White-light-emitting DNA (WED)

Article abstract of DOI:10.1002/chem.200901147

A study was conducted to show how to use the DNA backbone for the combination of a donor and an acceptor in such a way that a white-light-emitting DNA is formed upon hybridization. DNA1 bearing the blue-green emitter, ethynyl pyrene and DNA2 bearing the r

Full text of DOI:10.1002/chem.200901147

COMMUNICATION
DOI: 10.1002/chem.200901147
White-Light-Emitting DNA (WED)
Reji Varghese and Hans-Achim Wagenknecht*[a]
White-light-emitting organic molecules have received
red emitter, ethynyl nile red (600–750 nm). Both chromo-
phores were conjugated to the 5-position of 2’-deoxyuridine
to maintain the canonical dU:A base pairing. In DNA3-6
both chromophores were combined as an energy donor–ac-
ceptor couple and placed adjacent to each other in order to
obtain an efficient electronic interaction between them
(Figure 1). The phosphoramidite of 5-(1-pyrenylethynyl)-2’-
great attention for potential applications in full-colour light
emitting diodes (LEDs) and lighting purposes.[1] A common
and efficient approach to obtain these organic materials is
based on the partial energy transfer (dET) between donors
and suitable acceptors. The acceptor is either doped in the
donor scaffold or covalently attached to the donor and the
system simultaneously emits over the whole visible range
(l=400–750 nm). This approach has been successfully ap-
plied in polymers blended with luminescent dyes and other
systems.[2,3] Moreover, white-light-emitting ensembles have
been developed by making use of supramolecular recogni-
tion of specific non-covalent interactions encoded in the
donor and acceptor molecules.[4,5] Notably, in all these cases,
an efficient interaction and close proximity (Fçrster dis-
tance, <10 nm) of the donor and acceptor are essential for
the fluorescence resonance energy transfer (FRET).
The characteristic structural features of DNA like its in-
herent double helical conformation and the typical stacking
distance of ꢀ3.4 ꢀ between the bases offers a unique struc-
tural scaffold for the helical organization of covalently at-
tached chromophores with very efficient electronic interac-
tions between them. This has been successfully exploited for
the helical organization of various classes of p conjugated
chromophores, which can provide an efficient medium for
the migration of excitation energy for the development of
photoactive and functional nanomaterials.[6–13]
Herein, we demonstrate how to use the DNA backbone
for the combination of a donor and an acceptor in such a
way that a white-light-emitting DNA is formed upon hybrid-
ization. We synthesized DNA1 bearing the blue–green emit-
ter, ethynyl pyrene (400–600 nm) and DNA2 bearing the
Figure 1. Structure of pyrene- and nile-red modified deoxyuridines and
the DNA sequences used in this study.
deoxyuridine was commercially available. The correspond-
ing phosphoramidite of 2’-deoxyuridine modified with eth-
ynyl nile red was synthesized by palladium catalysed Sono-
gashira coupling of ethynyl nile red (which was synthesized
[a] Dr. R. Varghese, Prof. H.-A. Wagenknecht
Institute for Organic Chemistry
University of Regensburg
93040 Regensburg (Germany)
Fax : (+49)941-943-4617
Homepage: www-oc.chemie.uni-regensburg.de/Wagenknecht/
E-mail: achim.wagenknecht@chemie.uni-regensburg.de
according to the methodology in
a recent literature
report)[14] and 5’-O-(4,4’-dimethoxytrityl)-5-iodo-2’-deoxyuri-
dine followed by the phosphoramidite reaction under stan-
dard conditions.[15] Using both DNA building blocks, the
modified oligonucleotides DNA1-6 were synthesized by
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901147.
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automated phosphoramidite chemistry without major
changes to the standard procedures.[15]
The reference duplexes DNA1 and DNA2 showed the
characteristic optical behaviour of the corresponding chro-
mophores. DNA1 exhibited the absorption and fluorescence
maxima of pyrene at l=401 nm (e401nm =3.6ꢂ104 mÀ1 cmÀ1)
and l=440 nm (ff =0.15), respectively, and DNA2 showed
an absorption maximum at l=615 nm (e615nm =2.5ꢂ
104 mÀ1 cmÀ1) and emission maximum at l=665 nm (ff =
0.30), respectively.[15] The calculated spectral overlap inte-
gral (J(l)=1.5ꢂ1015 mÀ1 cmÀ1 nm4) from the emission spec-
trum of DNA1 and the absorption spectrum of DNA2 re-
veals the possible non-radiative transfer of excitation
energy. Moreover, the well separated absorption maxima
and the high extinction coefficient of the acceptor were
promising factors for the transfer of excitation energy. The
extinction coefficient of ethynyl nile red (e380nm
=
1400mÀ1 cmÀ1) at the excitation wavelength of ethynyl
pyrene is considerably low which diminishes the possibility
of the direct excitation of the acceptor.
Figure 2. a) Fluorescence spectra of DNA1 (blue), DNA2 (orange), ss-
The absorption spectra of the double labelled DNA3-6
showed the characteristic absorption of both ethynyl pyrene
and ethynyl nile red and thus ruled out the possibility of any
ground state interaction between them (single strand (ss)
and double strand (ds)).[15] Accordingly, circular dichroism
(CD) experiments showed no detectable signal in the ab-
sorption region of the chromophores, but confirmed a B-like
helical conformation.[15] The thermal melting (Tm) studies re-
vealed that the incorporation of a single chromophore desta-
bilises the duplex by À3.98C for DNA1 and À3.68C for
DNA2. However, double incorporation (DNA3-6) regained
the destabilization by the hydrophobic interactions between
the chromophores.[15]
Excitation of ss-DNA3 at l=380 nm resulted in 92%
quenching of the pyrene fluorescence at l=440 nm with a
concomitant formation of the nile red emission at 665 nm.
As expected, a FRET occurs from pyrene to nile red in a
non-radiative manner with a rate constant of 5.24ꢂ109 sÀ1.[15]
Very interestingly, the corresponding ds-DNA3 exhibited a
decrease in the FRET efficiency (83%) since only a partial
energy transfer (kET =1.69ꢂ109 sÀ1) occurs. The combined
effects, an increase at l=440 nm and a decrease at l=
665 nm (compared to the corresponding ss) yield a white
emission, owing to the almost equal intensity for the blue–
green and red emitting peaks (I440/I665 =0.96) (Figure 2a–c).
A schematic representation of this FRET controlled emis-
sion changes is illustrated in Figure 2d. Even though the dif-
ference in the FRET is only ꢀ10%, the significant drop of
the red emission intensity in the duplex is probably owed to
the large molar extinction coefficient and the high fluores-
cence quantum yield of ethynyl nile red. The efficiency of
dipole–dipole coupled Fçrster resonance energy transfer is
highly dependent on the relative orientation of the dipole
moments of the donor and acceptor.[16] The observed differ-
ence of the FRET efficiencies in the ss vs ds in the present
system is likely owed to the change in the relative orienta-
tion of the chromophore dipole moments upon duplex for-
DNA3 (red) and ds-DNA3 (grey) (c=2.5ꢂ10À6
250 mm NaCl, pH 7). lexc =380 nm for DNA1and DNA3 and lexc
m
in Na Pi buffer,
À
=
580 nm for DNA2. Photographs of the solution of DNA3 b) ss and c) ds
under illumination with l=380 nm UV light. d) A schematic representa-
tion illustrating the hydridization induced emission colour change.
mation (helically twisted conformation) which may prohibit
an efficient energy transfer in the case of ds-DNA3.
As the neighbour bases adjacent to the chromophores
affect significantly their photoluminescence behaviour,
owing to various side effects, such as charge transfer, exci-
plex formation etc., we synthesized DNA4-6 to investigate
the effect of the base pairs adjacent to the chromophores on
the hybridization induced white-light emission. As expected,
emission spectra of all three single strands (ss-DNA4-6) ex-
hibited a major peak at l=665 nm when excited at l=
380 nm, owing to an efficient energy transfer from pyrene to
nile red similar to ss-DNA3 (Figure 3a). Remarkably, the
fluorescence spectra of all three corresponding duplexes
both blue–green and red emitting peaks of almost equal in-
tensity (I440/I665 =0.6, 1.0, and 1.1 for DNA4, DNA5 and
DNA6, respectively) and hence yielded white emission (Fig-
ure 3b). Only when guanines are located adjacent to the
chromophores (DNA4) a low value of I440/I665 (0.6) was ob-
served even though the emitting light is still nearly white in
colour. Time resolved spectroscopic studies also support
non-radiative FRET from pyrene to nile red. The fluores-
cence decay profile of DNA3 (lexc =280 nm) when probed
at l=440 nm showed a biexponential decay with a major
component of ꢀ3 ns (91%). On the other hand, ss-DNA3
exhibited a triexponential decay with two major components
having time constants, t1 ꢀ1 ns (65%), and t2 ꢀ3 ns (25%).
This faster decay (t1) of the donor in the presence of the ac-
ceptor dye indicates the nonradiative energy transfer from
pyrene to nile red.[4a] Interestingly, ds-DNA3 also exhibited
a triexponential decay with almost same time constants but
with different amplitudes t1 ꢀ1 ns (52%) and t2 ꢀ3 ns
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White-Light-Emitting DNA (WED)
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Figure 3. Fluorescence spectra of DNA4-6 a) ss and b) ds (c=2.5ꢂ10À6 m,
À
in Na Pi buffer, 250 mm NaCl, pH 7, lexc =380 nm).
Figure 4. a) Temperature-dependent fluorescence spectra of ds-DNA3 re-
corded in an interval of 58C (c=2.5ꢂ10À6 m, in Na-Pi buffer, 250 mm
NaCl, pH 7, lexc =380 nm) and b) position of the fluorescent spectra of
ds-DNA3-6 in the CIE colour diagram.
(26%). This decreased amplitude of t1 can be attributed to
the decreased efficiency of the FRET in the duplex which
stands in agreement with the observed steady state spectra.
It should be noted that in all cases we observed a long lived
species with different life times and its nature is not clear at
present.
ciation of two oligonucleotides into the ds and back into the
ss yields fully reversible emission colour change by modulat-
ing the FRET efficiencies. Hence, this new example of a
DNA-based dynamic white-light-emitting assembly could be
an entry to the class of white-light-emitting materials. More-
over, such a system will be a promising candidate as a nucle-
ic acid probe to image DNA hybridisation by distinct emis-
sion colour changes upon duplex formation.
The major attractive feature of WED is the dependency
of the energy transfer efficiency with the association/dissoci-
ation of the duplex which can be controlled by temperature.
Accordingly, the intensity of the fluorescence peak of ds-
DNA3 at l=665 nm was found to increase, whereas the
peak at l=440 nm decreased at high temperature when
going from 258C to 758C. Concomitantly, the emission
colour of the DNA changes from white to red (Figure 4a).
Moreover, this colour change is completely reversible, as the
dissociated duplex at high temperatures can be reassembled
by cooling down the sample. Finally, the emitted white light
is assigned in photometric terms, as standardized by the
Commission Internationale de I’Eclairege (CIE). The CIE
coordinates of ds-DNA3-6 when their respective photolumi-
nescence spectra were converted into chromaticity coordi-
nates on a CIE 1931 diagram are (0.25, 0.23), (0.28, 0.23),
(0.26, 0.24) and (0.25, 0.24) for DNA3, DNA4, DNA5 and
DNA6, respectively (Figure 4b). The CIE coordinates for
absolute white light are (0.33, 0.33). It should be noted that
WED deviate slightly from the CIE coordinates for an abso-
lute white light, owing to the insufficient green emitting
region and experiments are progressing in our laboratory in
this direction.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft (GRK 640),
the Fonds der Chemischen Industrie, and the University of Regensburg
are gratefully acknowledged. RV thanks Alexander von Humboldt Foun-
dation for a post-doctoral research fellowship. We thank Prof. B. Dick
and Dr. U. Kensy of Institute for Physical Chemistry at the University of
Regensburg for lifetime measurements.
Keywords: DNA
·
fluorescent probes
·
FRET
·
multichromophores · white-light emission
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Received: April 30, 2009
Published online: August 13, 2009
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