A fluorescent probe for the 3′-overhang of telomeric DNA based on competition between two interstrand G-quadruplexes

Article abstract of DOI:10.1039/c3cc37504h

A 6-mer oligonucleotide containing a fluorescent ^BodU moiety has been used as a novel fluorescent probe for the 3′-overhang of telomeric DNA based on competition between non-fluorescent tetramolecular and fluorescent (3+1) intermolecular G-quad

Full text of DOI:10.1039/c3cc37504h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
A fluorescent probe for the 30-overhang of telomeric
DNA based on competition between two interstrand
G-quadruplexes†
Cite this: Chem. Commun., 2013,
49, 1717
Received 15th October 2012,
Accepted 16th January 2013
Ki Tae Kim and Byeang Hyean Kim*
DOI: 10.1039/c3cc37504h
www.rsc.org/chemcomm
A 6-mer oligonucleotide containing a fluorescent BodU moiety has been
used as a novel fluorescent probe for the 30-overhang of telomeric DNA
based on competition between non-fluorescent tetramolecular
and fluorescent (3+1) intermolecular G-quadruplexes.
Human telomeres, which are located at the ends of chromosomes,
play important roles in biological phenomena.1,2 The human
telomeres contain telomeric DNA featuring a very long (5–8 kb)
repeating duplex (TTAGGG)n/(CCCTAA)n with a long (ca. 200 nt)
G-rich single-stranded DNA, the so-called 30-overhang. Detection
and visualization of this 30-overhang of telomeric DNA are impor-
tant for understanding telomeres and many biotechnological
applications such as telomerase activity assay.3
An attractive feature of 30-overhang strands is formation
of a specific three-dimensional structure: the G-quadruplex.4
Previously, G-quadruplexes have been used as basic structure of
fluorescent DNA systems for detection of DNAs, enzymes, metal
ions, and biomolecules.5 Likewise, in the quest for simple,
sensitive, and selective detection methods for 30-overhang
Fig. 1 Fluorescent probe for the 30-overhang of telomeric DNA based on
formation of tetramolecular and (3+1) intermolecular G-quadruplexes.
strands, one approach would be to exploit structural differences Herein, we report that the significant fluorescence enhancement
among G-quadruplex structures.
of a short (6-mer) ODN (50-BodUGGGTT-30) containing a BODIPY–
Hamilton and co-workers reported a self-assembled tetra- phenyl-20-deoxyuridine (BodU) conjugate can be used to monitor
molecular G-quadruplex featuring G-rich sequences carrying the structural formation of a (3+1) intermolecular quadruplex.
planar aromatic 50-residues,6 which stabilize the tetramolecular Using this approach, we could selectively detect single-stranded
G-quadruplex through mutually attractive noncovalent inter- G-rich sequences, such as the 30-overhang of the human telomeric
actions. These systems exhibit remarkable variations in fluores- DNA, by fluorescence enhancement. We also found that the
cence upon structural changes of the G-quadruplex.7–9 The degree of fluorescence enhancement was closely related to the
(3+1) interstrand G-quadruplex,10 on the other hand, has also been length of the telomeric DNA.
used as a platform for DNA systems. Accordingly, we expected that
Firstly, we chose a BODIPY-phenyl moiety as the signaling
a short G-rich sequence presenting a fluorescent residue would fluorophore because its fluorescence is quenched through self-
display different fluorescence properties in its tetramolecular and aggregation11 or structural hindrance.12 We expected to observe
(3+1) intermolecular structures and could, therefore, be used as a fluorescence quenching of the BODIPY unit when the short
fluorescent probe for single-stranded G-rich sequences (Fig. 1). G-rich sequences formed a parallel tetramolecular G-quadruplex.
We prepared the fluorescent nucleoside BodU with the
BODIPY-phenyl unit attached to 20-deoxyuridine through an
acetylene linker. We synthesized a phenyl-BODIPY derivative
Department of Chemistry, BK School of Molecular Science, Pohang University of
Science and Technology, Pohang 790-784, South Korea.
from 4-bromobenzaldehyde and then conjugated it with
E-mail: bhkim@postech.ac.kr; Fax: +82 54-279-3399; Tel: +82 54-279-2115
20-deoxyuridine to give BodU (Scheme S1, ESI†). We examined
† Electronic supplementary information (ESI) available: Synthetic scheme, photo-
physical properties, and MALDI-TOF MS data. See DOI: 10.1039/c3cc37504h
the photophysical properties of BodU from its UV absorption
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1717--1719 1717

View Article Online
Communication
ChemComm
of BODIPY by energy transfer,13 as evidenced by broadening of
the UV absorption band (Fig. S1, ESI†).14 ODN-B exhibited
intense fluorescence when we added the K+ ion to a mixture
of ODN-B and ODN-1 (general experimental details, ESI†), which
features three repeating GGG sequences (G-rich sequence).
Evidence for the (3+1) interstrand G-quadruplex was apparent
from its CD spectrum (Fig. S2, ESI†) in the form of a CD
signature at 235 and 290 nm. This mixture exhibited a 7.3-fold
enhancement in fluorescence intensity without annealing, and a
10.5-fold enhanced fluorescence signal after annealing (Fig. 2),
as a result of no self-quenching effect in the (3+1) interstrand
G-quadruplex. The fluorescence of the (3+1) intermolecular
G-quadruplex was greater than ODN-B alone (Fig. 2, ODN-B).
We suspect that the ODN-B units interacted with each other
through aromatic–aromatic interactions even in the absence
of K+ ions.6 These noncovalent interactions among BODIPY-
phenyl moieties were precluded in the (3+1) intermolecular
G-quadruplex, leading to higher fluorescence. We expected that
the value of lmax fluorescence would undergo a bathochromic
shift as a result of the formation of an exciplex between the
BODIPY-phenyl unit and the G-quadruplex surface, stabilized
through hydrophobic stacking interactions (Fig. S5d and e,
ESI†).15,16 Fluorescence imaging allowed the tetramolecular
and (3+1) intermolecular G-quadruplexes to be discriminated
clearly by the naked eye (Fig. 2, inset). Accordingly, we expected
ODN-B to be a suitable candidate for probing (3+1) intermole-
cular G-quadruplex formation with a very low background signal.
These results led us to apply ODN-B to the detection of other
G-rich sequences. We employed a variety of G-quadruplex
sequences from the human telomeric DNA (Htelo1) and from
the promoter region (c-myc, vegf, c-kit, bcl2, pu18). Further-
more, we used the multimeric quadruplexes Htelo2 and Htelo3
to monitor the fluorescence change (Table 1). MALDI-TOF mass
spectrometry confirmed all of their sequences (Table S2, ESI†).
We expected that the longer human telomeric G-quadruplex
sequences would have a greater chance of donating their
T(GGGTTA)3T repeat sequences for the formation of (3+1)
interstrand G-quadruplexes.3c,17 In the presence of ODN-2, we
observed no fluorescence change because its single GGG repeat
sequence was unlikely to form a tetramolecular G-quadruplex
with ODN-B. On the other hand, the presence of a monomer-
type G-quadruplex sequence having four or more repeating
GGG units (Htelo1, c-myc, vegf, c-kit, bcl2, pu18) led to an
approximately two fold (1.4–3.5-fold) increase in fluorescence
(Fig. S4, ESI†). These increases were smaller than those
observed for the sequence containing three repeating GGG
units of ODN-1, presumably because of competition between
the formation of the (3+1) G-quadruplex and the intramolecular
G-quadruplex. Moreover, we also treated the solution of ODN-B
with the duplex formed from the G-rich sequences (Htelo1,
c-myc, vegf, c-kit, bcl2, pu18) and their C-rich sequences (ODN-3
to ODN-8) and with the single-stranded C-rich sequences to
evaluate the selectivity of ODN-B (Fig. S4, ESI†). In both cases,
we observed little or no fluorescence enhancement. It seems that
single-stranded C-rich sequences cannot form a duplex with
complementary ODN-B because of a big thermal difference
between the tetramolecular G-quadruplex and the duplex of
Table 1 ODN sequences investigated in this study
Name
Sequence
ODN-B
ODN-B2
ODN-1
50 BodU GGG TT
50 BodU TTT TT
50 T GGG TTA GGG TTA GGG T
50 T GGG TT
ODN-2
ODN-3 (Htelo1)
ODN-4 (c-myc)
ODN-5 (vegf)
ODN-6 (c-kit)
ODN-7 (bcl2)
ODN-8 (pu18)
c-myc
vegf
c-kit
bcl2
pu18
50 CCC TAA CCC TAA CCC TAA CCC T
50 TTC CCC ACC CTC CCC ACC CTC A
50 CCC GCC CCC GGC CCG CCC
50 CCC CTC CCT CGC GCC CGC CCG
50 CCC GCC CAA TTC CTC CCG CGC CC
50 CCC CAC CCT CCC CAC CCT
50 TGA GGG TGG GGA GGG TGG GGA A
50 GGG CGG GCC GGG GGC GGG
50 GGG CGG GCG CGA GGG AGG GG
50 GGG CGC GGG AGG AAT TGG GCG GG
50 AGG GTG GGG AGG GTG GGG
50 A(GGGTTA)3GGGT
Htelo1
Htelo2
Htelo3
50 A(GGGTTA)7GGGT
50 A(GGGTTA)11GGGT
and fluorescence spectra (Table S1, ESI†). The BodU unit exhibited
good quantum yields, even in DNA duplexes (quantum yields:
0.15–0.49; data not shown), in contrast to other types of BODIPY–
deoxyuridine conjugates that underwent significant fluorescence
quenching in DNA duplexes.12 Thus, our BodU nucleoside
appeared to be a good candidate for monitoring the structural
changes of ODNs.
Using phosphoramidite chemistry, we introduced BodU into
the 6-mer ODN-B (Table 1), which exhibited a fluorescence
quenching effect depending on the concentrations of K+ and
Na+ ions (Fig. 2 and Fig. S1, ESI†). From previous observations6
and its circular dichroism (CD) spectrum (Fig. S2, ESI†),
we confirmed that ODN-B formed a parallel tetramolecular
G-quadruplex, as evidenced by negative and positive CD signatures
at 235 and 265 nm, respectively. In contrast to ODN-B, ODN-B2 did
not exhibit any fluorescence quenching because it cannot form a
tetramolecular G-quadruplex (Fig. S3, ESI†). We suspect that the
fluorescence quenching of ODN-B resulted from self-quenching
Fig. 2 Fluorescence enhancement upon formation of a (3+1) intermolecular
G-quadruplex from ODN-B in the presence of ODN-1 and K+ ions (1.5 mM ODN-B;
10 mM trizma buffer, pH 7.2; total volume of the sample: 1 mL; 20 1C after
annealing; excitation wavelength: 500 nm). Inset: fluorescence of ODN-B under
irradiation with UV light in the (a) absence and (b) presence of ODN-1.
c
1718 Chem. Commun., 2013, 49, 1717--1719
This journal is The Royal Society of Chemistry 2013

View Article Online
ChemComm
Communication
a simple and sensitive method for the detection of the long
30-overhang in telomeric DNA.
We are currently optimizing this system to detect 30-over-
hangs in the telomeric regions of cellular DNA. As groundwork
towards this goal, we have evaluated (1) the kinetic versus
thermodynamic control of the system (Fig. S6, ESI†), (2)
potential interference by biomolecules (e.g. single stranded
DNA binding protein (SSB), full-length DNA, mRNA, Fig. S7,
ESI†), (3) nuclease resistance (Fig. S8, ESI†), and (4) cytotoxicity
of the oligonucleotides in cells (Fig. S9, ESI†).
In summary, we have developed a new selective fluorescence
probe for single stranded G-rich sequences, especially the
30-overhang in telomeric DNA, based on the formation of two
different G-quadruplexes, namely tetramolecular and (3+1)
intermolecular quadruplexes. The ability of this probe to detect
and visualize the 30-overhang can be applied to telomerase
activity assay and used in cells for further study.
This study was supported by the EPB center program of MEST/
KOSEF (20120000528) and the NRF project (2012R1A2A2A01047069).
We thank E. M. Jeon for cellular experiments.
Fig. 3 Fluorescence intensity of ODN-B in the presence of human telomeric
sequences of various lengths and concentrations (0.5 mM of ODN-B; 10 mM
trizma buffer, pH 7.2; 300 mM K+; total volume of the sample: 1 mL; 20 1C;
excitation wavelength: 500 nm).
ODN-B and ODN-3 (Fig. S5, ESI†). Thus, our system appeared to
selectively detect single-stranded G-rich sequences, but not their
double-stranded counterparts.
Notes and references
1 E. H. Blackburn, FEBS Lett., 2005, 579, 859–862.
2 W. E. Wright, V. M. Tesmer, K. E. Huffman, S. D. Levene and
J. W. Shay, Genes Dev., 1997, 11, 2801–2809.
3 (a) A. K. Pomerantz, W. E. Moerner and E. T. Kool, J. Phys. Chem. B,
2008, 112, 13184–13187; (b) J. Wang, L. Wu, J. Ren and X. Qu, Small,
2012, 8, 259–264; (c) L. Stefan, F. Denat and D. Monchaud, J. Am.
Chem. Soc., 2011, 133, 20405–20415.
We applied this selectivity to the detection of the telomeric
30-overhang region, because the telomeric 30-overhang may
exist in single-stranded form (or may form a G-quadruplex18
or a T-loop structure19), not the duplex. We observed 3.5-, 6.3-,
and 8.0-fold enhancement in the fluorescence of ODN-B in the
presence of one equivalent of Htelo1, Htelo2, and Htelo3,
respectively (Fig. S4, Htelo1, Htelo2, Htelo3, ESI†). Thus, a longer
30-overhang sequence led to higher fluorescence, implying that a
longer G-rich sequence has a greater chance of forming a (3+1)
G-quadruplex at a random position. In fact, sequences that can
form multimeric G-quadruplexes can feature several structural
equilibria,3c providing access to folding intermediates towards
the (3+1) intermolecular quadruplex.
To evaluate this system as a probe for the 30-overhang, we
monitored the fluorescence intensity with respect to the concen-
tration of the 30-overhang sequence (Fig. 3). As the length of the
telomere sequence increased, the probe exhibited a higher fluores-
cence intensity in the saturated state. The degree of fluorescence
enhancement by increasing concentration was clearly differentiated
among Htelo1, Htelo2, and Htelo3. Therefore, both the fluorescence
intensity and enhancement are significantly related to the
length of the G-rich sequence.
4 S. Burge, G. N. Parkinson, P. Hazel, A. K. Todd and S. Neidle, Nucleic
Acids Res., 2006, 34, 5402–5415.
5 (a) D. Hu, Z. Huang, F. Pu, J. Ren and X. Qu, Chem.–Eur. J., 2011, 17,
1635–1641; (b) C. Zhao, L. Wu, J. Ren and X. Qu, Chem. Commun.,
2011, 47, 5461–5463; (c) J. Zhao, L. Zhang, J. Jiang, G. Shen and
R. Yu, Chem. Commun., 2012, 48, 4468–4470; (d) K. Qu, C. Zhao,
J. Ren and X. Qu, Mol. Biosyst., 2012, 8, 779–782; (e) Y. Xu, Y. Suzuki,
K. Ito and M. Komiyama, Proc. Natl. Acad. Sci. U. S. A., 2010, 107,
14579–14584.
6 J. Jayawickramarajah, D. M. Tagore, L. K. Tsou and A. D. Hamilton,
Angew. Chem., Int. Ed., 2007, 46, 7583–7586.
7 D. Margulies and A. D. Hamilton, Angew. Chem., Int. Ed., 2009, 48,
1771–1774.
8 H. Zhu and F. D. Lewis, Bioconjugate Chem., 2007, 18, 1213–1217.
9 E. E. Merkina and K. R. Fox, Biophys. J., 2005, 89, 365–373.
10 N. Zhang, A. T. Phan and D. J. Patel, J. Am. Chem. Soc., 2005, 127,
17277–17285.
¨
11 I. Mikhalyov, N. Gretskaya, F. Bergstrom and L. B.-Å. Johansson,
Phys. Chem. Chem. Phys., 2002, 4, 5663–5670.
12 T. Ehrenschwender and H.-A. Wagenknecht, J. Org. Chem., 2011, 76,
2301–2304.
13 M. Dahim, N. K. Mizuno, X.-M. Li, W. E. Momsen, M. M. Monsen
and H. L. Brockman, Biophys. J., 2002, 83, 1511–1524.
14 Y. Tokoro, A. Nagai and Y. Chujo, Tetrahedron Lett., 2010, 51,
3451–3453.
15 T. Kawai, M. Ikegami and T. Arai, Chem. Commun., 2004, 824–825.
16 T. Ehrenschwender and H.-A. Wagenknecht, Synthesis, 2008,
3657–3662.
We also evaluated the sensitivity of this probe in terms of its
detection threshold, defined as the fluorescence signal that is
20% greater than that of the background signal. The detection
thresholds of the human 30-overhang sequences Htelo1,
Htelo2, and Htelo3 were 0.2, 0.05, and 0.02 mM, respectively
(Fig. 3); that is, they decreased upon increasing the length
of the G-rich sequence. In that respect, our system provides
ˆ
`
´
`
17 L. Bauer, K. Tluckova, P. Tothova and V. Viglask´y, Biochemistry,
2011, 50, 7484–7491.
18 H.-Q. Yu, D. Miyoshi and N. Sugimoto, J. Am. Chem. Soc., 2006, 128,
15461–15468.
19 A. J. Cesare and R. R. Reddel, Nat. Rev. Genet., 2010, 11, 319–330.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1717--1719 1719