Detection of three-base deletion by exciplex formation with perylene derivatives

Article abstract of DOI:10.1039/c1cc11041a

Here, we synthesized fluorescent DNA probes labeled with two perylene derivatives for the detection of a three-base deletion mutant. One such probe discriminated the three-base deletion mutant from the wild-type sequence by exciplex emission, and the dele

Full text of DOI:10.1039/c1cc11041a

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COMMUNICATION
Detection of three-base deletion by exciplex formation with perylene
derivativesw
Hiromu Kashida, Nobuyo Kondo, Koji Sekiguchi and Hiroyuki Asanuma*
Received 22nd February 2011, Accepted 19th April 2011
DOI: 10.1039/c1cc11041a
Here, we synthesized fluorescent DNA probes labeled with two
perylene derivatives for the detection of a three-base deletion
mutant. One such probe discriminated the three-base deletion
mutant from the wild-type sequence by exciplex emission, and
the deletion mutant was identifiable even by the naked eye.
deletions are called ‘‘in-frame’’ deletions, they do not cause a
frameshift, but rather amino acid deletions, and in some cases,
an essential residue is lost from a protein. Some in-frame
deletions are important as a crucial step in carcinogenesis.9
Thus, it is necessary to develop novel DNA probes for three-
base deletion polymorphisms.
Here, we synthesized two perylene derivatives (F, L in
Scheme 1B) and incorporated them into DNA probes for
the detection of three-base deletions. The F and L moieties
are better than the E moiety for the detection of three-base
deletion for several reasons. (1) Perylene derivatives can emit
excimer and exciplex emission.10–13 Thus, they are utilized as
fluorophores for our strategy. (2) These moieties are larger
than E so that they may form an excimer even in a five-base
bulge. (3) F and L have an extended p system so that the
absorption maximum should shift to a longer wavelength than
that of E. Consequently, the autofluorescence from the sample
should be suppressed. In addition, many commercial apparatus
are available for the detection of these longer wavelengths.
(4) The emission band of F substantially overlapped with the
absorption bands of L (vide infra) so that efficient FRET from
F to L led to enlarged apparent Stokes’ shift. In this communi-
cation, we first investigated spectroscopic behaviors of F and L
in DNA duplexes and then applied these fluorophores to the
detection of three-base deletion polymorphisms.
Recently, insertion/deletion polymorphisms (indels) have been
paid much attention as well as single-nucleotide polymorphisms
(SNPs) due to their frequent occurrence.1 Moreover,
small (1–4 bp) indels are more frequent than larger indels,
and some are related to genetic diseases.2 Although several
methods have been developed to detect indels,3–7 no fluorescent
probes have been developed to detect three-base deletion
polymorphisms.
Previously, we engineered perylene-labeled DNA probes
that could detect one-base deletion polymorphisms.8 The
general design of perylene-labeled probes that can detect
deletion mutants is shown in Scheme 1A. Two perylenes were
introduced into the middle of the probe on either side of the
target sequence. When the probe hybridized with wild-type
DNA, two perylenes (E in Scheme 1B) were intercalated
between base pairs, and the target base pair(s) interrupted
the interaction between the perylenes. However, when the
probe hybridized with a deletion mutant, a bulge that includes
two perylenes was formed, and the two perylenes, which were
in close proximity, interacted. Accordingly, monomer emission
from each perylene was quenched, and excimer emission with
a longer wavelength appeared. Perylene-labeled probes that
were able to detect two-base deletions were constructed by
changing the number of bases between perylenes (see Fig. S1A
in ESIw). However, three-base deletions could not be detected
using perylene-labeled DNA probes containing E (see Fig. S1B
in ESIw). Only monomer emission was observed from the
perylene-labeled DNA probes targeting three-base deletions
probably because two E perylenes were unable to establish
contact across five-base bulges including two dyes. Three-base
Phosphoramidite monomers with incorporated perylene
derivatives were synthesized through a phosphoramidite
method as shown in ESI.w14–16 Sequences synthesized in this
study are shown in Scheme 1B. First, we incorporated each
dye into the middle of 12 mer DNA oligonucleotides in order
to investigate the spectroscopic behaviors of these dyes
(F1a, L1a). In addition, the perylene moiety synthesized
previously was also introduced as a control (E1a).
Spectroscopic behaviors of these DNAs are shown in
Table 1. Absorption (lab.max) and emission maxima (lem.max
)
of the duplexes F1a/N and L1a/N were much longer than those
of E1a/N as we designed. In particular, lab.max of L1a/N was
as long as 484 nm. Interestingly, the F1a/N emission band
overlapped substantially with the L1a/N absorption band
(Fig. 1). Consequently, highly efficient FRET from F to L
was expected. Efficient FRET enables the enlargement of
apparent Stokes’ shift.17 Quantum yields (F) of these duplexes
are also shown in Table 1. F and L showed comparable
quantum yield to E; F of the duplexes F1a/N and L1a/N were
Graduate School of Engineering, Nagoya University, Furocho,
Chikusa-ku, Nagoya 464-8603, Japan.
E-mail: asanuma@mol.nagoya-u.ac.jp; Fax: +81-52-789-2528;
Tel: +81-52-789-2488
w Electronic supplementary information (ESI) available: Synthetic
schemes of phosphoramidite monomer tethering F and L, fluorescent
emission spectra of probes tethering E, UV-VIS spectra of duplexes.
See DOI: 10.1039/c1cc11041a
c
6404 Chem. Commun., 2011, 47, 6404–6406
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Scheme 1 (A) Schematic illustration of the detection of deletion polymorphisms. n represents the number of the bases deleted. (B) Sequences of
the modified DNA oligonucleotides synthesized in this study.
Table 1 Spectroscopic properties of duplexes containing perylene
derivatives
Sequence
lab.maxa/nm
lem.maxb/nm
Fc
Tmd/1C
E1a/N
F1a/N
L1a/N
452
467
484
462
479
502
0.65
0.56
0.32
46.1
49.7
37.8
a
b
Absorption maximum at 20 1C. Emission maximum at 20 1C.
Quantum yield determined from the quantum yield of perylene in
c
d
reference. Solution
N2-bubbled cyclohexane (0.78) used as
a
conditions: [DNA] = 2.0 mM, [NaCl] = 100 mM, pH 7.0 (10 mM
phosphate buffer).
Fig. 2 (A) Fluorescence emission spectra of FL/WT and FL/MUT at
20 1C. Excitation wavelength was 445 nm. Solution conditions were as
follows: [Probe] = 1.0 mM, [Target] = 1.2 mM, [NaCl] = 100 mM,
pH 7.0 (10 mM phosphate buffer). (B) A photograph of FL with wild
type (FL/WT) or three-base deletion mutant (FL/MUT) at RT.
Excitation wavelength was 400 nm. Solution conditions were as
follows: [Probe] = 1.0 mM, [Target] = 1.2 mM, [NaCl] = 100 mM,
pH 7.0 (10 mM phosphate buffer).
0.56 and 0.32, respectively. Accordingly, F and L moieties are
fluorophores with longer wavelength and without losing
detection sensitivity than E. Because F had a larger stacking
area than E, the melting temperature (Tm) of the duplex F1a/N
was higher than that of E1a/N.18 Although L is larger than F,
Tm of the duplex L1a/N was significantly lower than that of
the duplex F1a/N, probably due to steric hindrance.
when a probe hybridized with the mutant sequence (MUT), which
lacks three bases (50-AAG-30), the two fluorophores might interact
with each other. Thus, the wild-type sequence and deletion mutant
might be distinguished by monomer and excimer/exciplex
emissions, respectively, from probe–target duplexes.
Next, we attempted to detect a three-base deletion mutant
using DNA probes labeled with these fluorophores. The target
was the cystic fibrosis transmembrane conductance regulator
(CFTR) gene; a specific three-base deletion is responsible for
many cases of cystic fibrosis.4,19 The sequences of probes are
shown in Scheme 1B. We incorporated three bases (50-CTT-30)
with two fluorophores in each probe, and synthesized three
probes containing F and/or L (FF, LL, FL). We hypothesized
that when one of these probes hybridized with the wild-type
sequence (WT), the two fluorophores would intercalate within
the DNA duplex and monomer emission would occur, but
First, we evaluated the probes containing only F or L. The
results of hybridization of the FF probe with WT or MUT
sequences are shown in Fig. S2A. The duplex FF/WT showed
strong monomer emission of F at 476 and 509 nm, indicating
that the two Fs did not interact because of the intervening base
pairs. However, FF did not show excimer emission with MUT,
but decreased monomer emission due to quenching,
Fig. 1 Emission and absorption spectra of E1a/N, F1a/N and L1a/N at 20 1C. Each spectrum was normalized at the absorption or emission
maximum.
c
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Table 2 Detection ability and duplex stability of probe DNA
the spectral difference between wild-type and mutant samples
was easily visible to the naked eye.
a
Iex/Imon
Tmb/1C
This work was partially supported by a Grant-in-Aid for
Scientific Research (A) (21241031) and a Grant-in-Aid for
Young Scientists (B) (22750149) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
Partial support by SENTAN program, Japan Science and
Technology Agency (JST), is also acknowledged.
WT
MUT
WT
MUT
Probe
FF
LL
FL
0.11c,e
0.25d,f
0.25d,e
0.14c,e
0.33d,f
1.46d,e
57.5
41.0
49.2
40.6
34.1
42.6
a
Ratio of the intensity of excimer emission to monomer emission at
20 1C. [Probe] = 1.0 mM, [Target] = 1.2 mM, [NaCl] = 100 mM,
b
pH 7.0 (10 mM phosphate buffer). [Probe] = [Target] = 2.0 mM,
c
[NaCl] = 100 mM, pH 7.0 (10 mM phosphate buffer). I550/I476
.
Notes and references
d
e
. Excited at 445 nm. Excited at 455 nm.
f
I570/I507
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emission at 506 nm was observed with the duplex LL/MUT
(Fig. S2B). Thus, probes with homo FF or LL pairs did not
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exhibited exciplex emission from a five-base bulge (Fig. 2).
When FL was hybridized with WT, only monomer emission of
L was observed at 507 nm. These results indicated that F and L
were intercalated between base pairs and direct interaction
between F and L was suppressed. Furthermore, complete
disappearance of the emission band of F at 476 nm demon-
strates that highly efficient FRET occurred from F to L. In
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mutant (MUT), monomer emission of L decreased, and a
new band appeared at 556 nm concurrently.20 This new band
corresponded to the exciplex between F and L that formed
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that at 507 nm was 1.46 with the duplex FL/MUT whereas
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deletion was identifiable even by the naked eye (Fig. 2B); the
colors of FL/WT and FL/MUT were green and orange,
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Hetero-dimerization was also substantiated from the
UV-VIS spectrum; although spectra were rather complicated
due to overlaps of the absorption band of F and L (see Fig. S3
in ESIw), absorption spectrum of FL/MUT was completely
different from that of FL/WT. In particular, both hypo-
chromism and hyperchromism were observed at 450–500 nm
and 400–450 nm, respectively, with FL/MUT samples, indicating
that F and L were interacting excitonically to form a hetero-
dimer with FL/MUT. Spectral changes of FF/MUT and
LL/MUT were much smaller than that of FL/MUT. Only slight
hypochromism was observed. In addition, Tm of FL/MUT was
higher than those of FF/MUT and LL/MUT whereas Tm of
FL/WT was lower than that of FF/WT (Table 2). These results
indicate that F and L moieties in FL could form a dimer in the
bulge, but those in FF and LL could not. Consequently, strong
exciplex emission was observed only in FL/MUT.
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18 All the melting curves showed single transition and small
hysteresis. See Fig. S4 and S5 in ESIw.
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T. K. Cox, A. Chakravarti, M. Buchwald and L. C. Tsui, Science,
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20 Exciplex emission of F and L showed a maximum at about 25 nm
longer wavelength than excimer emission of E (see Fig. S6 in ESIw).
21 We believe that intervening bases between F and L are not flipped-
out from the duplex because natural nucleobases in five-base bulge
are accommodated within the DNA duplex. See: U. Dornberger,
A. Hillisch, F. A. Gollmick, H. Fritzsche and S. Diekmann,
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In conclusion, we engineered a fluorescent probe containing
size-expanded perylene derivatives, which have larger mole-
cular sizes than E. As a result, a three-base deletion poly-
morphism was successfully detected by monitoring exciplex
emissions from a perylene-labeled DNA probe. In addition,
22 The detection limit was estimated as 100 nM. This limit can be
improved by increasing the number of sequences. See Fig. S7 and S8
in ESIw.
c
6406 Chem. Commun., 2011, 47, 6404–6406
This journal is The Royal Society of Chemistry 2011