Development of a light-up fluorescent probe for HRAS G-quadruplex DNA

Article abstract of DOI:10.1016/j.tetlet.2017.07.032

The development of small-molecule G-quadruplex DNA probes has attracted significant attention in recent years. However, G-quadruplexes can display a wide variety of topologies, which process different structures and functions. Therefore, selective discrimination one G-quadruplex structure over another is promising. Herein, we reported the design, synthesis and biological evaluation of a long-chain fatty amine functionalized triphenylamine-quinolinium conjugate 1b. Significant enhancement of the fluorescence intensity (over 180 fold) was observed when 1b bound with HRAS G-quadruplex DNA, while much weaker enhancements were presented in the presence of other G-quadruplexes (45–90-fold) and single/double-stranded DNAs (less than 20-fold), indicating 1b had an excellent selectivity to HRAS. The details of the interactions were investigated by UV–Vis, FID and CD analysis. The results show 1b could interact and stabilize HRAS structure mainly by π-π stacking binding mode. The introduced amine chain of the structure core was found to be better in the terms of inducing selectivity toward G-quadruplex structure. In addition, the application of 1b as a fluorescent agent for living cell imaging was also demonstrated.

Full text of DOI:10.1016/j.tetlet.2017.07.032

Accepted Manuscript
Development of a light-up fluorescent probe for HRAS G-quadruplex DNA
Ming-Qi Wang, Zi-Yu Wang, Yi-Fan Yang, Gui-Ying Ren, Xiao-Ning Liu,
Shuo Li, Jian-Wei Wei, Lan Zhang
PII:
S0040-4039(17)30878-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.07.032
TETL 49114
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
26 May 2017
3 July 2017
8 July 2017
Please cite this article as: Wang, M-Q., Wang, Z-Y., Yang, Y-F., Ren, G-Y., Liu, X-N., Li, S., Wei, J-W., Zhang,
L., Development of a light-up fluorescent probe for HRAS G-quadruplex DNA, Tetrahedron Letters (2017), doi:
http://dx.doi.org/10.1016/j.tetlet.2017.07.032
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Development of a light-up fluorescent probe for HRAS G-quadruplex DNA
a,b
Ming-Qi Wang , Zi-Yu Wang , Yi-Fan Yang , Gui-Ying Ren , Xiao-Ning Liu , Shuo Li , Jian-Wei Wei , Lan Zhang
a
a
a
a
c,*
c
a
a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China
b
School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, P. R. China
c
College of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China
Abstract: The development of small-molecule G-quadruplex DNA probes has attracted significant attention in recent
years. However, G-quadruplexes can display a wide variety of topologies, which process different structures and functions.
Therefore, selective discrimination one G-quadruplex structure over another is promising. Herein, we reported the design,
synthesis and biological evaluation of a long-chain fatty amine functionalized triphenylamine-quinolinium conjugate 1b.
Significant enhancement of the fluorescence intensity (over 180 fold) was observed when 1b bound with HRAS
G-qudruplex DNA, while much weaker enhancements were presented in the presence of other G-quadruplexes (45-90 fold)
and single/double-stranded DNAs (less than 20 fold), indicating 1b had an excellent selectivity to HRAS. The details of the
interactions were investigated by UV-Vis, FID and CD analysis. The results show 1b could interact and stabilize HRAS
structure mainly by π-π stacking binding mode. The introduced amine chain of the structure core was found to be better in
the terms of inducing selectivity toward G-quadruplex structure. In addition, the application of 1b as a fluorescent agent for
living cell imaging was also demonstrated.
Keywords: G-quadruplex DNA, HRAS, fluorescent probe, living cell imaging
G-quadruplex DNAs are non-canonical secondary DNA structures formed by self-assembly of guanine-rich nucleic acid
sequences, the basic unit of which is the G-quartet, a planar motif generated from four guanine residues pairing together
1
with Hoogsteen hydrogen bonds . Depending on the direction of the strands or parts of a strand that form the G-quartet,
-3
4
the G-quadruplex structures display a wide range of topologies, such as parallel, anti-parallel and hybrid . These unique
5
,6
structures predominantly exist in the chromosomal telomeric sequences and the promoter regions of numerous genes . In
the recent years, G-quadruplex DNAs have drawn the immense attention of researchers because their structural features are
believed to be able to provide biological significance in gene regulation, replication and chromosomal stability, which are
7-9
highly associated with human diseases . Even though G-quadruplex DNAs have been studied for more than 20 years, the
exact nature of their biological significance is still poorly understood. Therefore, the surging interest in the area of
10,11
G-quadruplex research is calling for the development of specific quadruplex-targeting probe systems . Up to now, many
G-quadruplex probes have been designed and synthesized with high binding affinity and specificity toward the
1
2-18
G-quadruplex over genomic duplex DNA
. But few probes could specifically recognize G-quadruplexes with specific
. It is a challenging task to distinguish one G-quadruplex structure over another.
One important G-rich forming sequence is found in the ras gene family, present in the promoter of the HRAS oncogene,
1
9-21
structures
2
which is located immediately upstream of the transcription start site and form stable G-quadruplex structure . The HRAS
2

oncogene is reported to be associated with transcription regulation, and involve in the tumorigenesis of urinary bladder
2
cancer . Given the crucial role of HRAS oncogene in the cancer, one attractive therapeutic strategy could be to target the
3
G-quadruplex found in the region of the HRAS oncogene by using small molecules. Several classes of small-molecule
2
4, 25
probes have been reported to bind and stabilize HRAS G-quadruplex structure
. However, their selectivity for HRAS
G-quadruplex with a specific structure is not satisfactory. Therefore, the development of new chemical molecules, which
can selectivity recognize HRAS G-quadruplex structure, would provide a promising avenue in anticancer therapeutics.
Recently, our group has reported that triphenylamine-quinolinium derivatives can selectively interact with
G-quadruplexes, and the introduction of amine side chain would increase the binding affinities and selectivity of derivatives
2
6
for G-quadruplex DNA . For example, the derivative 1a with a 2-(dimethylamino)ethyl amino group can discriminate
G-quadruplex from duplex DNA (Figure 1). However, 1a has some limitations in analytical applications, such as low
selectivity toward a special type of G-quadruplex structure. In order to enhance the selectivity toward specific G-quadruplex
structure, in the present study, we designed a new triphenylamine-quinolinium dye 1b aiming to optimize its selectivity
through introducing different amine side chain (Figure 1). Its selectivity, sensitivity, binding performance and potential
application in living cells were investigated. This dye is an excellent targeting fluorescence probe for HRAS G-quadruplex
DNA.
Figure 1. The molecular structures of 1a and 1b.
The triphenylamine-quinolinium derivative, 1b, was synthesized following the procedure reported previously by our
2
group . The synthesis procedure is outlined in Scheme S1. The chemical structure of 1b was confirmed by H NMR,
6
1
13
C
NMR and MS spectra (See the Supporting information).
First, we investigated the changes in fluorescence of 1b in the presence of a variety of DNA forms. The excitation spectra
of 1b (2 µM) was recorded at 470 nm and the representative titrations with G-quadruplexes, duplex, and single-stranded
DNAs are shown in Figure 2A and Fig S1. Almost no fluorescence emission of 1b alone was observed in buffer solution. In
most cases, the fluorescence intensity of 1b was increased upon addition of G-quadruple DNAs including HRAS, 22AG,
Ckit1, Ckit3, CM22, Htg-21, C-myc, G3T3 and Src1. Very strong fluorescence was observed in the presence of HRAS DNA
(
~180-fold at saturation). Other G-quadruplex structures induced weaker enhancement of the fluorescence (45-90-fold at
saturation). In contrast, the addition of duplex (ctDNA, d(A-T) , d(G-C) , ds26) or single-stranded DNA (ss26) under the
9
9
same conditions resulted in a much smaller fluorescence enhancement (less than 20-fold). The degree of the fluorescence
enhancement revealed strong differences among various DNA forms, and these result indicated that 1b can be used as a

light-up fluorescence probe with good selectivity toward HRAS over other G-quadruplexes, duplex and single-stranded
DNAs (Figure 2B).
Fluorescence titration was carried out to study the response of 1b to HRAS G-quadruplex DNA. As Shown in Figure 2C,
with the addition of HRAS, the fluorescence intensity at approximately 595 nm was gradually enhanced. It can be seen that
2
there is a good linear relationship between the HRAS concentration (0-0.2 µM) and the fluorescence intensity (R = 0.998,
Figure 2D). Thus the sensitivity of 1b as a light-up HRAS probe was investigated and the limit of detection (LOD) was
2
7
estimated to be 1.12 nM according to the 3σ criteria (Experimental, see the Supporting information)
.
In order to examine whether the large fluorescence change of 1b was due to its interactions with G-quadruplex secondary
structures or simply just G-rich sequences, we then mixed HRAS and its complementary sequence in a ratio of 1:1 to form a
G-rich duplex structure. In contrast with the HRAS G-quadruplex, the G-rich duplex structure gave much lower fluorescence,
which demonstrated that 1b indeed interacts with G-quadruplex secondary structures instead of simple G-rich sequences
(
Fig. S2).
Furthermore, the binding affinity and stoichiometry between 1b and HRAS G-quadruplex were further fitted to an
independent-site model (eqn. (1), Experimental). As shown in Fig.S3, the titration date of HRAS could be well fitted by a
2
:1 (dye/DNA) binding mode (R = 0.997). The results were further confirmed by Job plot experiment (Fig. S4). The
9
7
-1
a
apparent binding constant (K ) between 1b and HRAS was derived to be 3.59 × 10 M .
Figure 2. (A) Fluorescence spectra of 1b (2 µM) in the absence/presence of different DNA forms (0.8 µM) in 10 mM Tris-HCl buffer (60 mM
KCl, pH 7.4). (B) Fluorescence titration curves and presence of different DNAs in buffer. (C) Fluorescence spectra of 1b (2 µM) upon titration
with prefolded HRAS G-quadruplex DNA in buffer. [HRAS]/µM: 0-0.4; (D) Linear plot of fluorescence intensity of 1b (2 µM) versus the
concentration (0−0.2 µM) of HRAS DNA.
G-quadruplex structures exist in the ocean of duplex DNAs, thus the competitive titrations were performed to further

confirm the selective fluorescence response of 1b binding to HRAS G-quadruplex DNA by adding HRAS to solution of 1b
containing excess of competing duplex DNA. As indicated from the screening results shown in Figure 3, we found that
duplex DNA (ctDNA/ds26, 20 µM) also slightly increased the fluorescence intensity of 1b, however, the gradual addition of
HRAS (0-0.4 µM) led to a much higher enhancement of the fluorescence, demonstrating the displacement of 1b from duplex
DNA and the formation of highly fluorescence complexes with HRAS. Collectively, the results showed that the promising
potential of 1b to serve as a HRAS probe even in a competitive environment.
Figure 3. Fluorescence titration of 2 µM 1b with the stepwise addition of HRAS DNAwithout and with 20 µM ctDNA or ds26 in 10 mM
Tris-HCl buffer and 60 mM KCl at pH of 7.4.
The dye 1b exhibited significant selectivity for G-quadruplexes, especially for HRAS. It is therefore interesting to
understand the details of their interactions. UV-Vis spectroscopy is an excellent technique for studying the extent of
molecule interaction with DNA. On this basis, we investigated the interaction of 1b with G-quadruplex DNAs using
absorption titrations. In buffer solution, 1b displayed its characteristic absorption profile with a maximum at 420 nm as
shown in Figure 4. Gradual addition of HRAS to 1b solution shifted the absorption band bathochromically to 452 nm (~32
nm shift) with a isosbestic point at 452 nm, pointing to strong interaction of 1b with HRAS DNA. Generally, the stacking
interaction between molecule and the base pairs of DNA would lead to bathochromic shifts and hypochromicity on the
2
8
electronic absorption spectra . It suggested that the quinolinium scaffold of 1b probably formed π-π stacking interaction
with guanine tetrads of HRAS DNA. Furthermore, we investigated the interactions between 1b and other G-quadruplexes.
Addition of these DNAs also led to significant hypochromisms and red shifts, which meant 1b could bind with these
G-quadruplex DNAs mainly through stacking interaction.
Figure 4. Electronic absorption spectra of 1b (8 µM) upon addition of HRAS. [HRAS] = 0-1 µM from top to bottom. Arrows indicate the change

in absorbance upon increasing the HRAS concentrations.
To gain further insight into the interaction between 1b and HRAS, the dye affinity for HRAS was tested by an indirect FID
(
(
fluorescence intercalator displacement) approach. This approach belongs to the family of the indicator-displacement assays
2
IDAs) , which is based on the competitive displacement of fluorescence probe TO (thiazole organe). TO is highly
9
fluorescent upon interaction with G-quadruplex DNA mainly by an end-stacking mode (~500- to 3000-fold exaltation)
whereas totally quenched when free in solution. Efficiency TO displacement would thus be an indication of an end-quarter
binding of our dye. For comparison, 1a with a shorter amine side chain was also studied. We performed the fluorimetric
titrations of TO-HRAS system with 1a and 1b in 10 mM Tris-HCl buffer, pH 7.4 containing 100 mM KCl and obtained
spectra were shown in Figure 5. The addition of 1a and 1b to HRAS pretreated with TO caused appreciable reduction in the
fluorescence intensity. Binding curves resulted from plotting fluorescence intensity of TO-HRAS versus dye concentration
were shown in Figure 5 (Insets) and subsequent Stern-Volmer analysis of fluorescence date gave the binding constants of
6
a (8.26 × 10 M ) and 1b (3.34 × 10 M ). As the TO interacts with G-quadruplex via end-stacking mode, these results
-1
6
-1
1
implied that there were stable end-stacking interactions between HRAS and the dyes and the lower Ksv value of 1b suggests
its weaker binding strength to HRAS than that of 1a. On the other hand, this study also revealed that amine side chain would
regulate the binding affinity and selectivity for G-quadruplex. The long amine chain1b had a lower binding affinity but
higher selectivity toward HRAS than the short one 1a. The exact mechanism of how this amine chain contributes to its
activity is unclear. However, the amine chains may dock onto the groove of G-quadruplex DNA and interact with the
3
G-quadruplex DNA by forming intermolecular hydrogen bond or electrostatic interaction . Dye 1b with longer length
0
amine chain could preferentially bind to HRAS structure form.
Figure 5. Fluorescence spectra recorded in FID experiment for 1a and 1b. Experimental conditions: [TO] = 1 µM, [HRAS] = 0.2 µM, [1a] or
[
0
1b] = 0-1.2 µM. Arrows indicate increasing concentration of dye. Inset: plot of F /F as a function of concentrations of dye 1a or 1b. λex = 504
nm.
The cation and quadruplex interaction is based primarily on ions which are located at the central channel formed due to
the quartet arrangement. The ions contribute to charge screening, interaction with the loops and the grooves, thus playing an
3
1
important role in G-quadruplex structure formation and their stability . To investigate the interactions between 1b and

HRAS G-quadruplex, we studied the effect of buffer cationic strength on the binding process. Fluorescence titrations were
carried out in different buffers with variation in potassium ion concentrations. As shown in Figure 6, the enhancement in
the fluorescence intensity of 1b with HRAS was found to show a slight decrease with increasing potassium ion concentration
of 20, 40, 60, and 100 mM. It demonstrates that as the cationic ion increases, it occupies the available binding sites on
G-quadruplex surface and reduces the possibility for molecule to bind exterior of G-quadruplex. And if the molecule mainly
3
2
shows a stacking mode of binding, the change is comparatively less . Thus, this indicated the fact that 1b mainly
underwent stacking binding mode with HRAS due to presence of quinolinium unit which may interact with G-quartet plane
by π-π stacking.
Figure 6. Change in fluorescence intensity of the 1b (2 µM) with increasing concentrations of HRAS at different ionic strengths of buffer
medium.
The effect on the binding and conformation of HRAS was investigated by recording the circular dichroism (CD) spectra
in the absence and presence of 1b. The CD spectrum of HRAS structure alone was of the typical antiparallel G-quadruplex,
4
with the positive peak at about 290 nm and a negative peak at about 250 nm .Gradual addition of 1b to HRAS solution only
made the characteristic CD peaks increased. This indicated the dye 1b did not cause significant conformation transition of
G-quadruplex structure (Figure 7). However, the remarkable enhancement of the positive peak attribute to the stabilization
of HRAS. Also, the results suggested that 1b was able to bind to HRAS, which was consistent with the observation of
fluorescent signal enhancements from the titration experiments.
Figure 7. CD spectra recorded for HRAS DNA(5 µM) solution, in 60 mM Tris-HCl buffer (60 mM KCl, pH 7.4) with 1b: (1) 0 equiv; (2) 0.5
equiv; (3) 1 equiv; (4) 2 equiv.

Besides possessing high affinity and selectivity for G-quadruplex DNA in vitro, probes should also possess ideal
3
3
biocompatibility and membrane permeability . Encouraged by the above findings, we further investigated the cellular
uptake and localization of 1b in live cells by using confocal laser scanning microscopy. As shown in Figure 8, confocal
imaging using Hep-G2 cell lines revealed that 1b could enter living cells. The co-staining with DAPI (a duplex DNA binder)
revealed that 1b gave red emission signals in the nucleus and mainly located in nucleoli. It is reported that this region
correspond to the nucleoli where the rDNA (ribosomal DNA) undergoes transcription. G-rich rDNA can also form a
3
4
G-quadruplex structure temporarily . Though the detail of whether the enhanced fluorescence signals of 1b was due to the
interaction with G-quadruplex is not clear, the staining images demonstrated that 1b was suitable for living cells
applications on the monitoring of nucleus activities.
Figure 8. Confocal fluorescence images of live Hep-G2 cells stained with 10 µM 1b (A) for 30 min and (5 mg/mL) DAPI (B) for 20 min. Scale
bar is 20 µm.
In summary, we have successfully developed a light-up fluorescent probe 1b for the detection of HRAS G-quadruplex.
This probe exhibited a high selectivity toward HRAS against other G-quadruplexes, duplex and single-stranded DNAs with
a detection limit of 1.12 nM. 1b binds to HRAS tightly via a stable π-π stacking interaction and could efficiently stabilize the
G-quadruplex structure. The results also hint that the introduced long-chain fatty amine side chain in the scaffold of 1b was
found to show high binding affinity and selectivity to a specific HRAS G-quadruplex structure. Considering the remarkable
properties of this probe, 1b also showed great potential in sensing intracellular G-quadruplexes, and further detailed
investigations on the intracellular and in vivo applications of 1b are now underway.
Authors Informations
*
Corresponding author.
E-mail addresses: wmq3415@163.com
Acknowledgements
This work was financially supported by the China Postdoctoral Science Foundation Funded Project (Grant No.
2
017M611704), Senior Talent Cultivation Program of Jiangsu University (Nos. 15JDG068). This research was also
supported by the Scientific and Technical Research Program for Chongqing Education Commission (KJ1600916,

KJ1709199).
Supplementary date
Supplementary date related to this article can be found at XXXXXX.
References and notes
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Highlights：
A light-up fluorescent probe 1b for HRAS G-quadruplex DNA was developed.
1
b could selective discrimination HRAS G-quadruplex structure over other DNA forms.
The detail interactions of BASQ with HRAS were invstigated.
The cellular application in living cells was demonstrated.