Molecular engineering of G-quadruplex ligands based on solvent effect of polyethylene glycol

Article abstract of DOI:10.1093/nar/gks578

Because various non-parallel G-quadruplexes of human telomeric sequences in K⁺ solution can be converted to a parallel G-quadruplex by adding polyethylene glycol (PEG) as a co-solvent, we have taken advantage of this property of PEG to study the covalent attachment of a PEG unit to a G-quadruplex ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC). The hybrid ligand with the PEG unit, BMVC-8C3O or BMVC-6C2O by substituting either the tetraethylene glycol or the triethylene glycol terminated with a methyl-piperidinium cation in N-9 position of BMVC, not only induces structural change from different non-parallel G-quadruplexes to a parallel G-quadruplex but also increases the melting temperature of human telomeres in K⁺ solution by more than 45°C. In addition, our ligand work provides further confidence that the local water structure plays the key to induce conformational change of human telomere.

Full text of DOI:10.1093/nar/gks578

Published online 26 June 2012
Nucleic Acids Research, 2012, Vol. 40, No. 17 8711–8720
doi:10.1093/nar/gks578
Molecular engineering of G-quadruplex ligands
based on solvent effect of polyethylene glycol
Zi-Fu Wang1,2 and Ta-Chau Chang1,2,
*
2
1Department of Chemistry, National Taiwan University and Institute of Atomic and Molecular Sciences,
Academia Sinica, Taipei 106, Taiwan, R.O.C.
Received April 17, 2012; Revised May 22, 2012; Accepted May 23, 2012
However, the G-rich sequence can adopt various G4
structures. For example, nuclear magnetic resonance
(NMR) analysis showed that human telomeric sequence,
d[AG3(T2AG3)3] (A-HT21), forms a basket anti-parallel
G4 structure (Scheme IA) in Na+ solution (4), whereas
X-ray crystallography showed that A-HT21 forms a pro-
peller parallel G4 structure (Scheme IB) in the presence of
K+ (5). In addition, the co-existence of different G4 struc-
tures of A-HT21 in K+ solution complicates the structural
analysis (13–16). To complicate matters further, telomere
sequences with slight differences can adopt other G4 struc-
tures, such as different hybrid forms of d[TAG3(T2AG3)3]
(TA-HT21) (Scheme IC) (13) and d[TAG3(T2AG3)3TT]
(TA-HT21-TT) (Scheme ID) (14) with three G-quartet
ABSTRACT
Because various non-parallel G-quadruplexes of
human telomeric sequences in K+ solution can be
converted to a parallel G-quadruplex by adding
polyethylene glycol (PEG) as a co-solvent, we have
taken advantage of this property of PEG to study
the covalent attachment of a PEG unit to a
G-quadruplex ligand, 3,6-bis(1-methyl-4-vinylpyrid-
inium) carbazole diiodide (BMVC). The hybrid
ligand with the PEG unit, BMVC-8C3O or BMVC-
6C2O by substituting either the tetraethylene
glycol or the triethylene glycol terminated with a
methyl-piperidinium cation in N-9 position of
BMVC, not only induces structural change from dif-
ferent non-parallel G-quadruplexes to a parallel
G-quadruplex but also increases the melting tem-
perature of human telomeres in K+ solution by
more than 45ꢀC. In addition, our ligand work
provides further confidence that the local water
structure plays the key to induce conformational
change of human telomere.
layers versus
a
basket form of d[G3(T2AG3)3 T]
(HT21-T) (Scheme IE) (17), with two G-quartet layers.
Unfortunately, it is not known which of these structures
are likely to be present in living cells so that the rational
design of selective ligands to G4 is challenging.
It has been argued that the different G4 structures
reported are because of different environmental condi-
tions in which the structures have been determined.
Miyoshi et al. (18–20) have used polyethylene glycol
(PEG) to mimic cellular crowding environment and
found that 2 M PEG can not only induce conformation
change but also increase the stability of G4 structure.
Chaires et al. (16) and Tan et al. (21) have showed that
the crowding agent PEG induces dramatic changes in the
G4 structure of human telomere in K+ solution. Recently,
Heddi and Phan (22) reported that the four different
non-parallel G4 structures are all converted to a propeller
G4 structure (Scheme I) under crowding PEG condition
due to water depletion. This finding has reinforced the
prevailing view that the parallel G4 structure is the form
present in living cells. However, Trent et al. (23) have used
50% v/v acetonitrile as a dehydrating agent, and
Dotsch et al. (24) have used either cell extract or
Ficoll70 as a crowding solvent; these results have sug-
gested that the parallel G4 structure of human telomeres
formed under PEG conditions is not because of the
crowding effect. Accordingly, the parallel G4 structure
formed under PEG might not be physiologically prevalent
INTRODUCTION
Telomeres, the ends of chromosomes, are essential for the
integrity of chromosomes by protecting them from
degradation and end-to-end fusion (1–3). Telomeres
contain guanine-rich DNA sequences. Of interest is that
a short 30-overhang with 100–200 bases of hexameric
repeats of TTAGGG single-stranded sequence could
adopt an intramolecular G-quadruplex (G4) structure
under physiological conditions both in vitro (4,5) and ex
vivo in the metaphase chromosome (6,7). Because the G4
structure is not a template of telomerase, the folding of
telomeric DNA into G4 structures may inhibit telomerase
activity (8,9). Such a structure might be a potential target
for therapeutic cancer intervention (10–12). Small mol-
ecules that can induce G4 structure and further stabilize
G4 structure have the potential to arrest tumor growth.
*To whom correspondence should be addressed. Tel: +886 2 2366 8231; Fax: +886 2 2362 0200; Email: tcchang@po.iams.sinica.edu.tw
ß The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

8712 Nucleic Acids Research, 2012, Vol. 40, No. 17
Scheme I.
(24). The question is whether concentrated PEG solutions
mimic the condition of molecular crowding in cells, or
whether the PEG-induced structural change arises from
other effects.
tolylphosphine, under the triethylamine/acetonitrile
solvent pairs in high-pressure system. After keeping the
system under ꢁ105ꢀC for 2 days, the precipitant was
collected and then extracted with H2O/CH2Cl2 (1:1, v/v)
twice. The insoluble solids in CH2Cl2 layer were filtered
and collected, washed with hot THF twice and then
dried by MgSO4. The crude powders were purified by
flash column chromatography with acetone/n-hexane
(1:1, v/v) as eluent to obtain the powders 4a. The
orange-red powders (BMVC-8C3O) were collected in
good yield after refluxing the compound 4a with excess
CH3I in acetone. The yield and NMR information are
listed later:
It is well known that water molecules play an important
role in DNA structure. At high concentrations, PEG is
expected to disrupt the water structure. Is it possible
that change in the water structure surrounding the DNA
is promoting the shift in the equilibrium of the G4 struc-
tures toward the parallel form? If so, covalent attachment
of a PEG unit to an existing G4 ligand may generate a
hybrid ligand with similar properties. To test this hypoth-
esis, a PEG unit, for example, tetraethylene glycol (TEG),
with a methyl-piperidinium cation, is covalently attached
to the G4 ligand 3,6-bis(1-methyl-4-vinylpyridinium) car-
bazole diiodide (BMVC), to generate the hybrid ligand,
BMVC-8C3O. Indeed, the design principle based on
PEG effect that can induce structural change and further
stabilize the G4 structure provides a basis for the design of
novel G4 ligands.
NMR data
BMVC-8C3O
1
(Yield: 86%, mp > 300ꢀC), H NMR (400 MHz, DMSO-
d6) d: 8.80 (d, J = 6 Hz, 4H), 8.68 (s, 2H), 8.23 (d, J =
16 Hz, 2H), 8.20 (d, J = 7.2 Hz, 4H), 7.90 (d, J = 8.8 Hz,
2H), 7.76 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 16 Hz, 2H),
4.64 (t, 2H), 4.24 (s, 6H), 3.82 (t, 2H), 3.71 (t, 2H), 3.47
(m, 4H), 3.38 (m, 10H), 2.97 (s, 3H), 1.67 (m, 4H), 1.43
(m, 2H).
MATERIALS AND METHODS
Sample preparation
13C NMR (400 MHz, DMSO-d6) d: 153.32, 145.24,
142.68, 142.28, 127.63, 127.21, 123.37, 123.14, 121.58,
121.04, 111.47, 70.48, 70.05, 69.87, 69.83, 63.87, 61.24,
47.21, 20.88, 19.71.
The syntheses of 9-substituted BMVC derivatives were
shown in Scheme II. Briefly, compound 2a was synthesi-
zed from 3,6-dibromocarbazole
1 (2 g, 6.15 mmole,
Aldrich) through 9-position substitution by sodium
hydride (0.295 g, 12.3 mmole, Aldrich) in Tetrahydrofuran
(THF) (20 ml) under nitrogen. A dibromo alkane repre-
sented by the formula Br-R-Br (R = -CCOCCOCCOCC-)
(100 mmole) was then added, and the mixture was refluxed
for 12 hours. After cooling and quenching the waste
sodium hydride with methanol, the solution was extracted
with H2O/ethyl acetate (1/1, v/v) twice, and the organic
layer was dried by MgSO4. The product 2a was collected
via flash column chromatography by a silica gel column
with hexane/ethyl acetate (2/1, v/v) as the eluent.
The dry powder of compound 2a, piperidine
(0.5 ml, Aldrich) and NaH (1.5 mmole) was refluxed in
DMF (20 ml) for 6 hours to obtain the compound 3a,
which was terminated by piperidine. The solvent was
evaporated in vacuum, and the residue was purified via
flash column chromatography by a silica gel column
with hexane/ethyl acetate (1/2, v/v) as the eluent to
collect the yellow product 3a.
BMVC-6C2O
1
(Yield: 83%, mp > 300ꢀC), H NMR (400 MHz, DMSO-
d6) d: 8.81 (d, J = 6 Hz, 4H), 8.68 (s, 2H), 8.23 (d, J =
16 Hz, 2H), 8.21 (d, J = 7.2 Hz, 4H), 7.92 (d, J = 8.8 Hz,
2H), 7.76 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 16 Hz, 2H),
4.68 (t, 2H), 4.33 (s, 6H), 3.85 (t, 2H), 3.68 (t, 2H), 3.51
(m, 2H), 3.42 (m, 6H), 2.99 (s, 3H), 1.67 (m, 4H), 1.43
(m, 2H).
13C NMR (400 MHz, DMSO-d6) d: 153.32, 145.30,
142.45, 142.38, 127.58, 127.21, 123.52, 123.32, 122.44,
121.68, 111.47, 70.38, 70.25, 69.97, 69.88, 63.92, 61.35,
48.20, 20.71, 19.66.
BMVC-8C
1
(Yield: 90%, mp > 300ꢀC), H NMR (400 MHz, DMSO-
d6) d: 8.82 (d, J = 6.4 Hz, 4H), 8.63 (s, 2H), 8.20 (d, J =
6.4 Hz, 4H), 8.19 (d, J = 16 Hz, 2H), 7.92 (d, J = 8.4 Hz,
2H), 7.77 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 16 Hz, 2H) 4.48
(t, 2H), 4.24 (s, 6H), 3.25 (m, 6H), 2.94 (s, 3H),1.80
Then the product 3a could couple with 4-vinylpyridine
at mixed powders of Palladium (II) acetate/tri-o-

Nucleic Acids Research, 2012, Vol. 40, No. 17 8713
Scheme II.
(m, 2H), 1.73 (m, 4H), 1.59 (m, 2H), 1.50 (m, 2H), 1.30 (m,
4H), 1.24 (m, 4H).
H2O/D2O (90%/10%), using
a
jump and return
sequence for solvent suppression. Strand concentration
was 0.1 mM; the solution contained 10 mM Tris-HCl
(PH 7.5) and 150 mM KCl, internal with reference
Sodium 4,4-Dimethyl-4-silapentane-1-sulfonate (DSS).
13C NMR (400 MHz, DMSO-d6) d : 153.29, 145.26,
142.35, 142.28, 127.53, 127.30, 123.34, 123.07, 121.66,
121.02, 111.12, 60.32, 47.18, 28.98, 28.84, 26.75, 26.16,
21.27, 21.07, 19.66.
Differential scanning calorimetry (DSC)
Chemical and sample preparation
DSC thermograms were measured using an N-DSC III
calorimeter (New Castle, DE). The data acquisition and
analysis were carried out through the built-in software
(NanoDSC Run version 3.6 and NanoAnalyze version
2.0). Each calorimetric experiment of 200 mM was per-
formed by scanning from the sample from 20ꢀC to
120ꢀC at 1.0ꢀC/min at 3 atm. This 3 atm prevents forma-
tion of gas bubbles and boiling of aqueous solutions above
100ꢀC (25,26). The corresponding baseline (buffer-buffer)
scans were subtracted from the buffer-sample scans before
their normalization and analysis.
All oligonucleotides were purchased from Bio Basic Inc.
and used without further purification. Solutions of 10 mM
Tris-HCl (pH 7.5) and 150 mM KCl mixed with each
DNA were heated to 95ꢀC for 10 min, cooled slowly to
room temperature and then stored overnight at 4ꢀC before
use.
Circular dichroism
The circular dichroism (CD) spectra were averaged 10
scans on a J-815 spectropolarimeter (Jasco, Japan), with
a 2 nm bandwidth at a 50 nm/min scan speed, and a 0.2 nm
step resolution. The CD spectra were measured under N2
over the range of 210–350 nm to ascertain the G4
structures.
Analytical ultracentrifugation
Sedimentation experiments were performed using a
Beckman Optima XLA analytical ultracentrifuge.
Sedimentation velocity experiments of 0.5 OD DNA at
260 nm were conducted at 20ꢀC using a rotor speed of
60 000 rpm. Data analysis was carried out with the
program Sedfit (16,27).
Polyacrylamide gel electrophoresis (PAGE)
The PAGE was conducted using 20% polyacrylamide
gels. Electrophoresis of the gels was carried out at
250 V/cm for 4 hours at 4ꢀC. They were then photo-
graphed under ultraviolet (UV) light at 254 nm using a
digital camera.
RESULTS
Spectral change induced by BMVC-8C3O
Imino proton NMR
CD has been applied to examine whether BMVC-8C3O
can induce the spectral changes from the non-parallel to
the parallel G4 structures of HT24 in K+ solution. The
Experiments were performed using 800 MHz Bruker spec-
trometers at 25ꢀC. 1D NMR spectra were measured in

8714 Nucleic Acids Research, 2012, Vol. 40, No. 17
CD has been widely used to distinguish parallel from
non-parallel G4 structures (28–33) because quartet
stacking and strand orientation are the determining
factors for the CD spectra. The positive CD band at
295 nm is characteristic of non-parallel G4 structure.
Under PEG, the 295 nm band is notably diminished, and
a strong positive band is detected at 265 nm, which is
attributed to the formation of a parallel G4 structure.
Figure 1a shows CD spectra of 20 mM HT24 in 150 mM
Structural conversion from different non-parallel types
to a parallel type induced by BMVC-8C3O
We have also examined whether BMVC-8C3O can
convert different non-parallel G4 structures of human
telomeres to the parallel G4 structure. Figure 2 shows
the CD and the imino proton spectra of TA-HT21,
A-HT21, HT21, HT21-T and
a mutant sequence
d[AG3(CTAG3)3] (22CTA) (34) in 150 mM K+ solution
and after addition of 5 eq. BMVC-8C3O followed by an-
nealing. The imino proton spectra of TA-HT21, HT21-T,
and 22CTA in K+ solution are consistent with the spectra
reported by Heddi and Phan (22). It is found that the
NMR spectra of TA-HT21, A-HT21, and 22CTA
induced by BMVC-8C3O are similar to each other,
although they have different G4 structures in K+
solution without the ligand. The NMR patterns of
TA-HT21, A-HT21 and 22CTA are slightly different
from the patterns of HT21-T and HT21, with an extra
single peak at ꢁ11.5 ppm. This difference may be
because of the 5’-flanking group in the former sequences,
but not in the latter sequences (22). In addition, the cor-
responding CD spectra induced by BMVC-8C3O are also
similar with each other. They all show a 265 nm positive
CD band and a 240 nm negative CD band, characteristic
of a parallel G4 structure.
K
+ solution after adding 1, 2, 3 and 5 eq. BMVC-8C3O
for 2 h at 25ꢀC (solid line), and their complexes after an-
nealing (dash line). The annealing was performed by
heating the solution to 95ꢀC for 10 min and cooling
slowly at 1ꢀC/min to room temperature, followed by
storage overnight. The CD spectra show no discernible
difference before annealing, but appreciable change after
annealing as a function of BMVC-8C3O concentration.
The spectral conversion from the non-parallel pattern to
the parallel pattern suggests that structural change of
HT24 induced by BMVC-8C3O can be achieved by
thermal activation.
To mimic physiological condition, we have examined
whether BMVC-8C3O can induce structural change at
37ꢀC. Figure 1b shows the CD spectra of 20 mM HT24
in 150 mM K+ solution and its complex with 5 eq.
BMVC-8C3O as a function of time at 37ꢀC, as well as
after annealing. After addition of BMVC-8C3O, the CD
spectra show the decrease of the 295 nm band, together
with the increase of the 265 nm band, as a function of
time. Eventually, the time-evolved CD spectra more
resemble the thermal-annealed spectrum. To confirm
this, Figure 1c shows the high-resolution imino proton
spectra of 100 mM HT24 in 150 mM K+ solution and 2,
4, 6 and 12 h after addition of 5 eq. BMVC-8C3O at 37ꢀC,
as well as after annealing. After addition of BMVC-8C3O,
the time-evolved NMR spectra show a rapid change
followed by gradual change to the thermal-annealed
spectrum. The rapid change may be because of interfer-
ence of BMVC-8C3O, and the gradual change is likely
because of structural change induced by BMVC-8C3O,
consistent with the CD results. The time-evolved and
thermal-activated NMR spectra suggest that the induced
structures of HT24 by BMVC-8C3O are eventually the
same. It appears that BMVC-8C3O can convert the
non-parallel form to the parallel form of HT24 at
moderate rates under physiological condition at 37ꢀC.
The structural conversion induced by BMVC-8C3O was
further confirmed by gel assay. The gel assays of HT24 in
150 mM K+ solution on addition of 5 eq. BMVC-8C3O
for overnight at 25ꢀC and 37ꢀC, as well as after annealing
are shown in Figure 1d. These assays show two
components with different migrations. The faster migra-
tion (lower) is the major component at 25ꢀC, and the
slower migration (upper) is the main component at
37ꢀC. Tan et al. (21) have reported the structural conver-
sion of HT21 induced by using 40% (v/v) PEG 200 and
assigned the faster migration in the gel assays to the
anti-parallel G4 structure and the slower migration to
the parallel G4 structure. Evidently, temperature plays a
critical role for structural conversion of HT24 induced by
BMVC-8C3O.
In addition, we have examined whether these parallel G4
structures induced by BMVC-8C3O are similar to those
converted under PEG. Figure 3a compares the imino
proton spectra of 100 mM TA-HT21 in 150 mM K+
solution after addition of 5 eq. BMVC-8C3O followed by
annealing and a control in the presence of PEG. The imino
proton spectrum of TA-HT21 in K+ solution containing
PEG is similar to that previously reported by Heddi and
Phan (22). Similar imino proton spectra are observed for
TA-HT21 between the addition of BMVC-8C3O and the
presence of PEG, except for the line broadening noted in
the latter, indicating that BMVC-8C3O can induce struc-
tural conversion of TA-HT21 from a hybrid G4 form to a
parallel G4 form. Similar results were also found for
A-HT21, HT21, HT21-T and 22CTA, as shown in
Supplementary Figure S1. Thus, BMVC-8C3O can
induce structural conversion of human telomeric sequences
from different types of non-parallel G4 structures to a
parallel type G4 structure in K+ solution.
The time course of the NMR spectral changes highlights
the evolution of the kinetic-driven products, and the
annealed spectrum corresponds to that of the equilibrium
structure. Supplementary Figure S2 summarizes the
time-evolved imino proton spectra of TA-HT21, HT21-T
and 22CTA, on addition of 5 eq. BMVC-8C3O at 37ꢀC.
They all showed rapid spectral change on addition
of BMVC-8C3O followed by gradual change to the
thermal-annealed spectra, consistent with the previous
results for HT24 (Figure 1c). The much faster conversion
rate for 22CTA than for HT21-T induced by
BMVC-8C3O deserves further study. Nevertheless, the
final structure is the same as the thermally activated
structure, indicating that the structural conversion
induced by BMVC-8C3O is feasible under physiological
conditions.

Nucleic Acids Research, 2012, Vol. 40, No. 17 8715
Figure 1. (A) CD spectra of 20 mM HT24 (solid line) and its complex with 1, 2, 3 and 5 eq. BMVC-8C3O (dash line) in 150 mM K+ solution, as well
as after annealing (dot). (B) Time-dependant CD spectra of 20 mM HT24 with 5 eq. BMVC-8C3O in 150 mM K+ solution at 37ꢀC. (C) High
resolution imino proton spectra of 100 mM HT24 in 150 mM K+ solution and 2, 4, 6 and 12 h after addition of 5 eq. BMVC-8C3O at 37ꢀC, as well as
after annealing. (D) UV shadowing and pre-stain of 0.2 nmol HT24 in 150 mM K+ solution after adding 5 eq. BMVC-8C3O for overnight at 25ꢀC (1)
and 37ꢀC (2), as well as after annealing (3).
Heddi and Phan (22) have further showed the conver-
sion of the hybrid G4 structure to the parallel G4 structure
can be reversed simply by quick dilution of the solution to
a negligible PEG concentration. In contrast, the corres-
ponding conformational transition of TA-HT21 induced
by BMVC-8C3O is not reversible by sample dilution even
overnight. Figure 3b shows the CD spectra of TA-HT21
induced by PEG and BMVC-8C3O followed by sample
dilution. This difference is because of the global solvent
effect versus the local ligand effect, which is important to
discriminate the possible effects between molecular
crowding and disruption of local water structure for con-
formational change.
In addition, Figure 4b shows the CD signals at 265 nm
for TA-HT21 and its complexes after adding BMVC-
8C3O for 12 h at 37ꢀC in 150 mM K+ solution. Here,
the Tm of TA-HT21 measured from the CD melting
curve is ꢁ67ꢀC, where the Tm of TA-HT21 measured
from the DSC is ꢁ70ꢀC. Slight deviation of the Tm was
also observed in the previous study of the bcl2mid under
different K+ concentration (35). Because the upper tem-
perature of the CD melting curve is limited at 95ꢀC, it
indicates that the Tm of the TA-HT21/BMVC-8C3O
complexes is much higher than 95ꢀC.
Furthermore, the Tm of the G4 structure generally
increases as a function of K+ concentration (35–37). In
accordance with this behaviour, DSC shows that the Tm
of the major transition of TA-HT21 is ꢁ45ꢀC in 5 mM
Melting temperature of G-quadruplex increased by
BMVC-8C3O
K+ solution and ꢁ97ꢀC after addition of
5 eq.
BMVC-8C3O at 37ꢀC overnight, as shown in Figure 4c.
Here, BMVC-8C3O also converts the G4 structure from
the non-parallel form to the parallel form and increases
the Tm of the G4 structure in 5 mM K+ solution by more
than 45ꢀC.
We have further used DSC to measure the melting tem-
peratures of TA-HT21 in 150 mM K+ solution, on individ-
ual addition of 40% (v/v) TEG, 40% (v/v) PEG and 5 eq.
BMVC-8C3O at 37ꢀC overnight, as well as after annealing.
These data are summarized in Figure 4a. The DSC shows a
monophasic transition with the melting temperature (Tm)
of ꢁ70ꢀC in 150 mM K+ solution and ꢁ97ꢀC after addition
of TEG (or PEG) overnight, a biphasic transition with the
Tm of ꢁ60ꢀC and ꢁ115ꢀC after addition of BMVC-8C3O
overnight at 37ꢀC and a transition with the Tm of ꢁ115ꢀC
with a shoulder at ꢁ95ꢀC after addition of BMVC-8C3O,
followed by annealing. Similar results were also found in
the DSC-melting curves of A-HT21, HT21-T, HT21 and
22CTA in 150 mM K+ solution and on addition of 5 eq.
BMVC-8C3O at 37ꢀC overnight, as shown in
Supplementary Figure S3.
Structural change of a longer telomeric sequence
induced by BMVC-8C3O
The 30-overhang single-stranded telomeric sequences are
generally of 50–200 bases in length. We have examined
the effects of BMVC-8C3O on the longer telomeric
sequence d(T2AG3)8 (HT48). Figure 5a shows the CD
spectra of HT48 in 150 mM K+ solution and after
addition of 10 eq. BMVC-8C3O at 37ꢀC for 2 and 12 h,
as well as after annealing. In addition, Figure 5b shows the
imino proton spectra of 100 mM HT48 in 150 mM K+

8716 Nucleic Acids Research, 2012, Vol. 40, No. 17
Figure 2. The CD spectra (left) of 20 mM (A) TA-H21, (B) A-H21, (C) 22CTA, (D) H21-T and (E) HT21 in 150 mM K+ solution (black) and after
addition of 5 eq. BMVC-8C3O followed by annealing (red). The imino proton NMR spectra (right) of 100 mM (A) TA-H21, (B) A-H21, (C) 22CTA,
(D) H21-T and (E) HT21 and 22CTA in 150 mM K+ solution (lower) and after addition of 5 eq. BMVC-8C3O followed by annealing (upper).
solution (lower), on adding 10 eq. BMVC-8C3O followed
by annealing (upper), in the presence of PEG with (upper
middle) and without (lower middle) addition of 10 eq.
BMVC-8C3O. Figure 5c shows the DSC melting curves
of HT48 in 150 mM K+ solution and on addition of 10 eq.
BMVC-8C3O at 37ꢀC overnight. Although the precise
structure of HT48 has not been determined yet, similar
spectral change in CD and NMR patterns suggest that
BMVC-8C3O can induce conformational change from a
non–parallel-like G4 structure to a parallel-like G4 struc-
ture of HT48. Moreover, BMVC-8C3O can increase the
Tm of HT48 in 150 mM in K+ solution from ꢁ60ꢀC to
ꢁ120ꢀC. It appears that BMVC-8C3O is a novel G4 sta-
bilizer for a long telomere sequence as well.
Structural change induced by BMVC-8C versus
BMVC-6C2O
To test whether the oxygen in BMVC-8C3O plays an
important role in the G4 structural conversion, we have
further modified BMVC by substituting either an
octane or a triethylene glycol in N-9 position with
a methyl-piperidinium cation to give BMVC-8C or
BMVC-6C2O, respectively. Figure 6a shows the CD
spectra of TA-HT21 in 150 mM K+ solution and its
complex with 5 eq. BMVC-8C at 37ꢀC for 12h, as well
as after annealing. We find that the structural conversion
induced by BMVC-8C is much slower than that by
BMVC-8C3O. In addition, NMR spectra show that the
imino proton signals at ꢁ11.2, ꢁ11.3 and ꢁ11.9 ppm of

Nucleic Acids Research, 2012, Vol. 40, No. 17 8717
Figure 3. (A) The imino proton spectra of 100 mM TA-HT21 in
150 mM K+ solution under 40% PEG condition (lower), followed by
addition of 5 eq. BMVC-8C3O (middle) for 2 h, and TA-HT21 in
150 mM K+ solution on addition of 5 eq. BMVC-8C3O followed by
annealing (upper). (B) The CD spectra of TA-HT21 in 150 mM K+
solution under 40% PEG and addition of 5 eq. BMVC-8C3O at
37ꢀC and followed by sample dilution after 12 h at 37ꢀC.
TA-HT21, after addition of BMVC-8C, disappeared in the
annealed spectrum, are clearly detected after addition of
BMVC-8C for 12h in the time-evolved spectrum at 37ꢀC
(Figure 6b), supporting that the unfolding of the hybrid G4
structure of TA-HT21 by BMVC-8C at 37ꢀC is slow. Of
interest is that the imino proton spectra of TA-HT21 on
addition of 5 eq. each BMVC-8C and BMVC-8C3O show
some differences, implying different structural conversions
induced by BMVC-8C and BMVC-8C3O. Figure 6c shows
the gel assays of TA-HT21 in 150 mM K+ solution on
addition of 5 eq. BMVC-8 C for overnight at 25ꢀC and
37ꢀC, where the component with slow migration induced
by BMVC-8C is even slower than that by BMVC-8C3O.
We have further applied the sedimentation for ultracentri-
fugation analysis (16,27) to characterize monomer, dimer
and higher order structures of TA-HT21. Figure 6d shows
the sedimentation results of TA-HT21 on interaction with 5
eq. BMVC-8C after annealing. Our results show that
BMVC-8C induces structural change of TA-HT21 from
monomers to dimers together with some high order
multimers.
Figure 4. (A) The DSC of TA-HT21 in 150 mM K+ solution (black),
adding 40% TEG (blue), adding 40% PEG (light blue) and addition of
5 eq. BMVC-8C3O at 37ꢀC overnight (red), as well as after annealing
(green). (B) CD signals at 265 nm for the measurement of melting tem-
perature of TA-HT21 (black) and its complexes of BMVC-8C3O (red)
in 150 mM K+ solution as a function of temperature. (C) The DSC of
TA-HT21 in 5 mM K+ solution (black) and 12 h after addition of 5 eq.
BMVC-8C3O (red) at 37ꢀC.
oxygen in both BMVC-6C2O and BMVC-8C3O plays
an important role in the structural transition of
TA-HT21 from a monomer hybrid conformation to a
monomer parallel conformation. It is not clear why differ-
ent conformational changes are induced by BMVC-6C2O
and BMVC-8C. The same non-parallel G4 structure con-
verted to a monomer and a dimer of parallel G4 structures
by these two hybrid ligands, is of interest and deserves
further studies.
We also studied the CD spectra, the imino proton
spectra, the gel assays and the sedimentation analysis of
TA-HT21 on addition of 5 eq. BMVC-6C2O, as shown in
Supplementary Figure S4. Our results show that the

8718 Nucleic Acids Research, 2012, Vol. 40, No. 17
of the G4 structure is accessible to water. Water depletion
favors the formation of the parallel form (22), as has been
found in x-ray crystallography (5). Thus, we believe that
the structural change induced by BMVC-8C3O is mainly
because of a local dehydration effect. Consistent with this,
no effects of sample dilution are expected because of the
strong association of the BMVC-8C3O to the G4 structure
(Figure 3b). Thus, our results provide further confidence
that the local water structure plays the key to induce the
various forms of the G4 structure to the parallel form.
Considering water molecules involved in DNA structure
formation, Sugimoto et al. (38) suggested that hydration
plays a major role for PEG in the stability of DNA, in
contrast to viscosity and dielectric constant, which have
little effects. In addition, the results reported by Trent
et al. (23) using 50% (v/v) acetonitrile and Dotsch et al.
(24), using Ficoll70 as a crowding solvent suggested that
the structural change under PEG conditions is not because
of the crowding effect. In this work, we have shown that
the 100 mM BMVC-8C3O, which is not sufficient to
generate
a crowding environment, can successfully
induce structural change of human telomeres to a
parallel form. In contrast, the use of 100 mM BMVC is
not able to induce various non-parallel G4 structures of
human telomeres to the parallel G4 structure (29). Thus, it
is the PEG unit in BMVC-8C3O that plays a major role in
disrupting the local water structure in the vicinity of the
G4 structure of human telomeres, and induces conform-
ational change. With our hybrid BMVC-PEG ligand, we
have discriminated between effects arising from molecular
crowding and disruption of local water structure for con-
formational change. This is more than a subtle difference
between our ligand work and earlier solvent studies.
Molecular engineering of G4 ligands
Figure 5. (A)Time-dependant CD spectra of 20 mM HT48 with 10 eq.
BMVC-8C3O in 150 mM K+ solution at 37ꢀC (B) The imino proton
spectra of 100 mM HT48 in 150 mM K+ solution (bottom) under 40%
PEG condition (lower middle) and 2 h after addition of 10 eq.
BMVC-8C3O (upper middle) together with TA-HT21 in 150 mM K+
solution upon addition of 10 eq. BMVC-8C3O followed by annealing
(upper). (C) The DSC of HT48 in 150 mM K+ solution (black) and
addition of 10 eq BMVC-8C3O at 37ꢀC (red) overnight.
The first G4 binding ligand is an anthraquinone derivative
reported by Sun et al. in 1997 (39). Since then, a growing
number of G4 ligands, such as TMPyP4 (40), BRACO-19
(41), telomestatin (42), BMVC (43), naphthalene diimide
(44) and pyridostatin (45) have been reported. Among
them, the pyridostatin can increase the Tm of human
telomere in 60 mM K+ solution up to 35ꢀC (45). Most of
the G4 ligands have common features, including a planar
heteroaromatic core for the p–p interaction, with a
terminal G-quartet and the presence of positive charge
at side arms for the electrostatic interaction with sugar-
phosphate backbone (46–48). However, none of them take
into account the possible solvent effect from surrounding
environment to further enhance the stability of folded
structure. To our knowledge, this is the first report
taking the advantage of the solvent effect to upgrade an
existing G4 ligand that can disrupt the local water struc-
ture, which in turn can convert all multiple G4 structures
of human telomeres to the parallel form and enhance the
stability of G4-ligand structure significantly. Specifically,
BMVC-8C3O can increase the Tm of d(T2AG3)8
(HT48) from ꢁ60ꢀC to ꢁ120ꢀC in 150 mM K+ solution
(Figure 5c) and that of TA-HT21 from ꢁ45ꢀC to ꢁ97ꢀC in
5 mM K+ solution (Figure 4b). This large increase in the
melting temperature of human telomeres could possibly be
DISCUSSION
Molecular crowding effect vs. local dehydration effect
In the past, the addition of 40% (v/v) PEG to the solu-
tions has been used to mimic the condition of molecular
crowding in cells. Although the PEG molecules are not
expected to bind to the G4 structure specifically, at these
high concentrations, they are sufficient to disrupt the
water structure in the vicinity of the G4 structure. At
the low concentration of several hundred mM
BMVC-8C3O, there should be no global or gross disrup-
tion of the solvent structure by the PEG pendant.
However, with the BMVC moiety bound to the G4 struc-
ture (29), the attached PEG is brought into the solvent
sphere of the G4 structure. Only the water structure in
the vicinity of the quadruplex-ligand complex is disrupted.
In this manner, a reduced amount of the molecular surface

Nucleic Acids Research, 2012, Vol. 40, No. 17 8719
Figure 6. (A) Time-dependant CD spectra of 20 mM TA-HT21 with 5 eq. BMVC-8C in 150 mM K+ solution at 37ꢀC (B) Imino proton spectra of
100 mM TA-HT21 in 150 mM K+ solution, 2 and 12 h after addition of 5 eq. BMVC-8C at 37ꢀC, as well as after annealing. (C) UV shadowing and
pre-stain of 0.2 nmol TA-HT21 in 150 mM K+ solution after adding 5 eq. BMVC-8C for overnight at 25ꢀC (1) and 37ꢀC (2). (D) Ultracentrifugation
of TA-HT21 in 150 mM K+ solution on addition of 5 eq. BMVC-8C after annealing.
exploited for inhibiting telomerase activity. Biological
studies and animal test of BMVC-8C3O are currently in
progress.
FUNDING
Academia Sinica [AS-98-TP-A04]; National Science Coun-
cil of the Republic of China [NSC-98-2113-M001-025].
Although the parallel G4 structure is unlikely the
physiologically prevalent conformation, the important
finding is that PEG can convert all non-parallel G4
forms to the parallel G4 form. To verify the possible
mechanisms between molecular crowding and water deple-
tion, we have taken advantage of this property of PEG to
study the covalent attachment of a PEG unit to the G4
ligand, BMVC. Consistent with our hypothesis, the local
water structure plays the key to induce conformational
change of human telomere. Aside from this finding, the
main thrust of our work is to outline a hybrid strategy
with taking into account the solvent effect toward the de-
velopment of an effective approach for drug design, and
have provided a demonstration of ‘‘proof-of-principle’’.
Conflict of interest statement. None declared.
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