS and TEMED, showing the formation of a reduced
product.
in reaction solution. This result is indeed intriguing, consider-
ing that the fluorescence spectrum of EtBr in aqueous solution
exhibits a maximum emission at 617 2 nm which is inde-
pendent from the time and the excitation wavelength as ex-
pected (Fig. S1).
In order to clarify that the new reduced product has distinct
characteristics from HE, both EtBr and HE in DMSO solu-
tions were excited at 350 nm and they exhibited emission
maxima at 640 and 420 nm respectively (Fig. S2). Figure 4
shows the fluorescence spectra of EtBr itself and after the
reagents added individually into the aqueous solution was
obtained by excitation at 430 nm after a waiting time up to
120 min to ensure that all interactions or reactions between the
species were ended (from Fig. S3 to Fig. S8).
Nevertheless, there seems to be change in spectrum of EtBr
after addition of APS and the peak maximum shifts to 542
2 nm (Fig. 4 curve 2 and Fig. S3). A new peak with a maxi-
mum of 525 nm appeared after addition of both APS and
TEMED (Fig. 4, curve 3). Moreover, the emission intensity
of peak at 525 nm, which might be ascribed to FS, gradually
increased depending on time (Fig. S4).
Addition of DMAA into this mixture results in a further
increase in emission intensity (Fig. 5, curve with squares).
This may be due to the two reasons: first, the concentration
of reduced product increases upon polymerization of DMAA
Fig. 7 Time dependent
fluorescence spectra of the
formation of fluorescent species
during reduction of EtBr in
aqueous solution containing
DMAA, APS, TEMED and
Laponite XLG upon three
different excitation wavelengths.
Time scales and excitation
wavelengths are indicated

J Fluoresc
Table 1 Photophysical properties
of EtBr in different environmentsa
Samples
Absorption maxima
(nm)
Emission maxima
(nm)
Quantum yield
EtBr
480
430
430
430
617
525
525
525
0.022
0.148
0.106
0.207
EtBr+APS + TEMED
EtBr+DMAA+APS
EtBr+DMAA+APS + TEMED
a: EtBr = 10 μM, DMAA = 1 M, APS = 3.51 mM, TEMED = 0.08 v/v%, Laponite XLG = 3 w/v %
and second, formation of poly(DMAA) creates a more rigid
environment by means of increasing the dynamic viscosity of
the reaction system, resulting in enhancement of fluorescence
emission intensity. Note that no emission peak observed
around 530 nm when excited at 430 nm (Fig. 5, curves with
triangles down and diamonds respectively) in the presence of
DNAwhether DMAA is included or not (Fig.S7 and Fig. S8).
This is evidence that the intercalation of EtBr with DNA does
not allow APS to reach EtBr for electron donation.
In the presence of a reducing agent, the change in the fluo-
rescence emission of EtBr can provide information to evaluate
the structure of DNA-intercalator complex. Therefore, we
used different excitation wavelengths in order to figure out
the environment of EtBr upon interaction with DNA
(Fig. 6). As seen, existence of emission at 600 nm upon exci-
tation at 530 nm indicates that polymerization of DMAA cre-
ates hydrophobic environment around EtBr and retards the
reduction of EtBr to some extent. On the other hand, binding
of EtBr by intercalating between DNA base pairs may also
inhibits the reduction process (Fig. 6).
XLS (Fig.S9) were recorded depending on time upon three
different excitation wavelengths. The existence of different
emission maxima in fluorescence spectra indicates that dis-
similar species exist in solution in the presence of the radical
producing agent APS or upon polymerization of DMAA.
When excited at 430 nm, maximum in fluorescence spectrum
(617 2 nm) belonging to EtBr in hydrophilic environment,
blue-shifted to 500 nm, showing that reduction of EtBr results
in creation of a fluorescent product. The emission intensity of
EtBr in the presence of both XLG and XLS at 500 nm en-
hanced by increasing the polymerization time (Fig. 8).
However, emission intensity of EtBr at 617 nm decreases first
and then increased depending on time. This might be due to
the charge neutralization of EtBr upon electrostatic interaction
with clays first and then starting of gel formation in the sys-
tem. When emission intensities were plotted against the poly-
merization time, turning point in curve is 40 min for XLG and
20 min for XLS (Fig. 8, inset).
The Fluorescence Quantum Yield of Treated Samples
of EtBr
Nanoclay Effect on FS during Reduction of EtBr
The quinine sulfate was used as a standard (Φref = 0.54 in
0.1 N H2SO4) in order to determine the fluorescence quantum
yield of EtBr in each sample. Fluorescence quantum yield was
calculated according to the following equation [26].
The fluorescence spectra of EtBr in pre-gel solution contain-
ing DMAA, APS, TEMED and nanoclays XLG (Fig. 7) and
!
ꢀ
ꢁꢀ
ꢁ
Fs
Fref
Aref
As
ns2
n2ref
Φs ¼ Φref
ð1Þ
where Fs and Fref are integrated area under the fluorescence
emission spectrum measured of sample and standard, respec-
tively; As and Aref are absorbances at the same excitation
wavelength of sample and standard, respectively; ns and nref
are refractive indexes of solvent used for sample and standard,
and Φref is quantum yield of standard. Fluorescence quantum
yields of each sample were shown in Table 1.
Maximum wavelength of EtBr at 617 2 nm in aqueous
solution at 25 °C shifted to 520 nm after completion of
DMAA polymerization by using only APS as an initiator at
55 °C (Fig. 9). This means that fluorescent product is also
formed by reduction of EtBr in the absence of TEMED. On
Fig. 9 Fluorescence spectra of 10 μM EtBr at 55 °C in the presence of
APS after polymerization of DMAA (1), with DNA added (2), with both
of DNA and Laponite XLG added (3)

J Fluoresc
Acknowledgements This work was supported by Istanbul Technical
University (ITU), BAP 39773. O.O. thanks to Turkish Academy of
Sciences (TUBA) for the partial support.
the other hand, when polymerization reaction was carried out
in the presence of DNA, fluorescence spectrum with double
emission maxima at both around 510 nm and 600 nm was
obtained. The emission peak at 600 nm shows the existence
of unreduced EtBr, whereas there was no any peak formation
at 510 nm in the presence of DNA when the polymerization
was carried out in the presence of both APS and TEMED at
25 °C. Meanwhile, addition of XLG to pre-polymer solution
in the presence of DNA caused to increase in peak intensity at
around 500 nm. These results revealed that polymerization
temperature and existence of XLG might tear off EtBr inter-
calated with DNA and lead to reduction of EtBr.
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We have found that EtBr was converted into a new fluo-
rescent signal (FS) during the radical polymerization of
DMAA. The photophysical property of product formed
is completely different from HE, which is two-electron
reduced product of EtBr. The results revealed that EtBr
partially reduced and FS formed in gel containing
nanoclays since each different excitation resulted in spe-
cific fluorescence spectra. However, there was no forma-
tion of reduced product in the presence of DNA, indicat-
ing that intercalative binding of DNA creates shielding
effect on EtBr to protect from reduction. Although DNA
protects EtBr from being reduced, the transfer of electron
to EtBr occurred by addition of nanoclay (Laponite XLG
or XLS) into the pre-polymer solution when polymeriza-
tion was carried out at 55 °C. The fluorescence measure-
ments after polymerization of DMAA in the presence of
DNA and nanoclay at 55 °C revealed that the conforma-
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tial destruction of double helix structure of DNA upon
heating. These findings might lead the way to future re-
search about elucidation of conformational change of
DNA depending on change in temperature or upon inter-
action with molecules. Besides, the nanoclay interacted
with EtBr by electrostatically could not protect EtBr from
being reduced. This indicates that electrostatic interaction
with DNA through phosphate backbone could not contrib-
ute the protection of EtBr from electron transfer process,
whereas intercalative binding of EtBr to DNA creates bar-
rier to shield EtBr from reduction process. The experi-
mental data generated through the spectrophotometric
techniques employed herein are of great importance since
they allow a better understanding of instant formation of
FS and can aid future studies on new systems specially
designed for investigation on structural change in confor-
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