Termini-free molecular beacon utilizing silylated perylene and anthraquinone attached to the C-5 position of pyrimidine nucleobase

Article abstract of DOI:10.1246/cl.2012.420

A termini-free molecular beacon type of DNA bearing a novel silylated perylene and an anthraquinone both at the C-5 position of deoxyuridine residues exhibits prominent fluorescence upon mixing with complementary oligonucleotide (24- to 50-fold increase in comparison with a single-stranded state) along with one base mismatch discrimination ability.

Full text of DOI:10.1246/cl.2012.420

doi:10.1246/cl.2012.420
420
Published on the web April 4, 2012
Termini-free Molecular Beacon Utilizing Silylated Perylene and Anthraquinone Attached
to the C-5 Position of Pyrimidine Nucleobase
Yuzuru Sato, Tomohisa Moriguchi, and Kazuo Shinozuka*
Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University,
1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515
(Received February 1, 2012; CL-120083; E-mail: sinozuka@chem-bio.gunma-u.ac.jp)
A termini-free molecular beacon type of DNA bearing a
still highly desirable. In recent studies3,4 to utilize novel silylated
fluorescent materials5,6 exhibiting enhanced fluorescence quan-
tum yield along with the bathochromic shift in absorption and
emission, due to the specific ·*-³* interaction,7 we have found
that a termini-free oligoDNA possessing a stem-loop structure8
as well as a modified nucleotide units bearing a silylated
perylene moiety (fluorophore) and an anthraquinone moiety
(quencher) in the middle of the stem-region exhibited efficient
fluorescence quenching (ca. 98%) in the absence of the target. In
the presence of the target, however, the fluorescence signal was
recovered prominently. In addition, the oligomer exhibited
discrimination ability of one-base-mismatched target through the
fluorescence signal.
The synthesis of modified perylene derivative 6 possessing
(aminomethyl)dimethylsilyl function was reported previously.9
Meanwhile, preparation of analogous compounds possessing
(aminopropyl)dimethylsilyl group and incorporation of that into
oligoDNA are summarized in Scheme 1. In brief, 3-bromoper-
ylene (1) was coupled with allylchlorodimethylsilane and the
resulting compound 2 was treated with 9-borabicyclo[3.3.1]-
nonane (9-BBN) followed by a mixture of EtOH/H2O2/NaOH/
H2O to give compound 3 bearing hydroxy function. The reaction
of 3 with phthalimide in the presence of diethyl azodicarbox-
ylate and triphenylphosphine10 gave the corresponding phthal-
imide derivative 4 which was further converted to a silylated
perylene derivative 5 bearing a primary amine group by
treatment with hydrazine hydrate. The silylated perylene
derivatives 5 and 6 were coupled with C-5 carboxymethyl
derivative of 2¤-deoxyuridine11 7 followed by the conversion to
the corresponding phosphoramidite derivatives 9. Incorporation
of phosphoramidite 9a into DNA was carried out with a standard
protocol. On the other hand, incorporation of 9b required rather
concentrated solution (0.2 M) with prolonged coupling period
novel silylated perylene and an anthraquinone both at the C-5
position of deoxyuridine residues exhibits prominent fluores-
cence upon mixing with complementary oligonucleotide (24- to
50-fold increase in comparison with a single-stranded state)
along with one base mismatch discrimination ability.
A molecular beacon (MB) is an oligonucleotide consisting
of a partial self-complementary sequence to form a stem-loop
structure along with a combination of a fluorophore at one end
and a quencher at the other end.1 Under the absence of the
complementary oligonucleotide (the target), the MB forms a
stem-loop structure and therefore fluorescent signal is quenched
because of the close proximity of the fluorophore and the
quencher. Hybridization of the MB with the target, however,
brings about the recovery of the fluorescence signal due to the
resolution of the stem-loop structure and separation of the
fluorophore and the quencher distally. Thus, the presence of the
target can be characterized by the increase in the fluorescence
signal of the MB. On the other hand, insufficient fluorescence
quenching of the original MB is a well-recognized drawback.
This would cause poor signal-to-noise ratio (S/N ratio) and the
reduction of detection limit because of the substantial back-
ground fluorescence signal even under the absence of the target.
Another significant problem of the MB is that the classical MB
cannot be coupled with modern microarray technology since
both termini (5¤- and 3¤-) of the MB are connected to a
fluorophore and a quencher. A number of studies have been
carried out to overcome these problems.2 In a practical point of
view to apply the MB methodology for high-throughput gene
analysis, development of new versatile and a straightforward
technique to overcome the above problems at the same time is
O
N
NH2
H3C Si CH3
OH
O
NH2
O
HN
H3C Si CH3
H3C Si CH3
EtOH/
NaOH/
H2O2
H3C Si CH3
H3C Si CH3
Br
O
H3C Si CH3
Cl
H2NNH2
DEAD, PPh3
9-BBN
n-BuLi
5
6
1
2
3
4
O
N
O
HN
n
O
OH
O
O
N
HN
n
HN
n
HN
HN
HN
HN
H3C Si CH3
O
NH
H3C Si CH3
O
O
O
O
N
O
DMTrO
O
O
DMTrO
O
O
N
DMTrO
(i-Pr)2N
O
O
DMTrO
5 or 6
into DNA
DMT-MM
(i-Pr)2N
OH
OH
7
O
O
8a (n=1)
8b (n=3)
P
O
9a (n=1)
9b (n=3)
P
10a (n=2)
10b (n=4)
NC
NC
O
O
Scheme 1. Synthetic route of the modified nucleoside phosphoramidite derivatives.
Chem. Lett. 2012, 41, 420-422
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

421
(a)
(b)
FDNA-1: 5'-CGGX(n=1)AC CTTGAGAAAGGGC GY(n=2)ACCG-3'
FDNA-2: 5'-CGGX(n=1)AC CTTGAGAAAGGGC GY(n=4)ACCG-3'
FDNA-3: 5'-CGGX(n=3)AC CTTGAGAAAGGGC GY(n=4)ACCG-3'
1
250
200
150
100
50
O
N
HN
H3C Si CH3
n
O
N
HN
FDNA-1
FDNA-1/tDNA-1
FDNA-1/tDNA-2
T G
n
NH
HN
O
O
T
A
HN
O
O
C
O
G
A
A
O
5'
TGGXAC
O
O
O
ACCAYG
3'
0.85
0
O
X
C
Y
A
O
20
40
60
80
20
40
60
80
G
G
G
O
Temperature/°C
Temperature/°C
tDNA-1: 3'-GAACTCTTTCCCG-5'
tDNA-2: 3'-GAACTGTTTCCCG-5'
tDNA-3: 3'-GAACTATTTCCCG-5'
tDNA-4: 3'-GAACTTTTTCCCG-5'
Figure 3. Thermal melting properties of FDNA-1. (a) UV
melting curves of FDNA-1 and duplexes with tDNA-1 and -2.
(b) Fluorescence melting curves of FDNA-1 (3.0 ¯M, 10 mM
sodium phosphate, 100 mM NaCl, pH 7.2).
Figure 1. Sequence and structure of the modified DNA used
in this study.
Table 1. Ralative fluorescence intensity ratios of FDNAs
a
b
Oligomer
Target
I
complex/Isingle
I
mis/Ifull
a
b
FDNA-1
FDNA-2
FDNA-3
FDNA-1
FDNA-1
FDNA-1
FDNA-1
tDNA-1
tDNA-1
tDNA-1
tDNA-1
tDNA-2
tDNA-3
tDNA-4
49.1
23.6
23.7
®
®
®
®
®
®
700
FDNA-1
FDNA-2
FDNA-3
FDNA-1/tDNA-1
FDNA-2/tDNA-1
FDNA-3/tDNA-1
1
0.30
0.35
0.17
®
A
B
aRatio of fluorescence intensity of duplexes (Icomplex) with that
of single strand (Isingle). bRatio of fluorescence intensity of
mismatched duplexes (Imis) with that of matched strand (Ifull).
0
440
520
Wavelength/nm
600
Figure 2. Fluorescent properties of FDNAs. (a) Fluorescence
spectra of FDNAs and duplex with tDNA-1 (3.0 ¯M, 10 mM
sodium phosphate, 100 mM NaCl, pH 7.2). (b) Photograph
displaying the emission behavior at room temperature of
FDNA-1 alone (A) and of the duplex with tDNA-1 (B) upon
illumination at 302 nm.
was around 71 °C indicating that the FDNAs form stem-loop
structure (Figure 3b). It is well known that the C-5 position of
pyrimidine nucleobase is facing the major-groove of nucleic
acid duplex and, therefore, both the fluorophore and the
quencher molecules are expected to be located in the groove.
These indicate that the observed fluorescence quenching could
be brought by the interaction of the fluorophore and the
quencher in rather hydrophilic major-groove since the molecules
are quite hydrophobic in nature. Fluorescence intensity shown in
Figure 2a also indicates that the efficiency of the quenching
depends on the length of the linker (alkyl) portion connecting
the fluorophore and/or the quencher to the nucleobase since
the signal of FDNA-1 possessing the shorter linker {X(n = 1),
Y(n = 2)} is quenched more effectively than those (FDNA-2
and FDNA-3) possessing longer linker portion.
Meanwhile, prominent recovery of the fluorescence signal
(24- to 50-folds) in the presence of the complementary DNA
(tDNA-1) was observed for all modified DNAs as those are also
depicted in Figure 2a. It should be noted that the fluorescence
quantum yield of all FDNAs under these conditions is about 0.5.
Thus, the change of the fluorescence signal in the presence and
the absence of the target was easily recognized by the naked eye
as it is shown in Figure 2b. The observed phenomenon is
presumably due to the formation of a double helical complex
between the loop-region of the modified DNAs and tDNA-1
causing the opening of the stem and relocation of the
fluorophore and the quencher to a distant position. The fluores-
cence intensity ratios under the presence and the absence of the
target DNA (Icomplex/Isingle) at 462 nm (emission maxima) are
listed in Table 1. Among the modified DNAs studied, FDNA-1
(360 s) to achieve satisfactory coupling yield (ca. 95%).
Compounds 10a and 10b (0.1 M) were also incorporated into
the same DNA in the same manner. After the usual work-up,
desirable modified DNAs (FDNA-1 to FDNA-3) were obtained
with satisfactory yields. The sequence and proposed stem-loop
structure of the modified DNAs are depicted in Figure 1 along
with the sequence of target DNA (tDNA-1 to tDNA-4)
complementary to the loop region of the modified DNAs. As
it is shown in Figure 1, modified nucleotide units described
above are incorporated into the stem region consisting of 6-bp
units so that they are facing each other.
The fluorescence spectra of the modified DNAs under the
presence and absence of target DNA were measured in near
physiological conditions (100 mM NaCl) and the obtained
fluorescence spectra are depicted in Figure 2. As it is clearly
shown in Figure 2a, the fluorescence signal of all modified
DNAs is strongly quenched in the absence of the targets. Under
the same conditions, the thermal-melting properties of the
FDNA-1 and the corresponding duplexes were examined. The
UV-melting profiles of the duplexes showed the identical curves
with the canonical duplexes, however, MB-type FDNA-1
showed no sigmoid curve (Figure 3a). Therefore, the Tm values
of the FDNA estimated by fluorescence-melting experiments
Chem. Lett. 2012, 41, 420-422
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

422
Table 2. Sequence discrimination ability of FDNA-1 under
lower salt concentration (50 mM NaCl)
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a
b
Oligomer
Target
I
complex/Isingle
I
mis/Ifull
FDNA-1
FDNA-1
FDNA-1
FDNA-1
tDNA-1
tDNA-2
tDNA-3
tDNA-4
16.1
3.0
6.9
3.0
1
0.19
0.43
0.19
aRatio of fluorescence intensity of duplexes (Icomplex) with that
of single strand (Isingle). bRatio of fluorescence intensity of
mismatched duplexes (Imis) with that of matched strand (Ifull).
exhibited the most impressive result showing the Icomplex/Isingle
ratio of 49. This means that in the absence of the target, about
98% of the fluorescence signal of the complex was quenched
and the number is close to the recently reported analogous MB
type of probe possessing two unmodified perylene moieties and
two anthraquinone moieties connected to non-nucleosidic scaf-
fold, namely D-threoninol.12
Since FDNA-1 exhibited drastic fluorescence-increment
effect in the presence of the complementary DNA, we further
examined discrimination ability of FDNA-1 to the one-base-
mismatched target through the fluorescence signal using
imperfectly complementary strands (tDNA-2 to tDNA-4) pos-
sessing one-base substitution to the native target DNA (tDNA-1)
and the obtained fluorescence intensity ratios between the full
matched complex (Ifull) and the mismatched complex (Imis) are
also listed in Table 2. Under certain conditions (50 mM NaCl),
FDNA-1 exhibited varied Imis/Ifull ratio depending on the
mismatched base in the complements. According to the results
listed in Table 1, FDNA-1 recognizes G/T mismatch more
nicely than other mismatches despite the fact that G/T mismatch
base pair is expected to form the most stable base pair of all
possible mismatch base pairs.
In conclusion, we developed the novel termini-free mo-
lecular beacon type of DNA bearing a novel silylated perylene
and an anthraquinone both at C-5 position of deoxyuridine
residues. These probe DNAs showed low noise emission for the
discrimination of the target DNA. Therefore, we anticipate that
these DNA probes will provide a practical tool capable of simple
visible detection.
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© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/