Post on 09-Oct-2020
Identification of a new Z-DNA inducer using SYBR green 1as a DNA conformation sensorJeong Hwan Hur1, Ae-Ree Lee2, Wanki Yoo1, Joon-Hwa Lee2 and Kyeong Kyu Kim1,3
1 Department of Molecular Cell Biology, Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University
School of Medicine, Suwon, Korea
2 Department of Chemistry and Research Institute of Natural Science, Gyeongsang National University, Jinju, Korea
3 Samsung Biomedical Research Institute, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center,
Sungkyunkwan University School of Medicine, Seoul, Korea
Correspondence
K. K. Kim, Department of Molecular Cell
Biology, Institute for Antimicrobial
Resistance Research and Therapeutics,
Sungkyunkwan University School of
Medicine, Suwon 16419, Korea
Tel: +82-31-299-6136
E-mail: kyeongkyu@skku.edu
(Received 30 April 2019, revised 13 June
2019, accepted 19 June 2019, available
online 10 July 2019)
doi:10.1002/1873-3468.13513
Edited by Miguel De la Rosa
Z-DNA, which is left-handed double-stranded DNA, is involved in various
cellular processes. However, its biological roles have not been fully evaluated
due to the lack of tools available that can control the precise conformational
change to Z-DNA in vitro and in vivo. Therefore, the need for identifying
new Z-DNA inducers is high. We developed an assay system to monitor the
conformational change in DNA utilizing the fluorescence of SYBR green I
integrated into a double-stranded oligonucleotide. By applying this assay to
screen for compounds that induce the B-DNA to Z-DNA transition, we iden-
tified the natural compound aklavin as a novel Z-DNA inducer.
Keywords: aklavin; B-to-Z transition; high-throughput assay; screening
system; SYBR green I; Z-DNA
Z-DNA, which is left-handed double-stranded DNA, is
induced and stabilized when alternating purine–pyrim-
idine repeats are exposed to positively charged mole-
cules, Z-DNA–binding proteins (ZBPs), or high salt
concentration [1]. Z-DNA can be also formed by nega-
tive supercoiling within cells in vivo to relieve transcrip-
tion-induced superhelical strain. Z-DNA is linked to
various cellular processes, including the innate immune
response [2] and transcriptional regulation [3]. More-
over, since Z-DNA formation can disrupt the confor-
mational and functional integrity of DNA, Z-DNA
formation is proposed to cause genomic instability and
deletions [4]. However, Z-DNA can be stabilized by Z-
DNA inducers that reduce the anionic repulsion cre-
ated by the close phosphate groups in its backbone [5].
Through intensive structural and biochemical analy-
ses, it has been well documented that ZBPs, including
ZBP1/DLM-1, ADAR1, E3L, and PKR-like protein
kinase, can induce the B-to-Z transition upon binding
and stabilize Z-DNA [6–9]. Moreover, it has been
reported that some metal chelate compounds, such as
([Ru(dip)2dppz]2+) [10], can efficiently push the equilib-
rium toward Z-DNA by reducing the electrostatic
repulsion in the phosphate backbone. Polyamines, such
as cobalt hexamine and spermine, can also induce the
formation of Z-DNA, presumably by a mechanism
similar to that of ruthenium complexes [11–13]. Z-
DNA can be also be stabilized in the presence of
monovalent or divalent cations, such as Mg2+, Zn2+,
Ni2+, Co2+, and Na+ (e.g. NaCl or NaClO4) at rela-
tively high concentrations in buffer [14–17]. Lysine-
containing peptide (Lys-Ala-Lys) can also transform B-
DNA to Z-DNA [18]. However, the precise control of
Z-DNA formation in vitro or in vivo requires a chemi-
cal inducer that can effectively and specifically trans-
form the DNA to a Z-DNA conformation.
To discover new Z-DNA inducers or probes, an effi-
cient and accurate high-throughput screening (HTS)
Abbreviations
HTS, high-throughput screening; SG, SYBR green I; ZBPs, Z-DNA–binding proteins.
2628 FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
system is necessary that identifies candidate chemicals
among many compounds in chemical libraries. In this
study, we developed a reporter system that can monitor
DNA conformational change by measuring the SYBR
green I (SG) fluorescence since SG fluorescence shows
a dramatic change during the B-to-Z transition. We
utilized this reporter to screen for and identify Z-DNA
inducers. To further separate real candidates from
false-positive candidates, we introduced a high-
throughput melting temperature analysis. The efficiency
of this screening system was validated using known Z-
DNA inducers, and it was ultimately used to identify a
new Z-DNA inducer from a natural compound library.
Methods and material
Purification of human ZaADAR1
The Za domain of human double-stranded RNA-speci-
fic adenosine deaminase 1 (hZaADAR1) was purified as
described previously. Briefly, the Za domain incorpo-
rated into pET28a was transformed into BL21 (DE3)
Escherichia coli, which were then cultured at 37 °C for
4 h, treated with 1 mM IPTG, and cultured for another
4 h. The cells were lysed using lysis buffer (50 mM Tris-
HCl, 300 mM NaCl, 30 mM imidazole, 0.1 mM PMSF,
0.5 lg DNase I, pH 8.0). The proteins were purified
using Ni-nitrilotriacetic acid and an SP column.
Purification of lac repressor protein
The DNA binding domain (1–331) of the lac repressor
protein was cloned into pVFT1S. The cloned plasmid
was transformed into BL21 (DE3), which were then
cultured at 37 °C for 4 h, treated with 1 mM IPTG,
and cultured for another 4 h. The cells were lysed
using lysis buffer (20 mM Tris-HCl, 300 mM NaCl,
20 mM imidazole, pH 7.5). The proteins were purified
using nitrilotriacetic acid followed by gel filtration.
Fluorescence measurement of SYBR green
I-labeled oligonucleotide
The single-strand synthetic oligonucleotides d(CG)6,
d(AT)6, and the lac repressor protein binding sequence
d(GAATTGTGAGCGCTCCTCTCACAATTC) (Cos-
mogenetech, Seoul, South Korea) were dissolved in
Buffer ‘A’ (5 mM HEPES, 10 mM NaCl, pH 7.5).
Then, the oligonucleotides were boiled at 97 °C for
5 min and slowly allowed to anneal at room tempera-
ture for at least 4 h. The oligonucleotides were stored
at 4 °C overnight followed by concentration via
centrifugation. The concentration of annealed DNA
was adjusted to 15 lM. The 10 0009 stock of SG
(Thermo Fisher Scientific, Waltham, MA, USA) was
mixed with the oligonucleotides to a dilution of 19.
The mixture of SG and the annealed DNA was again
boiled and annealed to incorporate SG into the DNA.
The mixture was stored at 4 °C before use. The fluo-
rescence intensities of the 15 lM SG-labeled DNA with
and without Z-DNA inducers were measured using a
Jasco FP-750 (Jasco, Easton, MD, USA). An excita-
tion scan was performed to determine the maximum
excitation wavelength. Then, an emission scan was per-
formed in the wavelength range of 500–700 nm after
exciting DNA at the maximum excitation wavelength.
The fluorescence intensity of the buffer was subtracted
from each dataset for normalization.
Screening of the Z-DNA–inducing chemicals
A natural product library containing 800 compounds
of diverse alkaloids, flavonoids, and sterols (NatProd
Collection; MicroSource Discovery System, Gay-
lordsville, CT, USA) was prepared in a 1 mM stock
solution in DMSO for screening. One microliter of
each chemical stock solution was introduced into a
well containing 29 lL of 15 lM DNA labeled with SG
in a 96-well plate. As a result, the total reaction vol-
ume was 30 lL and the final concentration of the
chemical was 333 lM. For screening, the plate was
sealed and inserted into a CFX Connect RT-PCR
machine (Bio-rad, Hercules, CA, USA), which has
excitation and emission ranges of 450–580 nm. Candi-
date chemicals from this first stage of screening were
chosen based on the fluorescence intensity of SG incu-
bated with DNA in the presence of each chemical. The
fluorescence intensity was measured by a Jasco FP-750
spectrofluorometer with excitation at 490 nm and
emission in the range of 500–550 nm (Fig. 2A). Then,
the melting temperature (Tm) of DNA was measured
in the presence of the chemicals selected in the first
stage (Fig. 2B). The Tm analysis was performed by
increasing the temperature from 10 to 95 °C in 0.5 °Cincrements with 5 s between each incremental increase.
Circular dichrosim
For 1 h, 15 lM d(CG)6 was incubated with purified
human ZaADAR1 protein at a ratio of 1 : 8 for
complete transition, and the CD spectra were mea-
sured in the wavelength range of 230–320 nm by a
Jasco-810 (Jasco). Three spectra were collected to
obtain the average spectra. In the CD experiment with
DNA at the high salt condition, both d(CG)6 and
2629FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
J. H. Hur et al. Searching for a new Z-DNA inducer with SYBR green
d(AT)6 were incubated in 4 M salt buffer for 1 h before
obtaining the spectra. CD signal from buffer and chem-
icals were subtracted from the obtained result.
Nuclear magnetic resonance
The oligonucleotides were prepared at a concentration
of 0.2 mM in buffer containing 5 mM HEPES and
10 mM NaCl at pH 7.5. For monitoring the imino pro-
ton region, the oligonucleotides were also dissolved in
buffer ‘A’ with 10% D2O and 90% DNase I-treated dis-
tilled water. All NMR experiments were performed
using a 700 MHz NMR machine (Agilent Technologies,
Santa Clara, CA, USA) at 28 °C. The WATERGATE
3919 NMR pulse program was used to monitor the
imino proton chemical shift (12.5–13.5 p.p.m.).
Statistical analysis
Statistical significance was assessed by two-tailed t-test
using GRAPHPAD software (GraphPad software, La
Jolla, CA, USA).
Results
SYBR green I displacement was observed during
B-to-Z transition
SYBR green I emits strong fluorescence around 520 nm
when it binds to double-stranded DNA, but gives a low
emission signal when it is present in solution [19,20].
The SG binding to B-DNA, the most stable double-
stranded DNA, has been well characterized, but its
binding to other forms of double-stranded DNA has
not been well studied. Because Z-DNA is also double-
stranded DNA with base pair stacking, we expected
that SG would also bind to Z-DNA and emit fluores-
cence. To verify this, we first examined the Z-DNA for-
mation of SG-bound B-DNA using CD spectrometry
(Fig. 1A). d(CG)6, double-stranded deoxyribonucleic
acid with hexa-repeats of deoxycytidylic-deoxyguanylic
acids, was incubated with SG to make SG-bound B-
DNA. The formation of Z-DNA was monitored in the
presence of the known Z-DNA inducer hZaADAR1, a Z-
DNA binding domain of human ADAR1, and under a
high salt condition (4 M NaCl), which is also known to
induce Z-DNA. We confirmed the Z-DNA formation
of d(CG)6 under both conditions by observing the typi-
cal CD spectra of Z-DNA, including a negative peak at
295 nm and incremented ellipticity in the wavelength
range of 250–255 nm (Fig. 1A). These results verified
that SG-bound B-DNA can be converted into Z-DNA
when exposed to Z-DNA–inducing conditions.
However, based on this evidence, it was unclear if
SG remained bound to Z-DNA or detached during the
B-to-Z transition. Therefore, we performed CD and
FRET experiment with d(CG)6 at various concentra-
tion (500 mM, 750 mM, 1 M, 2 M, 3 M, 5 M, 7 M, and
9 M) of sodium perchlorate (NaClO4) to see the corre-
lation between B-to-Z transition and the changes in flu-
orescence intensity emitted by SG during the transition
(See also Fig. S1). As the concentration of NaClO4
increased, d(CG)6 showed canonical B-to-Z transition
where the CD value at 255 and 293 nm become posi-
tive and negative, respectively (See also Fig. S1A),
while showing the significant decrease in fluorescence
intensity at 520 nm and slight red shift to 550 nm at
high concentration of NaClO4 condition (> 3 M; See
also Fig. S1B). The plot showing the CD value at
255 nm and the fluorescence intensity at 520 nm at
each NaClO4 concentration explicitly explains the fluo-
rescence decrement is deeply correlated with B-to-Z
transition (See also Fig. S1C). Moreover, we monitored
SG fluorescence by fixing the excitation wavelength at
475 nm and observing the emission spectra from 500 to
550 nm during the B-to-Z transition under fixed Z-
DNA–inducing conditions. Upon the B-to-Z transition
induced by hZaADAR1, kmax shifted from 520 to
525 nm, and the fluorescence intensity at 520 nm was
reduced up to 86.5% compared to the fluorescence of
SG-bound B-DNA (Fig. 1B). We conducted the same
experiment using d(AT)6 as a control because d(AT)6cannot be converted to Z-DNA, even in the presence
of hZaADAR1 (Fig. 1A). Although there was a slight
reduction in fluorescence, the reduction in intensity
caused by the incubation of d(AT)6 with hZaADAR1
was 39.4%, which was less than that of d(CG)6(Fig. 1B). Similarly, when SG-bound d(CG)6 was con-
verted into Z-DNA by 4 M NaCl, the reduction in SG
intensity was 51.1%, whereas d(AT)6 in 4 M NaCl
demonstrated only a 16.1% reduction in SG intensity
(Fig. 1B). Based on these results, we confirmed that SG
fluorescence was reduced when SG-bound B-DNA was
transformed into Z-DNA by either a protein inducer
or high salt conditions, although the reduction percent-
age varied depending on the inducing condition.
The fluorescence intensity of SG can also be dimin-
ished when dsDNA is denatured to ssDNA due to the
inability of SG to bind to ssDNA [19]. However, since
we observed that DNA remained in double-stranded
form based on the CD analysis during the transition
(Fig. 1A), the fluorescence reduction was likely caused
by the conformational change into Z-DNA rather than
the denaturation of dsDNA. However, it remained pos-
sible that protein binding to DNA caused SG fluores-
cence alterations. To examine this possibility, we
2630 FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
Searching for a new Z-DNA inducer with SYBR green J. H. Hur et al.
assessed the SG fluorescence change when the lac repres-
sor bound to its cognate dsDNA (See also Fig. S2B).
We found that the SG fluorescence was reduced by
21.4% upon protein binding (See also Fig. S2B), but
this reduction was less than that observed when hZaA-
DAR1 bound to d(CG)6. Notably, no conformational change
in DNA was observed upon lac repressor binding (See
also Fig. S2A). These results collectively suggest that the
change in SG fluorescence caused by hZaADAR1 binding
was mostly due to the subsequent conformational
change in DNA rather than the protein binding itself.
Therefore, we conclude that the altered fluorescence of
SG-bound DNA caused by a B-to-Z transition can be
distinguished from the fluorescence alterations observed
due to simple protein–DNA interactions or DNA-Na+
interactions.
SYBR green I is known to bind B-DNA by either
intercalation or surface binding depending on the ratio
of SG to DNA base pairs. Intercalation with DNA can
be achieved at a dye/base pair ratio (dbpr) of < 0.15,
whereas extra SG binds to the DNA surface when the
dbpr is higher than 0.15 [19]. Our experimental condi-
tions contained 175.0 lM base pairs of DNA and
66.2 lM SG, so the dbpr was calculated to be 0.38. There-
fore, it is highly probable that SG is bound on the surface
as well as intercalated in DNA based on the dbpr > 0.15.
Developed efficient and fast screening system for
identifying Z-DNA inducers
From the CD and fluorescence analyses of the SG-
bound DNA, we verified that DNA conformation
Fig. 1. CD spectra and fluorescence signal reduction showing changes in signal upon Z-DNA conformation. (A) CD spectra of SG-labeled d
(CG)6 and d(AT)6 in the presence of hZaADAR1 and 4 M NaCl (B) The measurement of fluorescence intensity changes upon the B-to-Z
transition. (C) The measurement of fluorescence intensity changes of SG d(CG)6 upon addition of a Z-DNA inducer (hZaADAR1) or control
(DMSO) in a 96-well set-up (***P < 0.0001).
2631FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
J. H. Hur et al. Searching for a new Z-DNA inducer with SYBR green
could be differentiated by change in SG fluorescence.
Therefore, this method had the potential to assess the
B-to-Z conformation transition and be useful in identi-
fying Z-DNA inducers or stabilizers. Based on this
hypothesis, we developed a screening system for identi-
fication of Z-DNA-inducing substances (Fig. 2). The
proposed assay method consists of three steps: (a) an
initial screen of the substances that can induce fluores-
cence change in SG-bound B-DNA; (b) Tm analysis of
DNA in the presence of the candidates selected in the
first step to verify whether they can stabilize the DNA
conformation; and (c) CD spectroscopic measurement
of DNA in the presence of candidate molecules to vali-
date the chemical-induced Z-DNA formation. The
measurement of Tm in the second step is necessary to
exclude any compounds that induce denaturation of
dsDNA, given that any fluorescence change observed
in the first step may also be caused by SG binding to
single-strand DNA. The first and the second steps can
be conducted simultaneously in 96-well plates using a
real-time PCR machine with a fluorescence reader.
Considering the short duration and simple setup of
these experiments, we believe that this method can be
employed as a HTS assay to discover new Z-DNA
inducer candidates.
Identified aklavin as Z-DNA inducer
To validate the utility of this method in a high-through-
put approach, we first tested whether known Z-DNA
inducers such as hZaADAR1 can be detected using this
method in a 96-well setup with a plate reader (CFX
connect machineTM; Bio-rad, Hercules, CA, USA). For
comparison, DMSO was used as a negative control.
Reduction in SG fluorescence was clearly detected when
hZaADAR1 was applied to the SG-bound B-DNA
(Fig. 1C) although, the scale of the fluorescence change
observed by the plate reader was different from that
obtained using the spectrofluorometer (Fig. 1B). This
discrepancy is likely due to the differences in excitation
and emission wavelengths of the spectrofluorometer
and the plate reader. Collectively, we determined that
the high-throughput approach could be used to identify
Z-DNA inducer candidates by monitoring changes in
SG fluorescence intensity. Therefore, we applied this
method to screen for Z-DNA inducers among 800 natu-
ral compounds by measuring the fluorescence intensity
changes of SG-bound DNA upon introduction of each
candidate. Because protein binding itself produces some
degree of reduction in fluorescence, we set our cut-off at
60.0% rather than 80.0%. Thirty-two compounds
demonstrated a > 60.0% decrease in SG fluorescence
intensity compared to the nontreated DNA control and
proceeded to the next step. The selected 32 candidate
compounds were then assessed by Tm analysis. This step
further selected seven of the 32 compounds that had a
measured Tm higher than the Tm (56.0 °C) of the
untreated DNA sample (See also Fig. S3A,B, Table S1).
These seven compounds were further validated as Z-
DNA inducers by CD spectrometry. Among the seven,
aklavin hydrochloride showed the CD spectra most rep-
resentative of Z-DNA (Fig. 3C, See also Fig. S3C).
Thus, we further characterized aklavin hydrochloride as
a Z-DNA inducer. When the Tm of DNA in the presence
of aklavin hydrochloride was analyzed again, more
accurately using the real-time PCR machine, we
observed that the absolute Tm of the DNA increased
from 56.0 to 69.0 °C (Fig. 3D). These results clearly sug-
gest that Z-DNA is highly stabilized when it interacts
with aklavin. Generally, Z-DNA can be characterized
by a distinct CD spectrum, with a negative peak around
290 nm and a positive peak around 255 nm; B-DNA
results in a positive peak at 290 nm and a negative peak
at 255 nm. We observed that d(CG)6 demonstrated a
positive peak (0.24 mdeg) at 300 nm and a negative
peak (�3.19 mdeg) at 255 nm, suggesting that it had a
B-DNA conformation in the absence of chemicals
(Fig. 3C). However, DNA in the presence of aklavin
hydrochloride at a 1 : 1 ratio resulted in a spectrum with
a negative peak (�0.66 mdeg) at 300 nm. In addition,
its ellipticity at 255 nm was positively shifted
(�1.65 mdeg) compared to B-DNA. When the ratio of
aklavin to DNA was increased to 2 : 1, the CD elliptic-
ity values at 300 and 255 nm were negatively
(�1.39 mdeg) and positively shifted (�0.94 mdeg),
respectively (Fig. 3C). These results demonstrate conver-
sion of B-DNA to Z-DNA in the presence of aklavin.
Verified aklavin as Z-DNA inducer using 1D1H-NMR
To further verify the Z-DNA–inducing activity of akla-
vin, we acquired the 1D 1H-NMR spectra of the 6-bp
DNA duplex d(CG)3 as a function of the [A]/[D] ratio
under both 10% D2O and 100% D2O conditions
(Fig. 4, right panel). Generally, the B-to-Z transition
can be confirmed by characteristic changes in the gua-
nine imino proton resonances. For example, the ability
of the ruthenium chemical compound to induce the Z-
DNA conformation of d(CG)12 was previously vali-
dated by line-broadening and downfield shifts of the
imino proton resonances of the guanine nucleotide at
around 13.0 p.p.m. followed by the treatment of ruthe-
nium [10]. Using a similar technique, we monitored the
change in imino proton resonances of the guanine
nucleotides (G2b and G4b) in d(CG)3 while adding
2632 FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
Searching for a new Z-DNA inducer with SYBR green J. H. Hur et al.
Fig. 2. Schematic representation of the HTS system for identification of Z-DNA-inducing molecules. (A) ‘Step 1’ measures the SG
fluorescence intensity reduction relative to the ‘control’ (black) to identify a ‘candidate’ (red). (B) ‘Step 2’ measures the increase in Tm of a
‘candidate’ (red) molecule compared to the ‘control’ (black). (C) ‘Step 3’ measures the ellipticity (mdeg) of d(CG)6 in the presence of
‘candidate molecules’.
2633FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
J. H. Hur et al. Searching for a new Z-DNA inducer with SYBR green
increasing concentrations of aklavin. We used the
WATERGATE 3–9–19 pulse sequence, which sup-
presses water resonance and enhances desired proton
resonances from nucleic acids. The intensities of the
imino resonances of the DNA decreased and broadened
as the [A]/[D] ratio increased up to 1.0 (Fig. 4). The two
imino resonances from the second guanine residue
(G2b) at 12.95 p.p.m. and the fourth guanine residue
(G4b) at 12.92 p.p.m., exhibited significant line-
broadening upon binding to aklavin in a concentra-
tion-dependent manner. In addition, the imino reso-
nances of G2b (G2z) and G4b (G4z) moved to 12.93
and 12.90 p.p.m., respectively. This represents a chem-
ical shift difference (Dd) of 0.02 p.p.m. (~ 14 Hz) for
(G4b) and 0.01 p.p.m. (~ 7 Hz) for G4b upon the
addition of with aklavin. This chemical shift differ-
ence was previously observed in high salt-induced
(Dd = 0.16 p.p.m.) [21] and ruthenium-induced
Fig. 3. Validation of aklavin hydrochloride as a Z-DNA inducer. (A) Representative data from a screen demonstrating a primary Z-DNA–
inducing candidate molecule (red bar) via observation of the reduction in SG intensity compared to the control (black bar), where each tick
on the x-axis represents each chemical compound in the set. The y-axis represents the SG fluorescence intensity, and the 60% reduction
cut-off is indicated by the red dotted line. (B) Chemical structure of aklavin. (C) CD spectra of d(CG)6 with increasing aklavin [A] to DNA [D]
molar ratio ([A]/[D]). (D) Tm analysis of d(CG)6 in the presence of aklavin (**P < 0.005).
2634 FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
Searching for a new Z-DNA inducer with SYBR green J. H. Hur et al.
Z-DNAs (Dd = 0.05 p.p.m.)[10]. In addition, this inter-
action represents a fast exchange mode rather than a
slow exchange mode. In the 100% D2O condition
(Fig. 4, left panel), significant peak broadening was
observed at the C1H6, C3H5’, and C5H5’ resonances,
indicating the interaction of aklavin with these residues.
These results suggest that d(CG)3 exists as a left-handed
Z-DNA after aklavin treatment (Fig. 4).
Discussion
Aklavin is known to have antibiotic activity as well as
antiphage activity [22], but its mechanism of action
remains unclear. It is also known that aklavin prevents
virus-mediated host cell lysis in a concentration-depen-
dent manner by altering the host–virus interaction and
suppressing viral growth [22]. Therefore, aklavin has
multiple functions in cells through binding to various
macromolecules via many functional groups. It is well
known that Z-DNA can be stabilized by reducing the
repulsive forces between negatively charged phosphate
groups in its backbone. It is possible that the tertiary
amine derivative of aklavin reduces the electrostatic
repulsion in the negatively charged phosphate back-
bone in this way, thus stabilizing the Z-DNA
(Fig. 3B). However, the detailed binding mode of akla-
vin to Z-DNA and the mechanism of the aklavin-me-
diated B-to-Z transition require further investigation.
Considering the important potential roles of Z-DNA
in various biological processes and diseases, there is a
need to identify Z-DNA inducers that can control Z-
DNA–relevant biological processes and can potentially
be used for therapeutic purposes. However, there is no
reliable screening system to identify Z-DNA inducers.
In this study, we developed an efficient and fast
screening system to identify Z-DNA inducers based on
the change in SG fluorescence and Tm, followed by
CD validation. We demonstrated the utility of this
screening system by identifying a novel Z-DNA indu-
cer from a natural compound library. This system
could be used to similarly identify more Z-DNA
inducers if applied to other chemical libraries. There-
fore, this screening system will play a pivotal role in
the discovery and validation of new Z-DNA inducers
with various chemical properties.
Acknowledgement
This work was supported by Samsung Science & Tech-
nology Foundation (SSTF-BA1301-01).
Author contributions
JHH and KKK wrote the manuscript under KKK’s
advisory. JHH performed HTS and CD-related experi-
ment, and prepared sample for NMR. ARL analyzed
and processed NMR data. WY purified lac repressor
proteins. JHL advised on the NMR experiment and
data. KKK advised on the entire project and the
manuscript.
Fig. 4. 1D 1H-NMR result of d(CG)3 upon titration with aklavin. 1D 1H-NMR spectra at the 10% D2O condition (right panel) showing the B-
to-Z transition upon addition of aklavin ([A]/[D] = 0, 0.25, 0.5, 0.75, and 1.0). The resonances from the B-form are labeled as G2b
(12.94 p.p.m.) and G4b (12.92 p.p.m.), and those from the Z-form are labeled as G2z (12.92 p.p.m.) and G4z (12.90 p.p.m.). 1D 1H-NMR
spectra at the 100% D2O condition (left panel) showing the B-to-Z transition upon addition of aklavin ([A]/[D] = 0, 0.25, 0.5, 0.75, and 1.0).
2635FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
J. H. Hur et al. Searching for a new Z-DNA inducer with SYBR green
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Supporting information
Additional supporting information may be found
online in the Supporting Information section at the end
of the article.
Fig. S1. The correlation between B-to-Z transition and
SG fluorescence intensity.
Fig. S2. CD spectra and fluorescence signal reduction
upon lac repressor binding.
Fig. S3. The physical characteristics of the candidate
molecules.
Table S1. Characteristics of selected compounds.
2636 FEBS Letters 593 (2019) 2628–2636 ª 2019 Federation of European Biochemical Societies
Searching for a new Z-DNA inducer with SYBR green J. H. Hur et al.