Intelligent fluorescent nucleoside in sensing cytosine base: Importance of hydrophobic nature of perylene fluorophore

Article abstract of DOI:10.1016/j.bmcl.2006.09.039

Fluorescence response upon hybridization of perylene labeled oligonucleotide probes depends on the microenvironment experienced by the perylene fluorophore. In mismatched duplex (^PerU-C), enhanced fluorescence was observed while in matched dupl

Full text of DOI:10.1016/j.bmcl.2006.09.039

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6338–6341
Intelligent fluorescent nucleoside in sensing cytosine base:
Importance of hydrophobic nature of perylene fluorophore
a
a
a
b
Subhendu Sekhar Bag, Yoshio Saito, Kazuo Hanawa, Satoshi Kodate,
b
a,
*
Isamu Suzuka and Isao Saito
a
Contribution From NEWCAT Institute, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
b
Department of Materials Chemistry and Engineering, School of Engineering, Nihon University, Koriyama,
Fukushima 963- 8642, Japan
Received 12 July 2006; revised 1 September 2006; accepted 5 September 2006
Available online 10 October 2006
Abstract—Fluorescence response upon hybridization of perylene labeled oligonucleotide probes depends on the microenvironment
experienced by the perylene fluorophore. In mismatched duplex ( U-C), enhanced fluorescence was observed while in matched
Per
Per
duplex ( U-A) fluorescence intensity decreased considerably. This observation will be a promising research effort in giving rise
to a new powerful tool in detection of SNP.
Ó 2006 Elsevier Ltd. All rights reserved.
The completion of the Human Genome Project, after 50
years of Watson–Crick, has set the stage to the biomed-
ical researchers toward the conceptual basis of develop-
ment of personalized medicine through the screening of
opposite to that of the corresponding pyrene or anthra-
5
cene derivatives. Being inspired by the hydrophobic
6
,7
nature of the perylene fluorophore, we thought that
it will be worthwhile to tether perylene carboxamide into
oligonucleotides and follow up the fluorescence behav-
ior in presence of complementary sequences. We
hypothesized that upon binding with its fully matched
complementary sequence, the fluorescence of the peryl-
ene chromophore would be quenched as a result of
exposing toward more polar aqueous environment.
But in the presence of mismatched sequence, however,
it would involve in intercalative stacking interaction in
the mispaired position (because of the lack of Watson–
Crick base pairing) of the duplex, facing more hydro-
phobic organic microenvironment and thus mismatch
containing duplex detection will be accomplished by
fluorescence enhancement. This concept is completely
opposite to the case of pyrene chromophore which upon
hybridization with mismatched target oligonucleotide
1
genetic mutations. Among all other mutations, single
nucleotide polymorphisms (SNPs) are the most common
form of genetic mutation. These genetic polymorphisms
are often diagnostic of particular genetic predispositions
2
toward diseases and drug responses. The detection and
targeting of such SNPs in an array of disease related
genes provides an avenue for the rational design and
the realization of new diagnostics and chemotherapeu-
tics. Recently, a number of methods based on oligonu-
cleotide probes sequence have been developed for SNP
3
typing, and all the research efforts are aimed to detect
the fluorescence signal generated upon hybridization
with complementary fully matched target oligonucleo-
tide sequences. Since last several years, we have also
devoted research efforts in designing base discriminating
fluorescence (BDF) nucleobases for the discrimination
of mismatched base and they are successfully utilized
4
a
showed only weak fluorescence. Thus, if we are able
to utilize a novel concept of detecting mismatch-contain-
ing duplexes with our designed perylene labeled nucleo-
side, it will be a promising research effort in giving rise
to a new powerful tool in the detection of SNP and in
4
in the detection of SNPs in homogeneous assay.
The fluorescence behavior of 3-perylene carboxaldehyde
and 3-acetyl perylene toward the solvent polarity is just
8
designing quencher-free molecular beacon.
To explore our concept, we tethered perylene to de-
oxyuridine and incorporated it into two different oligo-
nucleotide sequences. We synthesized perylene
carboxamide containing nucleoside 2 bearing a rigid
Keywords: Oligonuleotide probes; Fluorescence; Perylenefluorophore;
SNP typing.
*
Corresponding author. E-mail: saito@mech.ce.nihon-u.ac.jp
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.039

S. S. Bag et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6338–6341
6339
0
acetylenic linker via Sonogashira coupling of 5-iodo-2 -
deoxyuridine and the corresponding perylene carboxam-
ide 1 (Fig. 1). Nucleoside 2 was ultimately incorporated
into two oligonucleotide sequences ODN 3 and 4, differ
only by flanking bases near the labeled nucleoside [ODN
eroduplexes, 3/8 and 4/12, containing mismatched base
cytosine opposite to the labeled base, U, in the target
oligonucleotide sequences (Fig. 2).
Per
Three possible factors may be considered all of which
can explain these photophysical observations. These
3
(
contains C (G–C rich sequence), while 4 contains T
A–T rich sequence) as the flanking bases] via standard
1
0
10
are: (i) the intercalation, (ii) stacking interaction,
and (iii) the intercalative stacking interaction and the
DNA synthesis protocol, and were analyzed by MAL-
DI-TOF mass spectrometry (Table 1).
6
hydrophobic nature of perylene fluorophore. There
are several reports in which fluorescence of matched du-
plex is quenched via intercalation/stacking between the
Prior to incorporation into oligonucleotides, photo-
physical properties of nucleoside 2 were examined in dif-
ferent organic solvents of varying polarity. Increasing
the solvent polarity has only little effect on their absorp-
tion maxima but a blue shifted pattern was observed. In
contrast both the fluorescence emission intensity and the
wavelength are markedly affected by solvent polarity
1
1
fluorophore and the DNA bases inside the duplex.
This is only possible in case of flexible ethylenic linker
that can allow the fluorophore to come inside the du-
plex. But in the present case the linker is rigid acetylene
containing amide bond and thus the fluorophore is un-
able to come in between the bases inside the duplex
for matched case. Thus, the only possible way is that
perylene may reside outside the duplexes along the ma-
jor groove and hence more exposed toward the polar
aqueous microenvironment, and fluorescence quenching
is the result, which was exactly the case of completely
matched duplex (Fig. 2). This explanation is supported
by the nature of monomer fluorescence and absorption
behavior in solvents of different polarity as described
3
0
(
ET as a polarity parameter). In 1,4-dioxane, least po-
lar solvent tested nucleoside 2 displays relatively strong
emission with a maximum at 467 nm, while in methanol,
the most polar organic solvent tested, showed a weak
emission at around 486 nm. Solvents of intermediate
polarity display an intermediate behavior with a clear
bathochromic and hypochromism effects with increase
9
in solvent polarity.
9
above.
0
Per
The single stranded ODN 3 (5 -CGCAAC UCA
0
0
Per
(5 -CGCAAT UTAACGC-3 )
0
ACGC-3 ) and
4
On the other hand, the mismatched duplexes 3/8 and 4/
12 showed a strong fluorescence emission that can be ex-
plained if we consider a possible more hydrophobic
environment faced by perylene. As the opposite base is
cytosine, hence in that position of the duplex because
showed strong fluorescence emission at around 496
and 498 nm, respectively, when excited at 452 nm in
sodium phosphate buffer (pH 7.0). When hybridized to
their perfectly matched complementary ODNs to form
duplexes 3/5 and 4/9, the fluorescence intensity de-
creased considerably. The fluorescence intensities also
quenched in the case of duplex containing T or G base
Per
of impossible H-bonding between U and C bases, it
is quite reasonable that the rigid fluorophore upon get-
ting little flexibility and may slightly exposed itself to
the mispaired position and bind strongly via intercala-
tive stacking interaction and thus experienced more
hydrophobic organic microenvironment. As a result,
fluorescence enhancement was observed (Fig. 2).
Per
opposite to U in the complementary strands for both
A–T and G–C rich sequences. Most importantly, we ob-
served a strong fluorescence enhancement in case of het-
O
Per
The UV absorption of monomer U and ODNs, and
O
N
N
O
the thermal melting temperature behavior of duplex
ODNs support our proposed explanation. The melting
temperature experiments showed that the mismatched
duplexes 3/8 and 4/12 are almost as stable as
H
HN
N
H
O
HO
O
(
T = 56.4 °C, for 3/8 and T = 52.0 °C, for 4/12) the
m
m
1
2
OH
fully matched duplexes 3/5 and 4/9 (T = 59.9 °C, for
3/5 and T = 52.3 °C, for 4/9). Such stability is only pos-
sible if the perylene unit comes closer to the DNA bases
on the opposite strand and takes part in stacking inter-
action. The absorbance behavior of the mismatched
duplexes compared to that of the fully matched one also
supports such stacking interaction within the duplexes
m
5
'-C G C A A T X T A A C G C -3'
[A-T Rich ss DNA; X = U]
5
'-C G C A A C X C A A C G C -3'
G-C Rich ss DNA; X = U]
Mass Cald.for 4205.963, Found 4205.970 Mass Cald.for 4235.986,Found 4235.987
Per
m
Per
[
3
4
Figure 1. Synthesized perylene labeled nucleoside and oligo-
nucleotides.
Table 1. The sequences of oligonucleotides (ODNs)
(ODNs)
G–C Rich sequences
(ODNs)
A–T Rich sequences
0
0
Per
5 -d(CGCAAC UCAACGC)-3
0
0
0
Per
5 -d(CGCAAT UTAACGC)-3
0
3
5
6
7
8
4
9
0
0
5 -d(GCGTTG A GTTGCG)-3
5 -d(GCGTTG T GTTGCG)-3
5 -d(GCGTTA A ATTGCG)-3
5 -d(GCGTTA T ATTGCG)-3
0
0
0
0
0
0
0
0
10
11
12
5 -d(GCGTTG G GTTGCG)-3
5 -d(GCGTTG C GTTGCG)-3
5 -d(GCGTTA G ATTGCG)-3
5 -d(GCGTTA C ATTGCG)-3
0
0
0
0

6340
S. S. Bag et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6338–6341
Figure 2. Fluorescence behavior of 2.5 lM solution in a buffer (pH 7.0) containing different ODNs; ss denotes single stranded ODN and dsA, dsT,
dsG, and dsC denote double stranded ODNs containing A, T, G, and C bases opposite to U, respectively. Left side is for G–C and right side is for
A–T rich sequences.
Per
Per
along the major groove. The higher T value for U-C
nucleotide which upon hybridization with the comple-
mentary strands containing a mismatched or fully
matched sequence opposite to the base labeled with fluo-
m
mismatched duplexes suggests that perylene is involved
in stronger stacking interaction than in the case of other
mismatched duplexes (N = T and G) in both
Per
rophore is able to sense U-C mispair containing du-
1
2
sequences.
plex through the generation of enhanced fluorescence
signal. We explored, for the first time, that the hydro-
phobic nature of the perylene fluorophore is mainly
responsible for the selection of a particular duplex con-
Further support to our explanation comes from the
molecular modeling study. The optimized structures of
Per
Per
the
0
U
-d(CGCAAC UCAACGC)-3 /5 -d(GCGTTGNGTT
containing homo- and hetero-duplexes
0
taining U-C mismatch base pair. Selection of particu-
Per
0
5
GCG)-3 (N = A, C) and 5 -d(CGCAAT UTAAC
lar mismatched pair irrespective of sequence is highly
promising for the detection of SNPs. Discrimination
based on mismatched base pair recognition offers an
opportunity for selective targeting of mutated genes. Ef-
forts are underway to find even more efficient hydropho-
bic probes with longer emission wavelength for SNPs
genotyping.
0
0
Per
0
0
0
GC)-3 /5 -d(GCGTTANATTGCG)-3 (N = A and C)
were obtained using AMBER* force field in water
9
employing MacroModel vs. 9.0. In the energy mini-
mized structures for the fully matched duplexes in both
the A–T and G–C rich sequences, the perylene carbox-
amide chromophore was extruded to the outside of the
duplexes and thus exposed to a highly polar aqueous
microenvironment. On the other hand, the duplexes
containing
Acknowledgments
Per
U-C mismatched pairs bound strongly
via site-specific intercalative stacking interaction be-
tween the DNA bases and the aromatic rings of perylene
from the major groove side of the helix. The intercalated
perylene itself resembles another base pair, stacked be-
tween neighboring base pairs. Thus, in case of mis-
matched duplexes, perylene fluorophore is exposed to
a more hydrophobic microenvironment. Thus, it is clear
Author SSB is thankful to JST and JSPS for individual
fellowship and Shibpur Dinobundhoo Institution (Col-
lege), Howrah, India, for a study leave.
Supplementary data
Per
that the microenvironment around U is quite different
Synthetic Scheme, photophysical properties, Energy
minimized Structures. This material is available free of
charge via the Internet at http://www.sciencedirect.com.
doi:10.1016/j.bmcl.2006.09.039.
Per
Per
in matched ( U-A) and mismatched ( U-C) cases irre-
spective of flanking base pairs. As discussed earlier, per-
ylene emits fluorescence more strongly in hydrophobic
environment, thus, from both study it is quite obvious
Per
that U shows cytosine selectivity in fluorescence irre-
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