Monitoring DNA structures by dual fluorescence of pyrene derivatives

Article abstract of DOI:10.1021/ja053609e

We have developed a nucleotide modified by a pyrene derivative with dual fluorescence. The dual fluorescence of the fluorophore, which was incorporated into DNA, was effectively controlled at ambient temperature according to DNA structural status. Our nuc

Full text of DOI:10.1021/ja053609e

Published on Web 09/02/2005
Monitoring DNA Structures by Dual Fluorescence of Pyrene Derivatives
Akimitsu Okamoto,*,† Kazuki Tainaka,† Ken-ichiro Nishiza,† and Isao Saito*,‡
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto UniVersity,
Kyoto 615-8510, and NEWCAT Institute, School of Engineering, Nihon UniVersity, and SORST, Japan Science and
Technology Agency, Tamura, Koriyama 963-8642, Japan
Received June 2, 2005; E-mail: okamoto@sbchem.kyoto-u.ac.jp; saito@mech.ce.nihon-u.ac.jp
Dual fluorescence,1 originating from two relaxed singlet excited
states, a locally excited (LE) state, and an intramolecular charge
transfer (ICT) state, has often been observed for electron donor/
acceptor fluorophores, such as 1-arylpyrene2 and 4-dialkylami-
nobenzonitrile derivatives.3 The equilibrium between LE and ICT
states has usually been controlled by solvent temperature or
viscosity depending on the height of the activation energy to the
ICT state, Ea.4 However, a fluorophore with dual fluorescence has
not been used as a fluorescent probe for biomolecules, although
this unique character would be suitable for monitoring the change
Figure 1. The structures of novel N,N-dimethylaminophenyl-substituted
pyrene derivatives with dual fluorescence used in this study.
in the microenvironment of biomolecules by taking advantage of
the color change. Designing a system that can control the equilib-
rium between two excited states at ambient temperature without
changing the solvent properties is an unavoidable subject for
developing a dual fluorescence probe.
Here, we report the detection of DNA hybridization by monitor-
ing dual fluorescence. We tethered a pyrene derivative showing
dual fluorescence to a deoxynucleoside and incorporated it into
DNA. The dual fluorescence was effectively controlled by incor-
poration of the fluorophore into a duplex at ambient temperature.
The nucleoside with dual fluorescence serves as a conceptually new
fluorescent probe for monitoring hybridization of nucleic acids by
a color change.
Figure 2. (a) Fluorescence spectra of 7.3 µM 2 (black line), 25 µM single
strand 3 (red line), and 25 µM duplex 3/3′ (blue line) in 50 mM sodium
phosphate (pH 7.0) and 0.1 M sodium chloride at 5 °C. Excitation was at
380 nm. (b) Fluorescence image of the solutions of 25 µM 3 (left) and 25
µM 3/3′ (right) illuminated with a 365 nm transilluminator.
We synthesized a new pyrene derivative bearing a propargyl
linker, N-propargyl 8-(4′-(N′,N′-dimethylamino)phenyl)pyrene-1-
carboxamide (1) (Figure 1). Subsequently, we obtained a novel
fluorescent deoxyuridine 2 via Sonogashira coupling of 5-iodo-2′-
deoxyuridine with 1. Nucleoside 2 was converted to the phosphor-
amidite derivative, which was used for the synthesis of oligode-
oxynucleotides according to a conventional DNA synthesis protocol.
The fluorescence spectra of 2, a single-stranded oligodeoxy-
nucleotide 5′-d(CGCAAT2TAACGC)-3′ (3, MALDI-TOF calcd [M
- H] 4304.02, found 4304.93), and 3 hybridized with the
complementary DNA strand, 5′-d(GCGTTAAATTGCG)-3′ (3′),
were examined in sodium phosphate buffer (pH 7.0) on excitation
at 380 nm (Figure 2a). For nucleoside 2, a single fluorescence band
was observed at 540 nm. In contrast, for the single-stranded DNA
3 and the DNA duplex 3/3′, another fluorescence band at shorter
wavelength (440 nm) was observed in addition to a band at 546
nm. In particular, for 3/3′, the highest fluorescence intensity at 440
nm was observed. This dual fluorescence was observed not only
for a hybrid with a DNA strand but also for a DNA/RNA hybrid.5
The two fluorescence bands at 540 and 440 nm were assigned
to the ICT and LE fluorescence bands, respectively, by investigating
the photophysical behavior of the pyrene fluorophore 1. Only one
broad emission band at 533 nm was observed for 1 in water on
excitation at 380 nm. This emission band showed a strong
solvatochromicity, which can be assigned to an ICT fluorescence,
and shifted to longer wavelength in highly polar solvents (490 f
560 nm). From a solvatochromic plot of these data for the
wavenumbers of the fluorescence maxima against the solvent
polarity parameter f(ꢀ) - 1/2f(n2),6 a dipole moment in the excited
state of 18.1 D was obtained.5 The dipole moment greatly increased
from 4.8 D in the ground state, suggesting that a large conforma-
tional change by solvent relaxation is required for transition to an
ICT state.
Among three samples labeled by fluorophore 1, the fluorophore
of the duplex 3/3′ is located in a sterically restricted space. The
melting temperature (Tm) of 3/3′ suggests that the fluorophore is
bound to the minor groove. The duplex showed a surprisingly low
Tm (41 °C), which was much lower than that of a nonmodified
T/A duplex (60 °C) and close to those observed for mismatched
duplexes containing 2/N base pairs (N ) G, C, or T) (43-48 °C).5,7
It has been reported that the duplex containing a pyrene-tethered
nucleotide, PyU, forms a structure in which the glycosyl bond of
PyU is rotated to the syn conformation when PyU forms an unstable
base pair.8 The pyrene fluorophore of PyU is located in the minor
groove. If so, the degree of freedom of the fluorophore of the duplex
3/3′ would be greatly restricted. In the CD spectrum of 3/3′, a
positive CD was observed at 371 nm, suggesting that the fluoro-
phore binds to the minor groove.5
Two structural states of 3 showed different visible colors. An
orange-colored fluorescence was observed for the single strand state,
whereas the duplex 3/3′ emitted a pink fluorescence as a mixture
† Kyoto University.
‡ Nihon University.
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10.1021/ja053609e CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
The fluorescence change for the duplex 3/3′ was quite different.
The change in ln(Φ′(ICT)/Φ(LE)) behaved like that of the single
strand state at temperatures higher than the duplex Tm (41 °C). The
ln(Φ′(ICT)/Φ(LE)) value dropped steeply at approximately 45 °C,
and then, interestingly, linearly decreased with decreasing temper-
ature. These behaviors occurring by hybridization suggest that the
fluorescence emission was under low-temperature limits, where kd
is negligibly small compared with 1/τ′.11a The activation energy
0
for the forward ICT reaction, Ea, which was calculated from the
slope of the line, was estimated to be 9.9 kJ mol-1. For phenyl-
substituted pyrene derivatives, it is known that the viscosity effect
of solvents strongly affects the time-determining step of twisted
ICT (TICT) formation because of the irreversibility of TICT
formation at low temperature.12 In the present case, as a barrier to
the internal rotation of the fluorophore, a narrow free space in the
duplex structure acted instead of viscous solvents. The relaxation
of the fluorophore toward TICT would be difficult in the minor
groove of the duplex.
In conclusion, we have developed a conceptually new nucleoside
modified by the fluorophore with dual fluorescence. The dual
fluorescence was effectively controlled at ambient temperature by
incorporation of the fluorophore into a duplex. The TICT formation
of the fluorophore in the duplex 3/3′ was restricted by the
microstructure near the fluorophore. Our nucleoside with dual
fluorescence serves as a probe for monitoring hybridization with
DNA or RNA by the color change without multilabeling with
fluorescent dyes.
Figure 3. Stevens-Ban plot of the ICT-to-LE fluorescence intensity ratio
of 2, single strand 3, and duplex 3/3′ in 50 mM sodium phosphate (pH 7.0)
and 0.1 M sodium chloride. The temperature was changed from 5 to 75
°C; (a) 7.3 µM 2. The line through the data points (its slope equals -∆H/R
under the high-temperature limit condition) corresponds to a stabilization
enthalpy ∆H of -50.1 kJ mol-1; (b) 25 µM 3 (red squares) and 25 µM
3/3′ (blue triangles). Because the slope of the blue straight line is -Ea/R at
the low-temperature limit, the activation energy, Ea, was calculated to be
9.9 kJ mol-1
.
of two colors of orange ICT and blue LE fluorescence (Figure 2b).
The change in the fluorescence color vividly expresses the structural
change in the microenvironment around the fluorophore by
hybridization with the complementary strand. It has previously been
reported that the increase in the LE versus the ICT states is
connected with a conformational relaxation that can be frozen out
by lowering the temperature. Our observation is very similar to
this phenomenon. In our case, it is likely that the dual fluorescence
of the fluorophore was strongly controlled by the duplex formation.
The conformational restriction of the fluorophore in DNA induces
the LE fluorescence band.
To obtain information on an excited-state equilibrium between
LE and ICT states, we examined the temperature dependence of
the fluorescence intensities of 2 and 3 from 5 to 75 °C. The intensity
of the LE fluorescence of 2 increased with increasing temperature,
whereas the ICT fluorescence intensity gradually decreased. The
relation between reaction rate constants and fluorescence quantum
yields is as follows:9
Supporting Information Available: Detailed experimental data
on the synthesis and photochemical assays of the related DNA samples
(in PDF format). This material is available free of charge via the Internet
at http://pubs.acs.org.
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Φ′(ICT)/Φ(LE) ) (k′/kf) × {ka/(kd + 1/τ′)}
(1)
f
0
The Stevens-Ban plot, the plots of the natural logarithm of the
ICT/LE fluorescence quantum yield ratio versus the reciprocal
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data points tended to deviate from linearity. This indicates that the
(9) The ka and kd are the rate constants of the forward and reverse ICT reaction;
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kf′(ICT) are radiative rate constants.
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