Twin probes as a novel tool for the detection of single-nucleotide polymorphisms

Article abstract of DOI:10.1002/chem.200501526

Single-nucleotide polymorphisms (SNPs) are the most common form of DNA sequence variation. There is a strong interest from both academy and industry to develop rapid, sensitive and cost effective methods for SNP detection. Here we report a novel structura

Full text of DOI:10.1002/chem.200501526

FULL PAPER
Chem. Eur. J. 2006, 12, 3707 – 3713
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3707

DOI: 10.1002/chem.200501526
Twin Probes as a Novel Tool for the Detection of Single-Nucleotide
Polymorphisms
Erhan Ergen, Markus Weber, Josemon Jacob, Andreas Herrmann,* and Klaus Müllen*[a]
Abstract: Single-nucleotide polymor-
phisms (SNPs) are the most common
form of DNA sequence variation.
There is a strong interest from both
academy and industry to develop rapid,
sensitive and cost effective methods for
SNP detection. Here we report a novel
structural concept for DNA detection
based on fluorescence dequenching
upon hybridization. The so-called “twin
probe” consists of a central fluorene
derivative as fluorophore to which two
identical oligonucleotides are covalent-
ly attached. This probe architecture is
applied in homogeneous hybridization
assays with subsequent fluorescence
spectroscopic analysis. The bioorganic
hybrid structure is well suited for se-
quence specific DNA detection and
even SNPs are identified with high effi-
ciency. Additionally, the photophysical
properties of the twin probe were in-
vestigated. The covalent attachment of
two single stranded oligonucleotides
leads to strong quenching of the central
fluorescence dye induced by the nu-
A
N
ized by supramolecular aggregate for-
mation accompanied by red-shifted
emission and broad fluorescence spec-
tra.
Keywords: DNA detection · DNA ·
fluorescentprobes · hybridization
Introduction
beacons.[11,12] Instead of using interactions between two ex-
trinsic probes, interactions of one fluorophore with DNA
bases can be used for specific detection of nucleic acid se-
quences.[13,14] Besides the use of fluorescein,[15,16] very recent-
ly fluorene has also been incorporated into the loop struc-
ture of a hairpin architecture.[17] A single base mismatch was
detected by de-quenching upon hybridization with the fully
matched target and quenching upon hybridization with a
target containing a single base mutation.
In this paper, we report a novel structural concept for
DNA detection. The so-called “twin probe” consists of a
single central fluorophore to which two identical oligonu-
cleotides have been covalently attached. The twin probe is
applied in a homogeneous DNA detection assay. SNPs
could be detected upon hybridization of the twin probe and
a target sequence with high efficiency.
With the completion of the human genome project, maps of
human sequence variations have been reported and deposit-
ed to public databases.[1–3] Among these variations, single-
nucleotide polymorphisms (SNPs) are the most frequent
and according to the International SNP Map Working
Group, the occurrence of SNPs in human genes is approxi-
mately 1.42 million, 60000 of them being located in exons.[4]
Since SNPs are assumed to play an important role in deter-
mining the genetic predisposition towards inherited diseases,
disease diagnosis, and personalized medicine,[5] there is a
strong interest in developing rapid, sensitive, and cost effec-
tive detection methods. Various detection schemes are pres-
ently available for SNPs.[6,7] When utilizing hybridization in
homogeneous solution, a few formats have been established
which make use of fluorescence resonance energy transfer
(FRET).[8] The spatially close arrangement of a donor and
an acceptor is realized in TaqMan probes[9,10] and molecular
Results and Discussion
As a central emitter, the fluorene derivative 2 was chosen
for two reasons. First, it is an efficient blue emitter with a
high fluorescence quantum efficiency in organic solvents[18,19]
as well as water.[20] Second, at a later stage it will be advan-
tageous to extend this concept from monomeric to polymer-
ic emitters, which have already found applications as biosen-
sors.[21–23]
[a] E. Ergen, M. Weber, Dr. J. Jacob, Dr. A. Herrmann,
Prof. Dr. K. Müllen
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
Fax : (+49)6131-379-350
E-mail: andherrm@mpip-mainz.mpg.de
muellen@mpip-mainz.mpg.de
3708
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3707 – 3713

Twin Probes
FULL PAPER
Scheme 1. Synthesis of fluorescent core and oligonucleotide conjugates: a) 4-Carboxymethylphenylboronic acid, K2CO3, THF/H2O, [Pd(PPh3)4], 808C,
N
16 h, 73%; b) KOH, EtOH/H2O, reflux, 3 h, 88%; c) N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), DMF, RT, overnight; d) 5’-amino
modified oligonucleotide, DMF/sodium tetraborate decahydrate buffer (pH 8.5), RT, overnight.
The synthesis of the core fluorophore started from the
well-known fluorene derivative 1 (Scheme 1).
Compounds 4 and 5 exhibited a significant lower electro-
phoretic mobility than the amino modified oligonucleotide,
which was used as a starting material. With MALDI-TOF
mass spectrometry the identity of 5 was confirmed (Fig-
ure 1B).
2,7-Dibromo-9,9-di-n-octylfluorene (1) was treated in a
palladium-catalyzed Suzuki coupling[24] with 4-carboxymeth-
ylphenylboronic acid. Alkaline hydrolysis of the resulting
diester generated the biscarboxylic acid 2. Subsequentacit-
vation of the carboxylic acids was carried out with N-hy-
droxysuccinimide. The corresponding active ester 3 was
treated with a 17-mer oligonucleotide sequence (5’-CTCA-
GATCTGGTCTAAC-3’) carrying a 5’-amino linker to gen-
erate the desired twin probe 5 along with the monoconju-
gate 4 as a by-product. Purification of the target product 5
and monoconjugate 4 was performed by polyacrylamide gel
electrophoresis (PAGE) using a 20% denaturing gel. The
purity of 4 and 5 was proven by PAGE analysis (Figure 1A).
Two identical 5’-amino modified oligonucleotide sequen-
ces were used for coupling to the fluorophore core as these
modifications can be more easily accessed by automated
synthesis in contrast to 3’-modifications. Furthermore, 5’-
modified oligonucleotides are commercially available so
that the twin probe 5 can be synthesized in a standardized
fashion without the need for an oligonucleotide synthesizer.
To study the influence of the fluorene moiety and the at-
tached C6-alkyl spacers on duplex formation, the melting
temperature (Tm) of the twin probe 5 and its complementary
sequence with that of the free oligonucleotide with the same
sequence were compared. The melting temperature Tm of 5
was 588C; whereas, the free oligonucleotides exhibited a Tm
of 578C. This small difference in Tm, which lies within the
experimental error of the measurement, indicates that there
are only weak interactions between the chromophore and
the attached oligonucleotide sequence.
To test the suitability of twin probe 5 for SNP detection,
hybridization experiments were carried out (Table 1,
Figure 2). The nonhybridized twin probe 5 showed low fluo-
rescence intensity in buffer solution upon excitation with
350 nm. Hybridization of the twin probe 5 with the target
sequence ODN1, which fully matched the probe sequence,
led to a ten-fold increase in fluorescence intensity. Simulta-
Figure 1. A) Polyacrylamide gel electrophoresis analysis of
a) Oligonucleotide (5’-NH2-(CH2)6-CTCAGATCTGGTCTAAC-3’);
b) monoconjugate 4; c) twin probe 5. B) MALDI-TOF mass spectrum of
5 (calcd: 11241 gmolÀ1; found: 11187 gmolÀ1).
4 and 5.
Chem. Eur. J. 2006, 12, 3707 – 3713
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3709

A. Herrmann, K. Müllen etal.
A
from 432 to 417 nm. When 5 was incubated with a noncom-
plementary target ODN2 only a 1.6-fold enhancement of
the fluorescence intensity was observed, which is accompa-
nied by a hypochromic shiftof hte emission maxima from
432 to 439 nm. On the basis of the hybridization results of
the twin probe 5 with the fully matching and a noncomple-
mentary sequence, the possibility of detecting a single-nu-
cleotide mismatch was investigated. Accordingly, two target
sequences ODN3 and ODN4 containing an A to C and an
A to G mismatch, respectively, were examined. Upon hy-
bridization, the mismatch is located within the helix of the
probe and target sequences (Table 1). When 5 was hybri-
dized with the mismatched sequences ODN3 and ODN4,
the emission intensities dropped to 49 and 25%, respective-
ly, compared with the signal obtained with the fully matched
sequence ODN1. Upon duplex formation of the mismatched
sequences ODN3 and ODN4 with probe 5, the emission
maxima shifted hypsochromically from 432 to 420 nm
(Figure 1, Table 1). Very similar emission behavior of 5
upon hybridization was found for probe concentrations
Figure 2. Emission spectra of the twin probe 5 (1.7”10À7 m) and corre-
sponding duplexes of 5 with ODN1 to ODN4. All oligonucleotides have
been used in two-fold excess with respect to 5: a) nonhybridized twin
probe 5, b) noncomplementary ODN2 + 5, c) A to G mismatch ODN4
+ 5, d) A to C mismatch ODN3 + 5, and e) complementary ODN1 +
5. Fluorescence spectra were obtained using an excitation wavelength of
350 nm and were recorded at room temperature. Hybridization buffer
solutions contained 100 mm Tris base, 100 mm NaCl, and 50 mm MgCl2.
À8
ranging from 10À5 t o 10 m. Especially, the lower limit is of
importance since a typical polymerase chain reaction results
in equal amounts of DNA.[25] The fluorescence measure-
ments (Table 1), clearly demonstrated that the novel twin
probe architecture is well
suited for sequence specific
Table 1. Fluorescence spectroscopic analysis of the twin probe 5 and the corresponding hybridization experi-
DNA assays and even a single
base mismatch could be detect-
ed with a discrimination factor
of up to 4.
Hybridization experiments
were also carried out with the
fluorene derivative 4 connect-
ed only to a single-oligonucleo-
tide sequence. This compound
ments with different target sequences. Mismatches are in italic.
Sequence
Rel. fluo
G
Em. max. [nm]
5
probe: 5’NH2-CTCAGATCTGGTCTAAC-3’
comp: 3’-GAGTCTAGACCAGATTG-5’
noncomp: 3’-ACTCTTATCACATACGC-5’
A to C: 3’-GAGTCCAGACCAGATTG-5’
A to G: 3’-GAGTCGAGACCAGATTG-5’
432
417
439
420
420
ODN1
ODN2
ODN3
ODN4
exhibited high fluorescence intensity in buffer solution.
Upon hybridization with its complementary sequence, a de-
crease in fluorescence intensity was observed (data not
shown). Since the aim was to generate a system, which pro-
duces an increase in the fluorescence signal upon hybridiza-
tion, monoconjugate 4 was not suited for such detection pur-
poses. However, to gain further insight into the factors that
determine the fluorescence behavior of the central fluoro-
phore within twin probe 5, monoconjugate 4 is well suited
for comparative studies. To investigate the effect of the four
different nucleotides occurring in DNA on the optical prop-
erties of the fluorene moiety, additional quenching experi-
ments were performed with 4 and A16, G16, C16, T16, respec-
tively. Monoconjugate 4 has been utilized in the quenching
experiments because contrary to 2, itis readily soluble in
buffer solutions. The fluorescence intensities of the 4 (1.6”
10À7 m) in the presence of various 16-mer concentrations
ranging from 7.0”10À5 to 1.5”10À3 m were measured and ex-
pressed in Stern–Volmer plots.[26] With increasing concentra-
tions of A16, G16, C16, and T16, a decrease in the emission in-
tensity of 4 was observed. An exponential relationship in
the Stern–Volmer plot was obtained for quencher concentra-
tions higher than 5.0”10À4 m (data not shown), which can be
explained by sphere-of-action quenching also present in
other polyelectrolyte systems.[27] For oligonucleotide concen-
trations below 5.0”10À4 m, a linear dependence in the Stern–
Volmer plotwas obatined (Figure 3) from which KSV values
were calculated. The most efficient quencher was G16 with a
KSV of 5.1”10À4 mÀ1. The KSV values measured for C16 and
T16 were 3.8”10À4 mÀ1 and 3.5”10À4 mÀ1, respectively, the dif-
ference in magnitude being within the uncertainty of the
measurement. The oligonucleotide with the lowest effect on
the emission intensity of the fluorene moiety was A16 with a
KSV of 1.3”10À4 mÀ1. The data from the quenching experi-
ments were also in agreement with the results obtained
from hybridization. An A to G mismatch in ODN4 led to a
higher extent of quenching than the A to C mismatch in
ODN3 underpinning the fact that G was the most efficient
quencher for fluorene connected to oligonucleotides in con-
jugates 4 and 5.
Fluorescence quenching by nucleobases also referred to
as base quenching has been mentioned previously. Photoin-
3710
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3707 – 3713

Twin Probes
FULL PAPER
ther increase of the fluorescence intensity for the matching
ODN5 and no changes for ODN6. Equal amounts of avidin
with respect to biotin groups were used. More important,
the biotin–avidin interaction induced a significant hypso-
chromic shift of the emission wavelengths from 445 to
402 nm for the hybridization product of 5 and ODN5
(Table 2, Figure 4). For the noncomplementary oligonuleo-
tide ODN6 no changes of the fluorescence spectrum was ob-
served.
This shift of the emission wavelengths suggests that the
binding of biotin and avidin molecules in the hybridization
buffer interferes with the p-stacking and/or hydrophobic in-
teractions of the emitter units, supporting the hypothesis
that supramolecular aggregate formation plays an important
role for the twin probe nucleic acid detection system.
After elucidating the factors that determine the photo-
physical properties of the twin probe system, the generality
of the new detection system regarding variations in se-
quence composition needs to be demonstrated. It should be
Figure 3. Stern–Volmer plots measured in hybridization buffer for G16
!
~
&
( ), C16 (&), T16
( ), and A16 ( ) with the monoconjugate 4 (conc. 1.6”
10À7 m). The emission spectra were measured upon excitation at 350 nm.
duced electron transfer plays an important role for the
mechanism of quenching of the excited dyes. However,
other effects like coupled proton transfer and hydrophobic
interactions need to be considered as well in the context of
fluorescence quenching.[28] Due to the good electron donat-
ing properties of guanosine residues, this base has been re-
ported to quench the emission of other fluorescent dyes effi-
ciently.[15,29] The fluorescence measurements performed with
the monoconjugate 4 suggest that through the covalent at-
tachment of the second oligonucleotide chain, as realized in
the bisconjugate 5, the quencher concentration is becoming
high enough to induce a low fluorescence emission from the
fluorene emitter in the nonhybridized probe 5.
The broad fluorescence spectra of 5, and its hybridization
products, are a hint that besides base quenching, also aggre-
gate formation of the hydrophobic fluorene units influences
the emission behavior of twin probe 5. The aggregation of
fluorene units, leading to long wavelength emission and
broadened spectra, is well known and has been investigated
intensively in the context of polymeric materials.[30] To gain
further insight into aggregate formation of the central emit-
ter unit, avidin–biotin binding was applied. A biotin moiety
was introduced into the target strands, which are either fully
complementary or noncomplementary to the probe se-
quence of 5. Hybridization of twin probe 5 with the biotinyl-
Figure 4. Emission spectra of the twin probe 5 (2.8”10À7 m) and the cor-
responding hybridization products with biotinylated target sequences
without and in the presence of avidin. The target sequences were added
in twofold excess with respect to 5. a) Nonhybridized twin probe 5, b)
noncomplementary biotin modified ODN5 + 5, c) biotin-modified com-
plementary ODN6 + 5, d) biotin-modified complementary ODN6 + 5
in the presence of equimolar amounts of avidin with respect to biotin
groups. Fluorescence spectra were obtained using an excitation wave-
length of 350 nm and were recorded at RT. Hybridization buffer solutions
contained 100 mm Tris base, 100 mm NaCl, and 50 mm MgCl2.
ated oligonucleotides led in the
case of the fully matching se-
quence to a 5.4-fold increase of
Table 2. Fluorescence spectroscopic analysis of the hybridization experiments of the twin probe 5 and biotin-
B
the
fluorescence
intensity,
ylated target sequences in the presence of and without avidin. The biotin-modified bases are indicated by
.
whereas with the noncomple-
mentary sequence only a 1.6-
fold increase was obtained
(Figure 4, Table 2). The addi-
tion of avidin to both hybridiza-
tion solutions resulted in a fur-
Sequence
Rel. fluorescence int. [%] Em. max. [nm]
G
5
probe: 5’NH2-CTCAGATCTGGTCTAAC-3’
26
446
442
403
447
448
ODN6 comp-biotin: 3’-GAGTCTBAGACCAGATTG-5’
ODN6 comp-biotin+avidin: 3’-GAGTCTBAGACCAGATTG-5’
ODN5 noncomp biotin: 3’-AATACTBCACTTTACACT-5’
ODN5 noncomp biotin+avidin: 3’-AATACTBCACTTTACACT-5’
100
114
42
45
Chem. Eur. J. 2006, 12, 3707 – 3713
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3711

A. Herrmann, K. Müllen etal.
noted that the 17-mer oligonucleotide sequence (5’-CTCA-
GATCTGGTCTAAC-3’) of 5 can adopt a loop structure by
base pairing of the italicized oligonucleotides which might
influence the results of fluorescence measurements. There-
fore, twin probes 6 (5’-CGCTTCATTTGTTCTCCC-3’) and
7 (5-CTGACTGGGTTGAAGGCTCT-3’) were synthesized
by the same procedure as described above. The sequences
of 6 and 7 were designed to avoid any secondary structure
formation. The results of the hybridization experiments with
subsequentfluorescence specrtoscopic analysis are summar-
ized in Table 3.
Conclusion
In summary, a novel architecture for DNA detection was
presented, the so-called “twin probe”, where two identical
oligonucleotide sequences were attached to a central fluo-
rene emitter. The synthesis of the twin probe was carried
out in four straightforward synthetic steps. The twin probe
system appears to be well suited for sequence specific DNA
detection by means of fluorescence in a homogeneous hy-
bridization assay. The twin probe shows a remarkable selec-
tivity that even allows identification of different SNPs. The
general applicability of this nu-
cleic acid detection scheme was
Table 3. Fluorescence spectroscopic analysis of the twin probes 6 and 7 as well as the corresponding hybridiza-
demonstrated regarding se-
quence composition. Two fac-
tors seem to influence the pho-
tophysical properties of the
twin probe. First, the attach-
ment of two oligonucleotides to
the central emitter leads to effi-
cientquenching of ht e fluorene
by the nucleobases. Second, su-
pramolecular aggregate forma-
tion induced by the fluorene
leads to broad red-shifted emis-
tion experiments with different target sequences. Mismatches are in italic.
Sequence
Rel. fluorescence int. [%]
Em. max. [nm]
6
probe:5’NH2-CGCTTCATTTGTTCTCCC-3’
comp: 3’-GCGAGAACAAATGAAGCG-5’
C to A: 3’-GCGAAATAAACAAGAGGG-5’
noncomp: 3’-TGTACTGCCTCCAACA-5’
probe: 5’NH2-CTGACTGGGTTGAAGGCTCT3’
comp: 3’-GACTGACCCAACTTCCGAGA-5’
T to G: 3’-GACTGGCCCCAACTTCCGAGA-5’
noncomp: 3’-AAGTTAGGTGGGCCTAATTT-5’
48
100
66
50
20
100
62
38
436
438
442
444
421
430
439
421
ODN7
ODN8
ODN9
7
ODN10
ODN11
ODN12
For twin probes 6 and 7, a similar behavior as for 5 was
detected. When not hybridized, 6 and 7 exhibita low fluo-
rescence intensity. Upon base pairing with the fully comple-
mentary sequences, the emission increased two- and five-
fold, respectively. Addition of the noncomplementary se-
quences only slightly increased the fluorescence intensity.
As described for 5, SNPs were also detected with 6 and 7.
These measurements demonstrated that secondary struc-
ture formation of the attached ODNs to the fluorene emit-
ter only slightly influenced the performance of the detection
assay. More important, the general applicability of the novel
twin probe system with respect to sequence composition
was proved.
sion. And finally, the aggregates can be destroyed by the
strong biotin–avidin interaction.
To broaden the field of application of twin probes, such as
real-time PCR, the sites of oligonucleotide attachment will
be varied. Future work will also be dedicated to transfer our
novel detection concept to oligo- and polyfluorenes.
Experimental Section
Unless otherwise specified, materials were obtained from commercial
suppliers and used without further purification. 2,7-Dibromofluorene was
purchased from Aldrich and used as such. N-Hydroxysuccinimide (NHS)
was purchased from Acros, N,N’-dimethylformamide (DMF) from Fluka,
and 1,3-dicyclohexylcarbodiimide (DCC) from Merck. Dimethoxytrityl
(DMTr) protected phosphoramidites, trityl protected C6-amino linker
and polystyrene/divinylbenzene support were purchased from Perbio,
Glen Research and Amersham Biosciences, respectively. 1H and
In TaqMan probes[9,10] and molecular beacons,[11,12] donor
and acceptor dyes must be covalently attached to the termi-
ni of the probe sequences. Even though both methods are
applied commercially, each method suffers from dual-label-
ing of DNA. This ultimately results in low yields, while the
presence of impurities can adversely affect the sensitivity of
the assay.[31] Another drawback is that although design algo-
rithms for the sequences are well elaborated, both probe se-
quences require significant optimization and redesign. The
twin probe system offers several advantages, most noticea-
13C NMR spectra were recorded on
a Bruker AMX 250 (250 and
62.5 MHz, respectively) spectrometer. Molecular weights were deter-
mined using matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry (MS), using a Bruker MALDI-TOF
(Reflex-TOF) mass spectrometer. Field desorption (FD) mass spectra
were recorded on a VG Instruments ZAB2-SE-FPD spectrometer.
2,7-Dibromo-9,9-di-n-octylfluorene (1): Synthesized according to the lit-
erature.[33]
bly, the ability to introduce the fluorene moiety
3
2,7-Bis(4-phenylcarboxylic acid)-9,9-di-n-octylfluorene (2): Diacid func-
(Scheme 1) between virtually any sequence of choice. Also,
the need for dual labeling of an oligonucleotide at both the
5’- and 3’-ends can be eliminated, which makes the twin
probe assay relatively inexpensive. In addition, one of the
3’-ends in the twin probe, or one of the alkyl chains of the
fluorene moiety,[32] could be used for immobilization to
transfer the twin probe assay into a chip-based format.
tionalized fluorene
(Scheme 1).
2 was synthesized in two steps starting from 1
a) 2,7-Bis(4-phenylcarboxylic acid methyl ester)-9,9-di-n-octylfluorene:
2,7-Dibromo-9,9-di-n-octylfluorene (1; 1.06 g, 1.94 mmol), 4-carboxy-
A
3.88 mmol) were dissolved in THF (12 mL) and water (6 mL) in a
Schlenk flask. The solution was purged with argon for 15 min, tetrakis-
AHCTREUNG
3712
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3707 – 3713

Twin Probes
FULL PAPER
into dichloromethane, washed with brine, and dried over MgSO4. The
crude product was recrystallized from ethanol to generate the pure di-
Willey, S. E. Hunt, C. G. Cole, P. C. Coggill, C. M. Rice, Z. M. Ning,
J. Rogers, D. R. Bentley, P. Y. Kwok, E. R. Mardis, R. T. Yeh, B.
Schultz, L. Cook, R. Davenport, M. Dante, L. Fulton, L. Hillier,
R. H. Waterston, J. D. McPherson, B. Gilman, S. Schaffner, W. J.
Van Etten, D. Reich, J. Higgins, M. J. Daly, B. Blumenstiel, J. Bald-
win, N. S. Stange-Thomann, M. C. Zody, L. Linton, E. S. Lander, D.
Altshuler, Nature 2001, 409, 928.
A
CD2Cl2, 258C): d = 8.12 (d, J=8.5 Hz, 4H), 7.80 (m, 6H), 7.67 (m, 4H),
3.93 (s, 6H), 2.10 (t, J=8.9 Hz, 4H), 1.20–1.09 (m, 20H), 0.83–0.68 (m,
10H); 13C NMR (62.5 MHz, CD2Cl2, 258C): d = 167.53, 152.79, 146.63,
141.54, 139.80, 130.74, 129.70, 127.78, 127.05, 122.49, 121.09, 56.27, 52.74,
41.05, 32.51, 30.68, 29.94, 29.92, 24.59, 23.34, 14.56; FDMS: m/z: calcd:
658.93; found: 659.40; elemental analysis calcd (%): C 82.03, H 8.26;
found C 81.87, H 8.26.
[5] http://www.ornl.gov/sci/techresources/Human_Genome/project/jour-
nals/insights.html/
[6] M. M. Shi, Clin. Chem. 2001, 47, 164.
[7] P.-Y. Kwok, Annu. Rev. GenomicsHum. Genet. 2001, 2, 235.
[8] R. M. Clegg, MethodsEnzymol. 1992, 211, 353.
[9] K. J. Livak, S. J. A. Flood, J. Marmaro, W. Giusti, K. Deetz, PCR
MethodsAppl. 1995, 4, 357.
[10] M. M. Shi, S. P. Myrand, M. R. Bleavins, F. A. Iglesia, J. Clin. Pathol.
1999, 52, 295.
[11] G. Bonnet, S. Tyagi, A. Libchaber, F. R. Kramer, Proc. Natl. Acad.
Sci. USA 1999, 96, 6171.
[12] S. A. E. Marras, F. R. Kramer, S. Tyagi, Genet. Anal. 1999, 14, 151.
[13] O. Kohler, D. Venkatrao, D. V. Jarikote, O. Seitz, ChemBioChem
2005, 6, 69.
[14] T. Ishiguro, J. Saitoh, H. Yawata, M. Otsuka, T. Inoue, Y. Sugiura,
Nucleic AcidsRes. 1996, 24, 4992.
[15] A. O. Crockett, C. T. Wittwer, Anal. Biochem. 2001, 290, 89.
[16] C. P. Vaughn, K. S. J. E. Johnson, Am. J. Pathol. 2003, 1, 29.
[17] G. T. Hwang, Y. J. Seo, B. H. Kim, J. Am. Chem. Soc. 2004, 126,
6528.
[18] A. W. Grice, D. C. C. Bradley, M. T. Bernius, M. Inbasekaran, W. W.
Wu, E. P. Woo, Appl. Phys. Lett. 1998, 73, 629.
[19] C. Ego, D. Marsitzky, S. Becker, J. Y. Zhang, A. C. Grimsdale, K.
Müllen, J. D. MacKenzie, C. Silva, R. H. Friend, J. Am. Chem. Soc.
2003, 125, 437.
[20] M. Stork, B. S. Gaylord, A. J. Heeger, G. C. Bazan, Adv. Mater.
2002, 14, 361.
[21] L. H. Chen, D. W. McBranch, H. L. Wang, R. Helgeson, F. Wudl,
D. G. Whitten, Proc. Natl. Acad. Sci. USA 1999, 96, 12287.
[22] D. L. Wang, X. Gong, P. S. Heeger, F. Rininsland, G. C. Bazan, A. J.
Heeger, Proc. Natl. Acad. Sci. USA 2002, 99, 49.
[23] H. A. Ho, M. Boissinot, M. G. Bergeron, G. Corbeil, K. Dore, D.
Boudreau, M. Leclerc, Angew. Chem. 2002, 114, 1618; Angew.
Chem. Int. Ed. 2002, 41, 1548.
[24] N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457.
[25] J. Sambrook, D. W. Russel, Molecular Cloning, Cold Spring Harbour
Laboratory Press, New York, 2001.
[26] B. Valeur, Molecular Fluorescence: Principles and Applications,
Wiley-VCH, Weinheim, 2001.
[27] J. Wang, D. Wang, E. K. Miller, D. Moses, G. C. Bazan, A. J.
Heeger, Macromolecules 2000, 33, 5153.
[28] C. A. M. Seidel, A. Schulz, H. M. Sauer, J. Phys. Chem. 1996, 100,
5541.
b) 2,7-Bis(4-phenylcarboxylic acid)-9,9-di-n-octylfluorene (2): A mixture
of above diester (0.70 g, 1.06 mmol), KOH (0.80 g, 14 mmol), THF
(10 mL) and H2O (5 mL) was heated under reflux for 16 h. The reaction
was cooled to room temperature and then acidified with concentrated
HCl. The precipitated white solid was washed several times with water
and then ethanol, and dried under vacuum (0.59 g, 88%). 1H NMR
(250 MHz, [D6]DMSO, 258C): d = 13.03 (brs, 2H), 8.07 (d, J=8.5 Hz,
4H), 7.91 (m, 8H), 7.74 (d, J=7.9 Hz, 2H), 2.11 (m, 4H), 1.20–0.56 (m,
30H); 13C NMR (62.5 MHz, [D6]DMSO, 258C): d
= 167.10, 151.49,
144.49, 140.24, 138.06, 129.88, 129.36, 126.72, 125.99, 121.22, 120.64, 55.06,
40.45, 31.03, 28.99, 28.35, 28.33, 23.21, 21.91, 13.74; FDMS: m/z: calcd:
630.88; found: 630.70.
Disuccimidyl ester of 2,7-bis(4-phenylcarboxylic acid)-9,9-di-n-octylfluo-
rene (3): Diacid 2 (20 mg, 0.03 mmol) and NHS (18.25 mg, 0.15 mmol)
were dissolved in dry N,N’-dimethylformamide (DMF) (2 mL) under
argon. To this solution, DCC (18.55 mg, 0.09 mmol) dissolved in dry
DMF (1.5 mL) was added dropwise and stirred overnight at room tem-
perature. The completion of reaction was determined by TLC (methylene
chloride) and FDMS. FDMS: m/z: calcd: 825.0; found: 825.24.
5’-Amino-modified oligodeoxyribonucleotide (ODN): The amino modi-
fied oligonucleotide, 5’-NH2-C6-CTCAGATCTGGTCTAAC-3’ was syn-
thesized by standard solid-phase DNA chemistry using the phosphorami-
dite method[34] on a polystyrene/divinylbenzene support. After the syn-
thesis, deprotection of the oligonucleotides was carried out in a suspen-
sion of 37% ammonia at50 8C, overnight. The trityl group was deprotect-
ed under standard conditions by stirring in acetic acid/H2O (4:1) for 2 h
at room temperature. The resulting oligonucleotide was dried under
vacuum overnightand purified by ion-exchange HPLC.
ODN–fluorene conjugates: Active ester 3 (0.5 mg, 0.6 mmol) was dis-
solved in a small amountof dry DMF (25 mL) and added to the 5’-amino
modified oligonucleotide (ODN)[14] (19 mg, 3.6 mmol) in a total volume
of 250 mL of 0.10m sodium tetraborate buffer (pH 8.5) and shaken over-
night at room temperature. Purification of the products was performed
using a 20% denaturing polyacrylamide gel (100 V, 2 h) with trisborate/
EDTA buffer (90 mm Tris, 90 mm boric acid, 2 mm EDTA) as the running
buffer. Identification of conjugates 4 and 5 was achieved by UV shadow-
ing. The respective bands were excised from the gel and incubated in Tris
buffer (pH 7) overnightat37 8C. Products 4 and 5 were obtained after fil-
tration through a filter with 22mm pores.
[29] T. Heinlein, J. P. Knemeyer, O. Piestert, M. Sauer, J. Phys. Chem. B
2003, 107, 7957.
[30] S. Setayesh, A. C. Grimsdale, T. Weil, V. Enkelmann, K. Müllen, F.
Meghdadi, E. J. W. List, G. Leising, J. Am. Chem. Soc. 2001, 123,
946.
[31] J. P. Knemeyer, N. Marme, M. Sauer, Anal. Chem. 2000, 72, 3717.
[32] X. Chen, J.-L. Liao, Y. Liang, M. O. Ahmed, H.-E. Tseng, S.-A.
Chen, J. Am. Chem. Soc. 2003, 125, 636.
Acknowledgements
This work, as part of the European Science Foundation EUROCORES
Programme BIONICS, was supported by funds from the DFG and the
EC Sixth Framework Programme.
[33] M. Fukuda, K. Sawada, K. Yoshino, J. Polym. Sci. Part A: Polym.
Chem. 1993, 31, 2465.
[34] M. H. Caruthers, Acc. Chem. Res. 1991, 24, 278.
[1] http://genome.ucsc.edu/
[2] http://www.ensembl.org/
[3] http://www.ncbi.nlm.nih.gov/
[4] R. Sachidanandam, D. Weissman, S. C. Schmidt, J. M. Kakol, L. D.
Stein, G. Marth, S. Sherry, J. C. Mullikin, B. J. Mortimore, D. L.
Received: December 6, 2005
Published online: March 7, 2006
Chem. Eur. J. 2006, 12, 3707 – 3713
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3713