Fluorescent bioprobes: Structural matching in the docking processes of aggregation-induced emission fluorogens on DNA surfaces

Article abstract of DOI:10.1002/chem.200900778

Whereas most conventional DNA probes are flat disklike aromatic molecules, we explored the possibility of developing quadruplex sensors with nonplanar conformations, in particular, the propeller-shaped tetraphenylethene (TPE) salts with aggregation-induced emission (AIE) characteristics. 1,1,2,2-Tetrakis[4-(2- triethylammonioethoxy)-phenyljethene tetrabromide (TPE-1) was found to show a specific affinity to a particular quadruplex structure formed by a human telomeric DNA strand in the presence of K⁺ ions, as indicated by the enhanced and bathochromically shifted emission of the AIE fluorogen. Steady-state and time-resolved spectral analyses revealed that the specific binding stems from a structural matching between the AIE fluorogen and the DNA strand in the folding process. Computational modeling suggests that the AIE molecule docks on the grooves of the quadruplex surface with the aid of electrostatic attraction. The binding preference of TPE-1 enables it to serve as a bioprobe for direct monitoring of cation-driven conformational transitions between the quadruplexes of various conformations, a job unachievable by the traditional G-quadruplex biosensors. Methyl thiazolyl tetrazolium (MTT) assays reveal that TPE-1 is cytocompatible, posing no toxicity to living cells.

Full text of DOI:10.1002/chem.200900778

DOI: 10.1002/chem.200900778
Fluorescent Bioprobes: Structural Matching in the Docking Processes of
Aggregation-Induced Emission Fluorogens on DNA Surfaces
Yuning Hong,[a] Hao Xiong,[a] Jacky Wing Yip Lam,[a] Matthias Hꢀußler,[a]
Jianzhao Liu,[a] Yong Yu,[a] Yongchun Zhong,[b] Herman H. Y. Sung,[a] Ian D. Williams,[a]
Kam Sing Wong,[b] and Ben Zhong Tang*[a, c]
Abstract: Whereas most conventional
DNA probes are flat disklike aromatic
molecules, we explored the possibility
of developing quadruplex sensors with
nonplanar conformations, in particular,
the propeller-shaped tetraphenylethene
(TPE) salts with aggregation-induced
emission (AIE) characteristics. 1,1,2,2-
Tetrakis[4-(2-triethylammonioethoxy)-
phenyl]ethene tetrabromide (TPE-1)
was found to show a specific affinity to
indicated by the enhanced and batho-
chromically shifted emission of the
AIE fluorogen. Steady-state and time-
resolved spectral analyses revealed that
the specific binding stems from a struc-
tural matching between the AIE fluor-
ogen and the DNA strand in the fold-
ing process. Computational modeling
suggests that the AIE molecule docks
on the grooves of the quadruplex sur-
face with the aid of electrostatic attrac-
tion. The binding preference of TPE-1
enables it to serve as a bioprobe for
direct monitoring of cation-driven con-
formational transitions between the
quadruplexes of various conformations,
a job unachievable by the traditional
G-quadruplex biosensors. Methyl thia-
zolyl tetrazolium (MTT) assays reveal
that TPE-1 is cytocompatible, posing
no toxicity to living cells.
Keywords: aggregation-induced
a
particular quadruplex structure
emission
· biosensors · DNA ·
formed by a human telomeric DNA
fluorescent probes · G-quadruplexes
strand in the presence of K+ ions, as
Introduction
rich in guanine (G) base units can adopt a noncanonical
structure referred to as a G-quadruplex, a four-stranded
structure built from the stacking of several G·G·G·G tetrads
and stabilized by cationic species (e.g., K+ and Na+).[5] The
G-quadruplex structures have attracted much attention in
recent decades because of their presence in the genomic re-
gions of particular telomeres and their therapeutic applica-
tions as targets for anticancer drugs.[6,7] The surging interest
in the area of G-quadruplex research is calling for the devel-
opment of specific quadruplex-targeting probe systems.[8]
Analyses of G-quadruplex structures by X-ray crystallog-
raphy as well as NMR spectroscopy have revealed a diversi-
ty of topological conformations not seen in other DNA sys-
tems.[9] Depending on the length and sequence of a DNA
strand, a G-quadruplex structure can be tetra-, bi-, or unim-
olecular. From the orientation of the strands or the parts of
a strand that form G-tetrads, quadruplex structures can be
further divided into parallel, antiparallel, and mixed
types.[10,11] Scheme 1a depicts some examples of intramolecu-
lar G-quadruplex structures.
Fluorescent biosensors that can selectively identify biologi-
cally important macromolecules (e.g., DNA) are powerful
analytical tools for studying biological events.[1] A large vari-
ety of fluorescent dyes, such as ethidium bromide, SYBR
Green I, and YOYO, have been developed for specific tar-
geting of double-stranded DNA.[2–4] A single-stranded DNA
[a] Y. Hong, H. Xiong, Dr. J. W. Y. Lam, Dr. M. Hꢀußler, J. Liu, Y. Yu,
Dr. H. H. Y. Sung, Prof. I. D. Williams, Prof. B. Z. Tang
Department of Chemistry, Nano Science and Technology Program
The Hong Kong University of Science & Technology
Clear Water Bay, Kowloon, Hong Kong (P.R. China)
Fax : (+852)23581594
E-mail: tangbenz@ust.hk
[b] Dr. Y. Zhong, Prof. K. S. Wong
Department of Physics
The Hong Kong University of Science & Technology
Clear Water Bay, Kowloon, Hong Kong (P.R. China)
[c] Prof. B. Z. Tang
Department of Polymer Science and Engineering
Zhejiang University, Hangzhou 310027 (China)
A G-quadruplex offers multiple binding sites. A general
strategy for G-quadruplex targeting is to design a probe
molecule consisting of a large planar aromatic core prone to
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900778.
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FULL PAPER
Scheme 1. a) Representative unimolecular G-quadruplex structures:
I) propeller: a parallel structure with three double-chain reversal loops;
II) basket: an antiparallel structure with adjacent parallel strands and a
diagonal loop; III) hybrid-1: a mixed structure with three parallel and
one antiparallel strands; and IV) hybrid-2: a mixed structure with one an-
tiparallel and three parallel strands. b) Abbreviations and sequence of
the oligonucleotide strands used in this work.
p–p stacking with the G-tetrad platform and several cationic
side arms that facilitate the electrostatic interactions with
the anionic DNA strands.[12] Much effort has been devoted
to the search for fluoroescent ligands that can differentiate
quadruplex from duplex. Through structural tuning, biosen-
sors with high binding affinities to quadruplex over duplex
have been successfully developed.[8,13] The current challenge
is to develop new fluorescence probes with specificity for
particular individual quadruplex structures amongst the mul-
titude of potential quadruplex-forming sequences.[14]
Recently, we prepared a water-soluble fluorogen named
1,1,2,2-tetrakis[4-(2-triethylammonioethoxy)phenyl]ethene
tetrabromide (TPE-1).[15] This fluorogen is nonemissive
when dissolved but becomes highly emissive when aggregat-
ed due to the restriction to its intramolecular rotations by
the aggregate formation, thereby showing a novel effect of
aggregation-induced emission (AIE).[16] The AIE behavior is
“abnormal” because chromophore aggregation commonly
quenches light emission of a fluorophore and decreases its
efficiency of DNA detection in the traditional intercalating-
type biosensor systems (e.g., cyanine dyes).[17]
The extraordinary AIE effect of TPE-1 inspired us to uti-
lize it as a DNA probe. In our previous work, we found that
in an aqueous buffer, the emission of TPE-1 was turned on
when it was docked on the surfaces of DNA folding struc-
tures through electrostatic interaction, because the docking
process impeded its intramolecular rotations, which in turn
blocked the nonradiative channels and populated the radia-
tive relaxations of its excitons.[15,18] The strong interaction of
TPE-1 with DNA enabled rapid visualization of the DNA
bands in the poly(acrylamide) gel electrophoresis assays.[15]
The AIE probe discriminated the G-quadruplex structure
structures. The differentiation was signified by a redshift of
approximately 20 nm in the emission spectrum of TPE-1,
which permitted visual recognition and distinguishability of
the G-quadruplex from other DNA structures.[15]
In this work, we continued our study in this area of re-
search. We systematically designed and synthesized a series
of TPE derivatives with different lengths of side arms (TPE-
2) and alkyl units of the ammonium groups (TPE-3), and
with different numbers of side arms (TPE-4). The TPE de-
rivatives were mixed with the congeners of HG21 with vary-
ing sequences (Scheme 1b) in an effort to elucidate the
structural effects on the binding of the fluorogens with the
G-quadruplexes. Steady-state and time-resolved fluores-
cence studies revealed a high affinity of TPE-1 to a particu-
lar G-quadruplex structure formed by HG21, which is indi-
cative of the involvement of specific structural matching in
the binding process. A docking simulation study suggested
that the TPE-1 molecule prefers to bind to the grooves on
the quadruplex surface with the aid of electrostatic attrac-
tion. The specificity of the TPE-1 dye to the HG21/K+
quadruplex is utilized to monitor the conformational trans-
formations induced by cationic titrations.
Results and Discussion
Effect of DNA sequence: The oligonucleotides used in this
study are all G-rich DNA strands purchased from Invitro-
gen. PG12 is an oligonucleotide comprising solely of gua-
nine base units (dG12), whereas Ap15 is a thrombin-binding
formed by d[G
3ACHTUNGTRENNUNG(T2AG3)3] or HG21 (Scheme 1b), a mimic of
human telomeric DNA, from the random coil and duplex
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1233

B. Z. Tang et al.
aptamer
d[G2T2G2TGTG2T2G2]. Ox28 and CM22 are mimics of the
oxytricha telomeric repeat (d[G4A(T4G4)3]) and the G-quadru-
plex element in human c-myc promoter (d[TGA(G3T)2A-
(G3T)2A2]), respectively. The HG series mimic the human
telomeric sequences with a repeat unit of T2AG3. HG26 is
similar to that of the wild-type human telomeric sequence
and was chosen because of its well-solved, NMR-refined
structure in the presence of K+.[19]
The TPE derivatives were synthesized in our laboratories.
TPE-1 and TPE-4 were prepared by following our previous-
ly published experimental procedures.[15,20] TPE-2 and TPE-
3 were prepared according to the synthetic routes shown in
Scheme S1 (in the Supporting Information). All the reaction
intermediates and final products were characterized by stan-
dard spectroscopic methods, from which satisfactory analysis
data were obtained (see the Supporting Information for de-
tails).
We first studied photoluminescence (PL) behaviors of
TPE-1 in the presence of the G-rich DNA strands and K+
ions. The buffer solution of TPE-1 gives weak PL signals
(Figure 1), which indicate that TPE-1 is soluble in the aque-
with
a
nucleobase
sequence
of
(lex) is also observed at a longer wavelength (Supporting In-
formation, Figure S1).
C
It is known that all the DNA strands are capable of fold-
ing into G-quadruplex structures in the presence of K+ ions
but what is the cause for the different fluorescence behav-
iors of TPE-1? Analysis by circular dichroism (CD) spec-
troscopy offers some clues. The Ap15/K+ complex exhibits
positive and negative Cotton effects at 295 and 265 nm, re-
spectively, indicative of the formation of an intramolecular
antiparallel G-quadruplex structure (Supporting Informa-
tion, Figure S1). The PG12/K+ and CM22/K+ complexes
give positive CD bands at 265 nm and negative ones at
240 nm, typical of inter- and intramolecular parallel quadru-
plex structures.[21] The spectra of HG21/K+ and Ox28/K+
are similar (positive CD bands at 295 nm with shoulders at
265 nm), which suggests that they take similar conforma-
tions in the presence of K+ ions. The photographs shown in
Figure 1b manifest the differences in the interactions of the
AIE fluorogen with the quadruplex structures formed by
the different G-rich DNA strands. The intense light emission
of the TPE-1/HG21/K+ complex allows one to differentiate
the quadruplex structure formed by the human telomeric se-
quence from the other quadruplex structures with the naked
eye.
A
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
Effect of the flanking sequence: The remarkable enhance-
ment in the PL of TPE-1 suggests that the fluorogen has a
high binding affinity to the quadruplex structure formed by
human telomeric sequences. The orientations of the DNA
strands are geometrically governed by the glycosidic confor-
mations of the G units, which determine the morphologies
of the G-tetrad cores.[22] The polymorphism of the G-tetrad
cores arising from the different DNA sequences may ac-
count for the different association behaviors between the
TPE fluorogen and G-quadruplexes. Then what happens if
the DNA sequences are assembled into the same G-tetrad
structure? The human telomeric sequences (or HG series)
with different flanking sequences were thus scrutinized be-
cause of their identical G-quadruplex-forming motif. HG21
is a human telomeric sequence without any flanking units at
either the 5’- or 3’-end, HG22–HG24 are truncated DNA se-
quences with either 5’- or 3’-flanking units, and HG25 and
HG26 are capped with both 5’- and 3’-flanking units.
Among the AIE fluorogen/G-quadruplex complexes, the
TPE-1/HG21/K+ system displays the largest emission en-
hancement and bathochromic shift (Figure 2a). The changes
in the lem value and PL intensity against the HG sequences
are plotted in Figure 2b. The emission spectrum of TPE-1/
HG22/K+ peaks at 455 nm, which is blueshifted approxi-
mately 40 nm from that of TPE-1/HG21/K+. The lem values
of the TPE complexes with HG23/K+–HG26/K+ are ap-
proximately 460–470 nm, which is identical to those in the
absence of K+ ions. None of these complexes show emis-
sions stronger than that of TPE-1/HG21/K+.
Figure 1. a) Photoluminescence spectra of buffer solutions of TPE-1 in
the presence of K+ ions and G-quadruplexes with different conforma-
tions. [TPE-1]=4.5 mm, [DNA]=9 mm, [K+]=0.5m; lex =350 nm. b) Pho-
tographs of TPE-1 solutions in the presence of different G-quadruplexes
taken under UV illumination (365 nm).
ous medium. Addition of Ap15, PG12, Ox28, CM22, and
HG21 intensifies the PL signals by 2, 5, 7, 10, 12, and 45
times, respectively. The spectrum of the TPE-1/HG21/K+
complex shows an emission maximum (lem) of 492 nm,
which is redshifted by approximately 20 nm from those of
other DNA systems. Consistently, its excitation maximum
The CD spectra of HG21/K+–HG26/K+ show analogous
patterns, which suggests that all of them adopt a similar G-
quadruplex topology with indistinguishable strand orienta-
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Fluorescent Bioprobes
FULL PAPER
cationic TPE-1 molecule, which hampers its binding to the
G-quadruplex and hence weakens its emission.
Evidently, TPE-1 interacts strongly with HG21/K+ but
weakly with HG21/Na+, showing a binding specificity to the
K+-induced and -stabilized quadruplex of HG21. In other
words, TPE-1 has a strong affinity to a specific secondary
folding structure of the DNA, rather than its primary strand
sequence. Clearly, structure matching between HG21/K+
and TPE-1 is the cause for the strong binding preference
and unique fluorescence behavior. Although HG26 can
assume different G-quadruplex structures in the K+ and
Na+ buffers, the PL spectra of the complexes with TPE-1
are similar in profile (curve shape and peak position), which
is suggestive of similar binding behaviors (Supporting Infor-
mation, Figure S3).
Cation-driven conformational switching: By taking advant-
age of the spectral differentiation between the K+- and Na+
-driven quadruplex formation, we explored the possibility of
modulating DNA folding structures through cationic titra-
tion. Addition of a small amount of Na+ ions (0.1m) does
not completely quench the emission of TPE-1/HG21 at ap-
proximately 470 nm. Figure 3a shows the spectral change of
Figure 2. a) Photoluminescence spectra of TPE-1/HG complexes in the
presence of K+ ions in Tris-HCl buffer solutions. b) Changes in the emis-
sion maximum (lem) and peak intensity of TPE-1 with the variations in
the sequences of the HG DNA strands. [TPE-1]=4.5 mm, [DNA]=9 mm,
[K+]=0.5m; lex =350 nm.
tion and G-tetrad conformation (Supporting Information,
Figure S2). The flanking sequences at the 5’- and/or 3’-ends
of the DNA strands are critical for accommodation of the
loops, which lead to distinct groove dimensions.[22] The dif-
ferent PL profiles of TPE-1 in the presence of HG21/K+
suggest that the loop/groove on the G-quadruplex periphery,
rather than the G-tetrad core, plays an important role in the
accommodation of the AIE fluorogen.
Effect of cationic species: It is known that cationic species
play an important role in determining the structures of G-
quadruplexes.[23] K+ and Na+ ions can promote the forma-
tion of different quadruplex structures from DNA strands
with same G-rich sequences. Whereas the HG21/K+ com-
plex induces the TPE fluorogen to emit strongly at 492 nm,
faint PL is detected in the HG21/Na+ system (Supporting
Information, Figure S3a). The PL intensity is similar to the
isolated species of TPE-1, which indicates that almost no
fluorogenic molecules have bound to the HG21/Na+ com-
plex. The Na+ ions in the buffer solution compete with the
Figure 3. Changes in the emission spectra of buffer solutions of a) TPE-1/
HG21/Na+ ([Na+]=0.1m) and b) TPE-1/HG21/K+ ([K+]=0.1m) upon
titrations with a) K+ and b) Na+ ions. [TPE-1]=4.5 mm, [HG21]=4.5 mm;
lex =350 nm.
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1235

B. Z. Tang et al.
TPE-1/HG21/Na+ with K+ titration. The lem value progres-
sively shifts to the longer wavelength region, accompanied
with an increase in the emission intensity. Similar to the
main peak, the shoulder at approximately 400 nm is also en-
hanced and evolves into new peaks in the shorter wave-
length region.
conformational transitions between different quadruplex
structures. Further study of the binding modes and mecha-
nisms may help in the understanding of the kinetic processes
and folding pathways.
Effect of fluorogen structure: The above results suggest that
the unique green emission of TPE-1 arises from a specific
topology of the HG21 strand in the presence of K+ ions. Al-
ternation in the HG sequences dramatically affects the emis-
sion in the longer wavelength region, indicative of geometric
structure matching in the binding process of TPE-1 with the
quadruplex. To examine whether the molecular structure of
the AIE fluorogen is also crucial in the binding event, we
prepared a series of TPE derivatives with systematically
varied molecular structures.
Derivative TPE-2 contains longer alkyl chains in its side
arms than TPE-1. Unlike TPE-1, the dilute buffer solution
of TPE-2 shows a relatively strong PL at 475 nm (Fig-
ure 4b). The PL is boosted by the addition of HG21. TPE-2
contains a larger hydrophobic unit and its molecules may
form micellar aggregates in the aqueous buffer, thereby
emitting strongly upon photoexcitation. When bound to the
DNA strands, its intramolecular rotations are largely re-
stricted, thus leading to more efficient emission. Interesting-
ly, upon addition of K+ ions, its PL is weakened. TPE-2
may become better dissolved in the solution containing K+
ions and the disaggregation gives rise to the decrease in the
PL of the AIE fluorogen.[25]
Evidently, the alkyl chain lengths in the side arms of the
AIE fluorogen are important in its binding process to the
G-quadruplex. How about the ammonium chelating units?
To answer this question, we replaced the triethylammonium
groups in TPE-1 with less bulky trimethylammonium groups
in TPE-3. TPE-3 is even less emissive than TPE-1 in solu-
tion but its PL is greatly boosted when HG21 is added. Ad-
dition of K+ ions weakens the PL of TPE-3/HG21 but
brings little change to its spectral profile (Figure 4c). The
positively charged ammonium groups in TPE-3 are less ster-
ically shielded, which increases the solubility of the fluoro-
gen in the aqueous buffer and strengthens its interaction
with the negatively charged HG21 strand. When the K+
ions induce the DNA strand to fold into a G-quadruplex
structure, the TPE molecule is detached and driven into the
aqueous buffer, probably due to its unfavorable docking on
the G-quadruplex surface. The detachment results in the ob-
served PL quenching. Thus, besides the arm length, the mo-
lecular structure of the ammonium unit also plays a critical
role in the accommodation of the AIE fluorogen by the G-
quadruplex.
The peaks in the short-wavelength region may arise from
the TPE molecules bound to the quadruplex through one or
two ammonium chelating units. These AIE fluorogens can
undergo partial intramolecular rotations. Their twisted con-
formations decrease the extent of the electronic conjugation,
leading to the blueshifted emission. Because of the poor af-
finity of TPE-1 to HG21/Na+, most of the AIE molecules
may drift in the buffer solution and experience little interac-
tion with the G-quadruplex. Upon addition of K+ ions, the
Na+-driven quadruplex may gradually alter its conformation
to the K+-driven one, which attracts the TPE-1 molecule to
dock on its surface. After all the best binding sites are occu-
pied, the excess TPE-1 molecules may bind to the G-quad-
ruplex structure through a partial docking mode. The com-
petition between the sodium and ammonium ions may force
some of the bound TPE-1 molecules to release one or two
ammonium groups from the binding sites. Both of these two
events give rise to an increased blue emission at about
400 nm. The negative Cotton effect at 265 nm in the CD
spectrum of TPE-1/HG21/Na+ is weakened upon addition
of K+ and its sign is inverted at the high K+ concentrations,
which is indicative of a conformational transition from the
basket structure of HG21/Na+ to the mixed structure of
HG21/K+ (Supporting Information, Figure S4a).
In our previous work, we have demonstrated that TPE-1
can be used as a fluorescence probe to monitor the confor-
mational change of HG21/Na+ upon addition of K+ ions.
What will occur if the K+-stabilized quadruplex is titrated
with Na+ ions? As shown in Figure 3b, addition of Na+ ions
into the buffer solution of TPE-1/HG21/K+ steadily de-
creases the PL intensity. The Na+ ions drive the TPE-1 mol-
ecules chemisorbed on the quadruplex surfaces into the
aqueous buffer, leading to the observed PL attenuation. The
spectral profile, however, remains unchanged even at very
high Na+ concentrations, suggesting that despite the exis-
tence of large excess amount of Na+ ions in the buffer solu-
tion, the HG21/K+ quadruplex maintains its structural integ-
rity.
The G-quadruplex structure formed in the presence of K+
ions is undisturbed by the perturbation from the externally
added Na+ ions, as evidenced by the little variation in the
CD spectrum of the HG21/K+ complex (Supporting Infor-
mation, Figure S4b). The K+ ions preferentially stabilize the
G-quadruplex with folding structures different from those
formed in the presence of Na+ ions.[24] Only a low concen-
tration of K+ ions is required for the structural stabilization.
It is difficult for the Na+ ions to reverse the K+-stabilized
G-quadruplex structure due to their relatively weak induc-
tion power. These results indicate that the TPE-1 fluorogen
can readily reveal its transition through the change in its PL
signal, thus offering a convenient tool for monitoring the
TPE-4 has only two positively charged side arms. Its solu-
tion shows a dim PL, which is only slightly increased by
DNA addition (Figure 4d). Due to its lower density of posi-
tive charges, TPE-4 has a lower affinity to DNA strands and
thus it is difficult for it to emit efficiently in the presence of
HG21. TPE-4 has the same, but fewer side arms than its
congener TPE-1. It does not show the quadruplex-specific
wavelength when it is complexed with HG21/K+, similar to
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Fluorescent Bioprobes
FULL PAPER
tion, the intramolecular rota-
tions of the aryl rings around
the axes of the single bonds
linked to the central double
bond are active, which leads to
rapid annihilation of the TPE
excitons.
The fluorescence lifetime is
lengthened to nanoseconds
when the DNA is added to the
solution. The PL decay curve is
now better fitted by a double-
exponential function than the
single-exponential one. This in-
dicates that the TPE molecules
are now in two different envi-
ronments, with Ai denoting the
fraction of the molecules in
each environment. As shown in
Table 1, entry 2, upon addition
of HG21, 59% of the fluorogen
molecules decay slowly with a
lifetime of 2.30 ns, which is
>100-fold longer than that in
the solution without DNA. The
remaining 41% of the excited
fluorogen molecules decay in a
fast mode. The similar behav-
iors are observed when Ap15/
K+ and PG12/K+ are added
Figure 4. Photoluminescence spectra of HG21 complexes with a) TPE-1,[15] b) TPE-2, c) TPE-3, and d) TPE-4
in the presence and absence of K+ ions. [TPE]=4.5 mm; [DNA]=9 mm; [K+]=0.5m; lex =350 nm.
Table 1. Fluorescence decay parameters of dye/DNA complexes.
what was observed in the systems of TPE-2 and TPE-3.
These results indicate that the TPE-1 fluorogen possesses
the “right” structure for the G-quadruplex recognition.
[b]
[b]
Entry DNA/M+[a] Dye[a] A1
A2
t1 [ns][b] t2 [ns][b] <t> [ns][c]
1
2
3
4
5
6
7
8
–
TPE-1
1
–
0.02
–
0.02
1.52
0.43
1.62
1.06
1.35
3.26
1.46
1.18
1.35
1.99
1.63
0.55
1.68
0.76
0.62
1.40
HG21
TPE-1 0.41 0.59 0.42
TPE-1 0.65 0.35 0.02
TPE-1 0.63 0.37 0.11
TPE-1 0.33 0.67 0.02
TPE-1 0.21 0.79 0.02
TPE-1 0.25 0.75 0.32
TPE-1 0.37 0.63 0.19
TPE-1 0.55 0.45 0.13
TPE-1 0.28 0.72 0.02
TPE-1 0.40 0.60 0.02
TPE-1 0.35 0.65 0.17
2.30
1.19
4.20
1.57
1.71
4.23
2.21
2.47
1.86
3.32
2.41
1.97
2.75
1.75
2.44
1.81
Ap15/K+
PG12/K+
Ox28/K+
CM22/K+
HG21/K+
HG22/K+
HG23/K+
HG24/K+
HG25/K+
HG26/K+
Time-resolved fluorescence: Time-resolved PL spectra can
offer valuable information that is lost during the time-aver-
aging processes of the steady-state spectra measurements.
To collect information on the interactions of the fluorogens
in the excited states with the environment, their fluores-
cence lifetimes (t) were measured. The PL decay traces as
well as their fitting curves are shown in Figures S6–S9 (Sup-
porting Information), from which the dynamic parameters
were obtained and are summarized in Table 1.
In the absence of DNA, TPE-1 in the aqueous buffer
decays in a single-exponential way with a lifetime of 20 ps,
which is nearly the resolution limit of the streak camera
system (Table 1, entry 1). It is known that t can be ex-
pressed as 1/t=1/tr +1/tnr, in which tr and tnr denote the ra-
diative and nonradiative lifetimes, respectively. Here tr is
the intrinsic property of the fluorogen and hence a constant.
The short lifetime and low PL efficiency of TPE-1 in the
buffer solution suggests that an efficient nonradiative pro-
cess is involved in the relaxation of the excited states. It is
well known that vibrational and torsional motions can non-
radiatively deactivate the excited states. In the buffer solu-
9
10
11
12
13
14
15
16
17
HG21/Na+ TPE-1 0.73 0.27 0.02
HG26/Na+ TPE-1 0.43 0.57 0.23
HG21/K+
HG21/K+
HG21/K+
TPE-2 0.59 0.41 0.08
TPE-3 0.79 0.21 0.13
TPE-4 0.26 0.74 0.24
[a] [TPE]=4.5 mm; [DNA]=9 mm; [M+]=0.5m (M+ =K+, Na+). [b] De-
termined from Equation (8), in which A and t are the fractional amount
and fluorescence lifetime of shorter- (1) and longer- (2) lived species, re-
spectively. [c] Weighted mean lifetime determined from Equation (9).
into the TPE-1 solution (entries 3 and 4). In the systems of
Ox28/K+ and CM22/K+, however, the majority of the TPE
molecules decay by means of the slow relaxation channel
(entries 5 and 6).
Chem. Eur. J. 2010, 16, 1232 – 1245
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1237

B. Z. Tang et al.
To better understand the dynamic processes, we need to
interpret the origins of the two components. The fast decay
component has a lifetime similar to that in the pure solution
(in the absence of DNA). This component represents the
fraction of the free fluorogen molecules in the isolated state,
which relax nonradiatively and are thus associated with the
weak emission. The slow decay component, on the other
hand, is assignable to the bound fluorogen molecules. Our
previous mechanism investigation has proved that the re-
striction to the intramolecular rotations of the fluorogen
molecules is the main cause for the AIE effect.[16,20] Upon
complexation with DNA, it becomes difficult for the bound
molecules to undergo intramolecular rotations. Impeding
the molecular motions blocks the nonradiative channel and
populates the radiative decay, thereby making the TPE mol-
ecules emissive.
The PL maximum of the TPE-1/HG21/K+ complex is lo-
cated at approximately 490 nm. We thus measured its fluo-
rescence lifetimes at this long wavelength. Two components
were observed, with lifetimes of 0.32 and 4.23 ns, respective-
ly (Table 1, entry 7). The longer-lived species predominate
(A2 =75%), from which an average lifetime of 3.26 ns is ob-
tained. Close examination of the steady-state PL spectrum
of TPE-1/HG21/K+ reveals that there is a shoulder band at
approximately 400 nm (cf. Figure 1). The PL decay at this
short wavelength also relaxes in a biexponential mode. By
using the same excitation power (2 mW) as in the above
measurement, it is found that half of the excited species
relax through the slow decay pathway (t2 =5.49 ns), while
another half decays fast with a lifetime of t1 =40 ps.
In an effort to gain insight into the dynamic process in the
TPE-1/HG21/K+ system, power-dependent lifetime meas-
urements were carried out. The lifetimes recorded at 400 nm
are shortened with increasing excitation power, while those
recorded at 490 nm remain practically unchanged (Figure 5).
The results for the PL decays are summarized in Table S2
(Supporting Information). All the decays are comprised of
two components. Careful analysis of the dynamic parame-
ters reveals that both the fraction and lifetime of the longer-
lived species largely decrease with an increase in the excita-
tion power when the decay curves are measured at 400 nm
(Table S2, entries 1–5). The lifetimes of the shorter-lived
species, however, do not change much with the power varia-
tion.
In contrast, neither the fast component nor the slow com-
ponent shows a power-dependent lifetime when the decay
curve is measured at 490 nm (Table S2, entries 6–10). The
fraction of the shorter-lived species is slightly increased with
increasing excitation power. The lex-dependent steady-state
PL measurements reveal the same trend as that observed in
the PL decay experiments. Thus, continuous scanning by
changing the lex from 370 to 300 nm progressively intensifies
the emission at 400 nm but causes little change in the PL in-
tensity at 490 nm (Supporting Information, Figure S7).
These results are understandable. A G-quadruplex can
offer several sites for the fluorogen molecules to bind. Here
we define the one at which a TPE-1 molecule can dock with
Figure 5. Time-resolved fluorescence decay curves of TPE-1/HG21/K+
recorded at a) 400 and b) 490 nm; excitation power was changed in the
range of 0.6–6 mW.
the lowest energy as the most favorable binding site. The
fluorogen in this locus may contribute to the emission at ap-
proximately 490 nm. The unalterable photophysical proper-
ties in this region suggest that the best binding sites are all
occupied by the fluorogen molecules in this system. Enhanc-
ing the excitation power or shortening the lex value barely
changes the population of the excited species in this state.
On the other hand, the emission at the shorter wavelength
of approximately 400 nm is from those unbound and/or par-
tially bound molecules. In terms of fluorescence lifetime,
these species correlate to the shorter-lived component.
Stronger power and/or shorter wavelength can pump more
molecules from the ground state to the excited state, there-
by increasing the population of the excited species. The
amount of the shorter-lived species is thus increased by ad-
justing the experimental conditions.
To the best of our knowledge, no fluorescence probes
have been found that differentiate HG21/K+ from HG21/
Na+ through steady-state PL measurements. Only has a
small difference in their decay times been observed in time-
resolved measurement.[26] Intriguingly, in terms of lifetime,
TPE-1 also behaves differently upon interacting with HG21/
K+ and HG21/Na+. The majority of TPE-1 molecules in the
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Fluorescent Bioprobes
FULL PAPER
presence of HG21/Na+ relaxes by means of the fast channel,
opposite to the case of HG21/K+ (Table 1, entry 13). The
average lifetime is calculated to be 0.55 ns, which is much
shorter than that in the presence of HG21/K+ (3.26 ns). The
great difference between the two lifetimes offers us with an-
other means to distinguish the two G-quadruplex conform-
ers.
The lifetimes of TPE-2 to TPE-4 complexed with HG21/
K+ were also measured. All the complexes show shorter
lifetimes than TPE-1/HG21/K+ does (Table 1, entries 15–
17). For TPE-2 and TPE-3, the shorter-lived species domi-
nate the PL decay processes, whereas the relaxation of
TPE-4 is mainly by means of the slow pathway. Because of
their low binding affinity, most of the TPE-2 and TPE-3
molecules are stripped off from the DNA by the competitive
cations and thus undergo the fast PL decay. Since TPE-4
has the same appendage as TPE-1, the fractional amounts
of shorter- and longer-lived species are similar (cf. Table 1,
entries 7 and 17). This result verifies our hypothesis that
structural matching is the key factor that determines the
photophysical behavior of TPE-1 in the quadruplex-binding
events.
Figure 6. Job plot for determination of the stoichiometry in the binding
process of TPE-1 to the G-quadruplex of HG21 formed in the presence
of K+ ions. The total concentration of TPE-1 and HG21 was kept at
10 mm.
Determination of binding constant: The above experimental
data obtained from the steady-state and time-resolved PL
measurements suggest that the unusual specificity originate
from a geometric fit between TPE-1 and HG21/K+. In an
effort to understand the complexation process, we evaluated
the binding stoichiometry and constant of the TPE-1/HG21/
K+ complex. We conducted continuous variation analysis by
changing the mole fractions of fluorogen and DNA in the
buffer solution, with the sum of the fluorogen and DNA
concentrations kept constant.[27]
According to the AIE principle, in the presence of K+
ions, only the bound fluorogen molecules emit, whereas the
free fluorogen molecules in the buffer solution are practical-
ly nonluminescent. The emission intensities of the solutions
were recorded and correlated according to a Job plot as
shown in Figure 6. The plot has a peak at 0.5, thus giving a
1:1 binding ratio for TPE-1 to HG21 in the presence of K+.
In the absence of K+ ions, however, the TPE-1/HG21 com-
plex is formed in an approximately 3:1 ratio (Supporting In-
formation, Figure S11). The interaction between TPE-1 and
HG21 is mainly electrostatic in nature, as proven in our pre-
vious publications.[15,18,20] The K+ ions, however, promote
HG21 to fold into a G-quadruplex structure, which provides
a specific site for the TPE-1 fluorogen to bind or dock.
To obtain the binding constant of TPE-1 to the HG21/K+
quadruplex, we conducted a PL titration experiment by
adding a solution of HG21 into a solution of TPE-1 with K+
. The emission was turned on and gradually increased with
increasing G-quadruplex concentration, [GQ] (Figure 7a).
The binding constant (Kb) for the TPE-1/G-quadruplex
complexation can be estimated by analyzing the changes in
the PL intensity with the variations in [GQ].[28] According
to the 1:1 ratio obtained from the Job plot, the equilibrium
can be represented by Equation (1):
Figure 7. a) Fluorescence titration of HG21 to TPE-1 in the presence of
K+ ions in 5 mm Tris-HCl buffer. b) Change in the emission intensity at
490 nm with variation in the G-quadruplex concentration and curve fit-
ting using Equation (7). [TPE-1]=4.5 mm, [K+]=0.15m; lex =350 nm.
Chem. Eur. J. 2010, 16, 1232 – 1245
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1239

B. Z. Tang et al.
TPE þ GQ Ð TPE ꢀ GQ
ð1Þ
(hence [GQ]) is varied. The Kb value obtained by fitting the
titration data with Equation (7) is (7.8ꢂ0.7)ꢂ105 mꢀ1 for the
TPE-1/HG21/K+ complex (Figure 7b), from which a Gibbs
energy (DG8) of ꢀ8.0 kcalmolꢀ1 is obtained from the Gibbs
equation.
in which GQ denotes the G-quadruplex structure formed by
HG21 in the presence of K+ ions. Because the K+ concen-
tration is in a large excess amount relative to the HG21 con-
centration, [GQ] is equal to [HG21]. Kb for the equilibrium
reaction given in Equation (1) can thus be expressed by
Equation (2):
Thermodynamic parameters: The PL results described
above tell us that TPE-1 interacts differently with the
HG21/K+ and HG21/Na+ G-quadruplexes. In an attempt to
understand the difference in terms of thermodynamics, iso-
thermal titration calorimetry (ITC) traces were measured.
Figure 8a shows the thermograms obtained from the ITC ex-
periment of HG21/Na+ with TPE-1 buffer solution. The
negative signals indicate an exothermic process. Integration
of the area of each injection peak by subtraction of the heat
of dilution of TPE-1 furnishes a differential enthalpic bind-
ing curve, as shown in Figure 8b.
½TPE ꢀ GQꢁeq
Kb ¼
ð2Þ
½TPEꢁeq½GQꢁeq
in which [TPE–GQ]eq is the equilibrium concentration of
the complex for a given G-quadruplex concentration. By
using [TPE] and [GQ] to denote the total concentrations of
the fluorogen and G-quadruplex, respectively, we get Equa-
tion (3):
The isotherm for TPE-1/HG21/Na+ titration is virtually
identical to that of the dilution of TPE-1 (data not shown),
which suggests that there is practically no interaction be-
tween TPE-1 and HG21/Na+. The Kb and DG8 values are
determined to be 4.3ꢂ102 mꢀ1 and ꢀ3.5 kcalmolꢀ1, respec-
tively. Such small values agree well with our
½TPEꢁ ꢀ ½TPEꢁeq
Kb ¼
ð3Þ
½TPEꢁeqð½GQꢁ ꢀ ½TPEꢁ þ ½TPEꢁeqÞ
which can be rearranged to give Equation (4):
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
earlier findings and indicate a weak binding
affinity.[15] In fact, the small Kb value for
TPE-1/HG21/Na+ is almost to the detection
limit of the apparatus. The titration of TPE-1
ꢀ½Kbð½GQꢁ ꢀ ½TPEꢁÞ þ 1ꢁ þ ½Kbð½GQꢁ ꢀ ½TPEꢁÞ þ 1ꢁ þ 4Kb½TPEꢁ
½TPEꢁeq
¼
2Kb
ð4Þ
into HG21/K+, however, is very different, indicating that
TPE-1 molecules interact strongly with HG21/K+ (Fig-
ure 8b). Nonlinear fitting of the data gives Kb and DG8
values of 2.4ꢂ105 mꢀ1 and ꢀ7.3 kcalmolꢀ1, respectively. The
data are consistent with those obtained from the fluores-
cence titration experiment (Kb =7.8ꢂ105 mꢀ1 and DG8=
ꢀ8.0 kcalmolꢀ1), which indicates a binding affinity three
orders of magnitude higher than that in the TPE-1/HG21/
Na+ system.
Since PL intensity (I) is proportional to fluorogen concen-
tration, I can thus be considered as a composite of that con-
tributed from the bound and unbound (or free) fluorogen
molecules.[28] The following relationship is therefore estab-
lished [Eq. (5)]:
½TPEꢁeq
½TPEꢁ
½TPE ꢀ GQꢁeq
½TPEꢁ
ð5Þ
I ¼ If;max
þ Ib;max
Parallel ITC titration experiments were performed to
evaluate the interactions of TPE-1 with HG26/K+ and
HG26/Na+. As shown in Figure 9a, injections of TPE-1 ali-
quots into the HG26/K+ solution generate exothermic
peaks. Integration of the binding isotherms for TPE-1 with
HG26/K+ and HG26/Na+ give similar profiles (Figure 9b):
complexation takes place instantly with the injection of the
first few aliquots. Further injections cause a progressive
merge of the binding isotherm with the dilution curve. This
indicates that all the binding sites on the G-quadruplexes
are gradually occupied by the TPE-1 molecules and eventu-
ally no binding sites are available for accommodation. By
fitting the isotherms, the Kb values for the TPE-1/HG26/K+
and TPE-1/HG26/Na+ complexations
in which If,max is the initial, maximum emission intensity of
the free fluorogen molecules in the absence of the DNA
and Ib,max is the maximum intensity when all the fluorogen
molecules are bound to the DNA. On account of their AIE
nature, the free TPE molecules are nearly nonfluorescent in
the aqueous solution. In other words, If,max is negligibly small
relative to Ib,max. Equation (5) can thus be simplified to give
Equation (6):
½TPEꢁ ꢀ ½TPEꢁeq
ð6Þ
I ¼ Ib;max
½TPEꢁ
Combining Equations (4) and (6) gives Equation (7):
"
#
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
are both found to be around 3ꢂ
2
Kb½TPEꢁ ꢀ Kb½GQꢁ ꢀ 1 þ ðKb½GQꢁ þ Kb½TPEꢁ þ 1Þ ꢀ 4Kb2½GQꢁ½TPEꢁ
104 mꢀ1.
I ¼ Ib;max 1 ꢀ
2Kb½TPEꢁ
ð7Þ
The thermodynamic parameters
for the fluorogen/G-quadruplex bind-
ing processes are summarized in Table 2. In the titration ex-
periment of TPE-1 into the HG21/K+ solution, a negative
enthalpy change (DH8) is involved, which suggests that the
In the titration experiment, the fluorogen concentration
([TPE]) is kept constant, while the HG21 concentration
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Fluorescent Bioprobes
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Figure 9. a) Calorimetric curves for titration of HG26 in a Na+-Tris
Figure 8. a) Calorimetric curves for titration of HG21 in a Na+-Tris
buffer with serial injections of TPE-1 at 258C. b) Binding isotherm as a
buffer with serial injections of TPE-1 at 258C. b) Binding isotherm as a
function of [TPE-1]/ACTHNUTRGNEUNG
[HG26] molar ratio in K+-Tris or Na+-Tris buffer.
function of [TPE-1]/ACHTUNGTRENNUNG
[HG21] molar ratio in K+-Tris[15] or Na+-Tris buffer.
binding is enthalpy-driven. The binding process is further fa-
vored by a positive entropy change (DS8>0). This seems
surprising but can be understood: it may be caused by the
release of the counterions upon electrostatic attraction of
TPE-1 to the HG21/K+ ensemble. The data resembles those
published by other groups, in which the researchers used the
ITC technique to determine the binding constants of the
cationic ligands to the G-quadruplex.[27]
The thermodynamic data offer some insights into the driv-
ing forces behind the binding process. Various noncovalent
interactions such as electrostatic, hydrophobic, and van der
Waals forces as well as hydrogen bonding can be involved in
a bimolecular complexation process. Judging from the struc-
ture of TPE-1, hydrogen bonding can be ruled out. Al-
though hydrophobic interactions are usually characterized
by small positive DH8 and DS8 values, van der Waals inter-
actions often involve a negative DH8 value.[29] The negative
DH8 and positive DS8 values involved in the formation of
TPE-1/HG21/K+ complex imply that both hydrophobic and
van der Waals interactions are involved in the binding pro-
cess. Moreover, as revealed by our previous study, electro-
static attractions play an essential role in the fluorogen/G-
Table 2. Thermodynamic parameters for TPE-1 binding with different
quadruplexes.[a]
DNA/ion
Kb [mꢀ1
]
DH8
DG8
TDS8
[kcalmolꢀ1
]
[kcalmolꢀ1
]
[kcalmolꢀ1
]
HG21/K+
HG21/Na+
HG26/K+
HG26/Na+
2.4ꢂ105
4.3ꢂ102
3.6ꢂ104
3.2ꢂ104
ꢀ5.4ꢂ0.6
ꢀ7.3ꢂ0.1
ꢀ3.5
1.9
n.d.[b]
ꢀ0.3
ꢀ4.7
n.d.[b]
ꢀ1.6ꢂ0.3
ꢀ2.6ꢂ0.2
ꢀ6.2ꢂ0.1
ꢀ6.2ꢂ0.3
[a] Obtained from ITC measurements at 258C and calculated from Equa-
tion (10). [b] n.d.=not determined (binding interaction too weak to be
determined accurately).
quadruplex association process.[15] The binding affinity of
TPE-1 to HG21/Na+, on the other hand, is so weak that
their thermodynamic parameters are very difficult to deter-
mine accurately. Apparently, the topology of the G-quadru-
plex is vital in the binding event and determines the binding
affinity of the fluorogen molecule.
In the titration experiments of TPE-1 with HG26/K+ and
HG26/Na+, the DH8 and DS8 values in both processes are
negative, which suggests that the association is enthalpy-
driven but entropically unfavorable. Here, the van der
Chem. Eur. J. 2010, 16, 1232 – 1245
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1241

B. Z. Tang et al.
Waals interaction and electrostatic attraction may be the
main driving forces for the complexation events. The larger
negative DH8 values arising from the van der Waals interac-
tions compensate for the smaller entropy losses, resulting in
the negative DG8 values in these systems.
Modeling of the binding mode: In an effort to further eluci-
date the interactions between the fluorogen and quadruplex,
we conducted a modeling study. The Autodock software
package[30] was adopted for the computation simulation with
the aim of understanding two main issues: how the fluoro-
gen molecule binds to the quadruplex and how the binding
interaction alters the photophysical behaviors of the fluoro-
gen. Unfortunately, the folding structure of the HG21/K+
G-quadruplex in solution is not available. We thus chose the
NMR-refined structure of the HG26/K+ G-quadruplex for
the docking simulation. The folding structure, which is be-
lieved to be similar to that of HG21/K+, was retrieved from
the protein data bank (PDB) maintained by the Research
Collaboratory for Structural Bioinformatics (RCSB).[19]
In the modeling process, a flexible ligand docking meth-
odology of Autodock[30] was employed, which allows TPE-1
to enjoy full torsional freedom.[31] From the 100 configura-
tions generated by the docking simulation, the best binding
configuration with the lowest energy was identified (Fig-
ure 10a). Distinctly different from the “conventional” end-
stacking motif to the G-tetrads, the TPE-1 molecule docks
on the surface of the folding structure of the DNA strand.
The aromatic core is stacked on the deoxyribose regions of
the DNA backbone through carbohydrate–p interactions
(gray region).[32] The ethylene chains are located above the
grooves formed by phosphates and sugars, with the alkyl
groups of the ammonium groups extruding into the valley
between the adjacent phosphate ions (red region). A simpli-
fied illustration of the proposed fluorogen/quadruplex bind-
ing mode is shown in Figure 11.
Figure 11. Cartoon illustration of the proposed binding mode. The struc-
ture of TPE-1 is simplified as a solid tetragon (tetraphenylethylene core)
with four lines (alkyl side arms) and solid crosses (terminal ammonium
cations). Phosphates on the DNA backbone are shown as open octagons.
The noncovalent interaction between carbohydrates and
aromatic rings has been described as a hydrophobic effect,
[33]
ꢀ
dispersion force, or C H···p interaction.
The simulated
binding model is in good agreement with our thermodynam-
ic study, which indicates that both the hydrophobic and van
der Waals interactions are involved in the binding process.
As revealed by the crystalline lattices of the AIE molecules
ꢀ
in our previous investigation, the C H···p interactions can
rigidify the conformations of the fluorogen molecules and
hamper their intramolecular rotations, thereby making them
highly emissive.[33]
ꢀ
Without exerting any constrains on the C C single bonds
of the TPE core, the phenyl rings have sufficient flexibility
to adjust and optimize their conformations. The torsion
angles between the aryl planes and the central olefinic
double bond determine the lex and lem values.[22] Through
extraction from the docking results, the torsion angle of the
fluorogen molecule in the simulated conformation is found
to be approximately 518. The geometry of the HG21/K+
quadruplex may differ from that of the HG26/K+ quadru-
plex within the dimensions of the groove. To achieve an op-
timal binding configuration, the phenyl rings of TPE-1 may
have adopted smaller torsion angles, which lead to the ob-
served stronger emission at the longer wavelength in the
TPE-1/HG21/K+ system.
The positively charged ammonium groups also play an es-
sential role in the binding process. The ammonium units
impart good water solubility to the fluorogen molecules and
facilitate their approach to the DNA strands. Their electro-
static attractions are critical to the selective binding. The nu-
cleotides involved in the binding region include A1, A2, A3,
G17, G18, and G21 (cf. Figure 10b). The negatively charged
phosphate units in the DNA backbone assemble perfectly
around the positively charged ammonium units with the aid
of electrostatic attraction.
Figure 10. Docking arrangement of TPE-1 on human telomeric G-quad-
ruplex folded by HG26 in the presence of K+ (PDB entry of the NMR-
refined DNA structure: 2HY9). a) Surface contour depicting the binding
locus (TPE-1 shown in sticks). The model is colored for clarity: C in
gray, O in red, and P in magenta. b) Stereostructure showing the binding
region of the DNA (shown in sticks) for TPE-1 (shown as transparent
surface in gray) with labels indicating the residues involved in the binding
interaction. Spheres showing the electrostatic potential of the DNA back-
bone with negative charge in red and neutral in gray.
Furthermore, the size and shape of the side arms of the
AIE fluorogen are part and parcel of the specific recogni-
tion. Different from those in the crystalline state, the termi-
nal G-tetrads of the G-quadruplexes are unexposed and
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Fluorescent Bioprobes
FULL PAPER
buried by the loops and phosphate backbones in the solu-
tion state. The quaternized ammonium groups of the fluoro-
gen may be too bulky to thread through the loop regions
and to put the TPE core on top of the G-tetrad. The
grooves formed by phosphates and sugars may perfectly ac-
commodate the side arms with spacer lengths of two methyl-
ene units. Consistent with the experimental data, changing
either the spacer length or the size of the ammonium group
results in the disappearance of the characteristic PL features
(cf. Figure 4).
tion to the unique quadruplex binding selectivity, cytocom-
patibility is another merit of the TPE-1 fluorogen system.
The excellent biocompatibility of TPE-1 encouraged us to
utilize it for cell staining. The HeLa cells were imaged by
TPE-1 using a standard cell-staining protocol. The living
cells were incubated with TPE-1 (10 mm) for 3 h and then
washed three times with phosphate buffered saline (PBS)
solution. As shown in Figure 13a, the living cells grow
The docking models were also simulated on other DNA
strands, whose structures could be retrieved from the PDB
of RCSB. Table 3 summarizes the binding energies for TPE-
Table 3. Binding energy for TPE-1 to G-quadruplex DNA obtained from
the docking simulation.
Figure 13. a) Phase contrast and b) fluorescence image of HeLa cells
stained with TPE-1. 100ꢂ magnification; scale bar=10 mm; [TPE-1]=
10 mm.
DNA/ion
HG26/K+
(2HY9)
HG22/Na+
(143D)
Ap15/K+
(1C32)
CM22/K+
(1XAV)
(PDB entry)
binding energy
ꢀ5.31
ꢀ2.71
ꢀ2.16
ꢀ1.49
[kcalmolꢀ1
]
healthily after being treated with TPE-1. Whereas some
commercially available fluorescence dyes, such as CellTrack-
er Green,[35] stain the entire cells, only the cytoplasmic re-
gions of the cells have been stained by TPE-1, as can be
clearly seen from the image taken under UV illumination
(Figure 13b). The TPE fluorogen molecules pass through
the cell membrane and cluster in the organelles outside the
nucleus. The TPE-1 fluorogen may thus be used to probe or
monitor biologically important processes in vitro as well as
in vivo.
1 to all these quadruplex structures. The docking energy of
TPE-1 on the HG26/K+ quadruplex is ꢀ5.31 kcalmolꢀ1,
close to the experimental data (ꢀ6.2 kcalmolꢀ1). The bind-
ing energies for other G-quadruplexes, however, are much
smaller, indicative of unfavorable docking and consistent
with the fluorescence results.
Cytotoxicity assay: For a fluorescent bioprobe to be useful
practically, it should not interfere with the metabolism of
the living system. In other words, it should neither suppress
nor stimulate cell-growth process. To examine the biocom-
patibility of TPE-1, cytotoxicity tests were performed with
HeLa cells based on the reduced activity of methyl thiazolyl
tetrazolium (MTT).[34] The HeLa living cells were exposed
to different concentrations of TPE-1 buffer solution for
48 h, after which the percentages of the viable cells were
quantified. The MTT assay reveals that the cell viability is
not significantly altered even when up to 80 mm TPE-1 is
added to the culture medium (Figure 12). Therefore, in addi-
Conclusion
Recent years have witnessed a surge of interest in G-quad-
ruplex studies for diagnostic and therapeutic applications.
This calls for the development of highly selective ligands
that can specifically bind to the intended target, because of
the presence of a large number of G-quadruplex-forming se-
quences in human genomic DNA and the abundant varia-
tions in the G-quadruplex folding structures.
In this work, a series of TPE derivatives with AIE charac-
teristics were synthesized and their potential as G-quadru-
plex bioprobes was explored. Among the fluorogens, TPE-1
shows excellent selectivity to a particular G-quadruplex
structure formed by human telomeric DNA. This specific
binding is characterized by a unique redshifted quadruplex
emission, offering a convenient visualization method for dis-
criminating not only G-quadruplex from duplex but also be-
tween various quadruplex structures. Both experimental and
computational results indicate that the specificity arises
from a preferred structural matching. With the capability of
distinguishing different quadruplex structures adopted by
the same DNA sequence under different conditions and by
an analogue DNA series with minor differences in the flank-
ing sequences, the TPE-1 fluorogen represents a novel
ligand that functions by means of a docking mechanism that
Figure 12. Growth of HeLa cells in the presence of different concentra-
tions of TPE-1 after 2 d incubation.
Chem. Eur. J. 2010, 16, 1232 – 1245
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1243

B. Z. Tang et al.
and 150 mm NaCl) buffer at pH 7.50, as required. The buffer solutions of
HG21 and HG26 were heated to 858C and cooled slowly to ensure the
folding of the DNA strands into G-quadruplex structures. For a typical ti-
tration run, a series of aliquots (10 mL) of TPE-1 solution was injected
into the DNA/M+ solution at an interval of 240 s. The heat for each in-
jection was determined from the integration of the peak area in the ther-
mogram with respect to time. A blank titration was conducted by injec-
tion of TPE-1 solution into the sample cell containing the buffer under
identical conditions. The heat of interaction of the TPE-1/HG21 titration
was corrected by subtraction of the value from the blank experiment
(control). The values of Kb and DH8 were derived by fitting the isother-
mal curves by using the Origin 5.0 software.[36] The DG8 and DS8 values
were calculated from Equation (10):
differs from the well-known tetrad-stacking mode. Further
studies on the exploration of biomedical applications of the
fluorogenic probe are ongoing in our laboratories.
Experimental Section
General information: All oligonucleotides were purchased from Invitro-
gen (Carlsbad, CA) in desalted quality and used without further purifica-
tion. Concentrations of the DNA strands were determined by measuring
their absorptivity (e) values at 260 nm in a 100 mL quartz cuvette. The e
values of the oligonucleotide strands are given in Table S1 (Supporting
Information). Water was purified with a Millipore filtration system. The
buffer solution was prepared by titrating 5 mm tris(hydroxymethyl)ami-
nomethane (Tris) with 1n HCl until its pH value reached 7.50. All ex-
periments were performed at room temperature (ꢃ238C) unless speci-
fied otherwise. Further experimental details can be found in the Support-
ing Information.
DGꢄ ¼ ꢀRT ln Kb ¼ DHꢄ ꢀ TDSꢄ
ð10Þ
Cell culture: HeLa cells were cultured in minimum essential medium
containing 10% fetal bovine serum and antibiotics (100 unitsmLꢀ1 peni-
cillin and 100 mgmLꢀ1 streptomycin) in a 5% CO2 humidity incubator at
378C.
G-quadruplex formation: Complexes of the oligonucleotides and the
TPE derivatives were prepared by mixing a DNA strand (10 mL, 0.1 mm)
and a TPE derivative (5 mL, 0.1 mm) in 5 mm Tris-HCl buffer solution in
a 1.5 mL Eppendorf cup. The solution was incubated at 48C for 30 min.
G-quadruplex formation was induced by adding a 1.0m KCl or NaCl so-
lution (10 mL) into the Eppendorf cup. The final concentrations of the
TPE derivative and DNA strand were kept at 4.5 and 9.0 mm, respective-
ly.
MTT assay: HeLa cells were placed into a 96-well plate at 5ꢂ103 cells
per 0.1 mL per well and treated for 48 h at 378C with different concen-
trations of TPE-1 solutions (5, 10, 20, 40, and 80 mm). After the treat-
ment, PBS (20 mL) containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT; 5 mgmLꢀ1) was added to each well and the
plate was incubated at 378C for 2–4 h. To dissolve intracellular formazan
produced by active mitochondria in the living cells, detergent solution
(10% w/v sodium dodecyl sulfate in 10 mm HCl; 100 mL per well) was
added and the plate was incubated overnight in the dark at room temper-
ature. The absorbance at 595 nm was measured with a spectrophotomet-
ric plate reader and used for the calculation of relative cell viability,
whereas the cells treated with PBS only were used as the control (100%
viability).
Lifetime measurement: Fluorescence decay curves were recorded with
an Edinburgh Instruments FLS920 combined with steady-state and time-
resolved spectrofluorometers. A femtosecond Ti:sapphire oscillator was
used as the excitation laser source (200 fs pulse width and 76 MHz repeti-
tion rate). The second harmonic (358 nm) of the oscillator output at
716 nm was used for the PL measurement. Time-resolved PL decay
curves were measured with a Hamamatsu model C4334 streak camera
coupled to a spectrometer. The time resolution was 20 ps. The PL signals
were taken at an emission peak of 470 nm, except for the TPE-1/HG21/
K+ system (490 nm). The laser energy level for excitation is 2 mW.
Decay of the fluorescence intensity (I) with time (t) was fitted by a
double-exponential function as shown in Equation (8):
Cell imaging: HeLa cells were grown overnight on a plasma-treated
25 mm round cover slip mounted onto a 35 mm Petri dish with an obser-
vation window. The living cells were stained with TPE-1 (10 mm) for 3 h.
The cells were imaged under an inverted fluorescence microscope
(Nikon Eclipse TE2000-U) using a combination of excitation and emis-
sion filters: lex =330–380 nm, diachronic mirror=400 nm. The images of
the cells were captured using a computer-controlled SPOT charge-cou-
pled device (CCD) camera (SPOT RT SE 18 Mono).
-t=t1
-t=t2
ð8Þ
I ¼ A1e
þ A2e
in which t1 and t2 are the lifetimes of the shorter- and longer-lived spe-
cies, respectively, and A1 and A2 are their respective amplitudes. The
weighed mean lifetime (<t>) was calculated according to Equation (9):
Computational modeling: The structure of the TPE-1 molecule was opti-
mized with the Gaussian 03[37] package using the B3LYP method with the
6-31G* basis set. The DNA structure files were retrieved from the RCSB
PDB, which include NMR-refined structures of solutions of HG26 with
K+ (2HY9), HG22 with Na+ (143D), Ap15 with K+ (1C32), and CM22
with K+ (1XAV).[19,38] Docking studies were performed by using the Au-
todock 4.0[30] software package in a Core2 Duo 2.4 G CPU Linux termi-
nal.
A1t1 þ A2t2
ð9Þ
hti ¼
A1 þ A2
Job plot: The continuous variation method of analysis was employed to
determine the binding stoichiometry for the TPE-1 and K+-driven forma-
tion of the G-quadruplex.[27] The concentrations of TPE-1 and HG21
were varied, while the sum of the two concentrations was kept constant
at 10.0 mm. Solutions of TPE-1 and HG21 were prepared in an all-potassi-
um (K+-Tris: 5 mm Tris-HCl, 150 mm KCl) aqueous buffer (pH 7.50). In
the sample solutions, the mole fraction of the ligand (cL) was varied from
0 to 1.0 in 0.1 ratio steps, while the mole fraction of DNA (1ꢀcL) was
varied accordingly from 1.0 to 0 also in 0.1 ratio steps. Each mixture was
incubated at 48C for 30 min. PL spectra were recorded at 258C. Fluores-
cence intensities taken at 490 nm (lem for the quadruplex-bound TPE-1)
were plotted against the mole fraction of the total ligand present. The
binding stoichiometry was obtained from the c intercept (cint mole frac-
tions of ligand) after linear least-squares fits to the left- and right-hand
Acknowledgements
The authors are grateful to the Research Grants Council of Hong Kong
(603008 and 602706), the Ministry of Science & Technology of China
(2009CB623605), and the National Science Foundation of China
(20634020) for financial support. B.Z.T. thanks the support from Cao
Guangbiao Foundation of Zhejiang University.
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Received: March 25, 2009
Revised: October 8, 2009
Published online: December 2, 2009
Chem. Eur. J. 2010, 16, 1232 – 1245
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www.chemeurj.org
1245