Insulator base pairs for lighting-up perylenediimide in a DNA duplex

Article abstract of DOI:10.1002/chem.201001638

Quenched: Perylenediimide (PDI) is highly quenched by nucleobases, which greatly restricts its application as a fluorescent probe. Here, we propose insulator base pairs tethering cyclohexane ring through d-threoninol. When insulator base pairs were inserted between PDI and nucleobases, the quantum yield of PDI drastically increased several thousand-fold. The insulator base pairs reported here also have the potential to increase the quantum yields of other fluorophores.

Full text of DOI:10.1002/chem.201001638

DOI: 10.1002/chem.201001638
Insulator Base Pairs for Lighting-up Perylenediimide in a DNA Duplex
Hiromu Kashida,[a] Koji Sekiguchi,[a] and Hiroyuki Asanuma*[a, b]
Fluorescent-labeled oligonucleotides are powerful tools
for biochemical and biological research.[1] However, some
fluorophores, even those with high potential, cannot be uti-
lized for the labeling of DNA or RNA due to their strong
quenching by natural nucleobases.[2] In order to protect
these fluorophores from severe quenching, a new methodol-
ogy that can shield these fluorophores from natural nucleo-
bases is required. Kool et al. first reported an “insulator
molecule” which enhances the emission of pyrene (Y) in a
single strand.[3] They synthesized a trimer which had 5,6-di-
hydro-2’-deoxythymidine (DHT) inserted between pyrene
and thymine (5’-TACHTUNGTRENNUNG(DHT)Y-3’). This trimer showed a quan-
tum yield about 70 times higher than that of a directly-con-
jugated dimer (5’-TY-3’). However, structural fluctuations in
the single-stranded state still prevented complete recovery
of the quantum yield of pyrene.
Here, we propose “insulator base pairs” that can shield a
fluorophore from nucleobases in order to achieve a high
quantum yield in a duplex. We designed two insulator mole-
cules: trans-isopropylcyclohexane (H) and 4-cyclohexylben-
zene (I) moieties as shown in Figure 1, each of which has
one rigid cyclohexane ring with no p electrons.[4,5] The ab-
sence of p electrons should efficiently suppress electron
transfer. A biphenyl moiety (J) containing no cyclohexane
rings was used as a control. These molecules were intro-
duced into both strands of a DNA duplex to form tentative
“base pairs”. Because these molecules have high hydropho-
bicity, they should face to the inside of the duplex and form
“base pairs”. These “insulator base pairs” would therefore
be expected to shield a fluorophore from quenching by nu-
cleobases.
Figure 1. Sequences of the modified DNAs synthesized in this study.
We selected the fluorophore perylenediimide (PDI: D) to
test the shielding effect of these “insulator base pairs”. PDI
has been widely applied to material use because of its high
electron affinity, high brightness, strong p–p stacking inter-
action and photostability.[6–8] Furthermore, PDI has also
been used for the functionalization of DNA.[9–18] However,
even though the PDI monomer shows a high quantum yield
and photostability, the quenching of PDI by nucleobases,
due to electron transfer from nucleobases, has severely lim-
ited its application as a fluorophore.[10,17,19] Thus, we intro-
duced “insulator base pairs” between PDI and nucleobases
in order to enhance the fluorescence of PDI. Sequences of
modified DNA are shown in Figure 1. Two or six “insulator
base pairs” were introduced between PDI and nucleobases
on d-threoninol as a scaffold.[20] DNA containing one PDI
but no insulator was also synthesized as a control (D1).
For the hybridization of D1 with a complementary strand
(N), almost no fluorescence emission was detected from
[a] Dr. H. Kashida, K. Sekiguchi, Prof. Dr. H. Asanuma
Graduate School of Engineering, Nagoya University
Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan)
Fax : (+81)52-789-2488
E-mail: asanuma@mol.nagoya-u.ac.jp
[b] Prof. Dr. H. Asanuma
CREST (Japan) Science and Technology Agency
4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001638.
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Chem. Eur. J. 2010, 16, 11554 – 11557

COMMUNICATION
PDI (see the orange line in the inset of Figure 2A). This ex-
tremely low emission is attributable to the almost complete
quenching of PDI by electron transfer from nucleobases to
PDI.[17] However, when two H–H pairs were inserted be-
tween PDI and its neighboring nucleobases (H2AD/H2B),
distinct emission at 550 nm was observed from PDI. As
summarized in Table 1, the quantum yield (F) of H2AD/
H2B was 0.020 whereas that of D1/N was less than 0.001.
Table 1. Effect of duplex sequence on the quantum yield (F) and melt-
ing temperature.
Sequence
F[a]
Tm [8C][b]
D1/N
<0.001
41.0
H2AD/H2B
I2AD/I2B
J2AD/J2B
0.020
0.025
0.009
44.2
48.2
46.9
H6AD/H6B
I6AD/I6B
0.59
0.56
62.0
61.8
H2AD/N
0.006
39.7
Figure 2. Fluorescence emission spectra at 208C of A) H2AD/H2B,
H2AD/N, D1/H2B, single-stranded H2AD and D1/N and B) H2AD/
H2B, I2AD/I2B, J2AD/J2B, H6AD/H6B and I6AD/I6B. Solution condi-
tions were as follows: [DNA]=1.0 mm, [NaCl]=100 mm, pH 7.0 (10 mm
phosphate buffer). Excitation wavelength: 500 nm. Note that spectra in
B) were measured at low sensitivity mode whereas those in A) were
measured at medium sensitivity mode.
D1/H2B
<0.001
39.8
[a] Quantum yield was determined using the quantum yield of rhodamine
6G in ethanol (0.94) as a reference. [b] [DNA]=5.0 mm, [NaCl]=100 mm,
pH 7.0 (10 mm phosphate buffer).
Since the value of F remained still lower than 0.1 follow-
ing the introduction of two insulator pairs, we determined if
insertion of additional insulator pairs might enhance PDI
emission. Indeed, the emission intensity of H6AD/H6B was
dramatically higher than that of H2AD/H2B (Figure 2B).
The F value of H6AD/H6B was as high as 0.59, which is
about 30 times higher than that
of H2AD/H2B (See Table 1).
Consequently, quantum yield of
PDI increased approximately
several thousand-fold compared
These results clearly demonstrated that the “insulator base
pairs” successfully suppressed electron transfer from the nu-
cleobases to PDI. On the other hand, the emission intensity
of duplexes containing two H moieties in only one strand of
the duplex (H2AD/N and D1/H2B) was far lower than that
of H2AD/H2B (Figure 2A). Furthermore, the emission in-
tensity of single-stranded H2AD was about half that of
H2AD/H2B. Although incorporation of insulators into
single-strand raised quantum yield of PDI, these data indi-
cated that “base pairing” of insulators is desirable for the
enhancement of PDI fluorescence. Incorporation of PDI
into the “base-paired” insulator molecules might also lead
to shielding from water as well as adjacent nucleobases.
We next examined the importance of the cyclohexane
ring for the enhancement of PDI emission. I2AD/I2B, which
has an I–I pair between PDI and nucleobases, showed
almost the same emission intensity as H2AD/H2B (compare
the blue line with the red one in Figure 2B). Hence, F of
I2AD/I2B was almost identical to that of H2AD/H2B. Thus,
I–I pairs show almost the same “insulating ability” as H–H
pairs. In contrast, introduction of J–J pairs did not much en-
hance the fluorescence emission (green line in Figure 2B),
indicating that at least one cyclohexane ring is necessary for
the insulating effect.
to that of D1/N without insula-
tors. The F value of H6AD/
H6B is comparable to those of
common fluorophores,[21] indi-
cating that this duplex can be
utilized for labeling. This dra-
matic increase in quantum yield
can also be detected even by
Figure 3. A photograph of A)
the naked eye (see Figure 3).
The solution exhibits bright
orange color by incorporating
six “insulator base pairs”. Simi-
larly, incorporation of six I–I
D1/N, B) H2AD/H2B and C)
H6AD/H6B at RT. Solution
conditions were as follows:
[DNA]=1.0 mm,
[NaCl]=
100 mm, pH 7.0 (10 mm phos-
phate buffer). Excitation wave-
pairs also increased F to 0.56 length: 520 nm.
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H. Kashida et al.
(RP)-HPLC and were characterized by MALDI-TOFMS (Autoflex,
Bruker Daltonics).
(I6AD/I6B). These results clearly show that incorporation
of multiple “insulator base pairs” between PDI and nucleo-
bases is effective for enhancement of the quantum yield. In
addition, emission intensity of H6AD/H6B was still higher
than that of single-stranded H6AD (see Figure S1 in Sup-
porting Information), demonstrating that “base pairing” of
insulators is also effective for the enhancement in quantum
yield.
The MALDI-TOFMS data for the DNA were as follows: m/z: H2AD:
calcd for [H2AD+H+]: 4865; found: 4864; H2B: calcd for [H2B+H+]:
4283; found: 4281; H6AD: calcd for [H6AD+H+]: 6142; found: 6141;
H6B: calcd for [H6B+H+]: 5560; found: 5560; I2AD: calcd for
[I2AD+H+]: 4933; found: 4935; I2B: calcd for [I2B+H+]: 4351; found:
4351; I6AD: calcd for [I6AD+H+]: 6346; found: 6346; I6B: calcd for
[I6B+H+]: 5764; found: 5764; J2AD: calcd for [J2AD+H+]: 4921;
found: 4921; J2B: calcd for [J2B+H+]: 4339; found: 4341; D1: calcd for
[D1+H+]: 4227; found: 4227.
The melting temperatures (Tm values) of the duplexes are
also summarized in Table 1. Incorporation of insulator pairs
into DNA unexpectedly stabilized the duplex even though
insulators have non-planar structures. For example, the Tm
of H2AD/H2B was 44.28C, which was 3.28C higher than
that of D1/N. Furthermore, the introduction of six H–H
pairs greatly enhanced the thermal stability of the duplex:
the Tm of H6AD/H6B was as high as 62.08C. It can be con-
cluded that H–H pairs strongly stabilize the duplex probably
due to hydrophobic interactions. This high stability of H–H
pairs supports the idea that insulator moieties form “base
pairs” in a DNA duplex and disturb p–p stacking between
PDI and the nucleobases. Similarly, incorporation of I–I and
J–J pairs showed similar stabilization compared to D1/N.
Shielding of PDI from natural nucleobases was also substan-
tiated by the UV/Vis spectra; absorption maximum of
H2AD/H2B was 535 nm whereas that of D1/N was 546 nm
(See Figure S2 in Supporting Information). Concurrently,
absorbance of H2AD/H2B was larger than that of D1/N.
These blue-shift and hyperchromic effect show that interac-
tions between PDI and nucleobases were disturbed by insu-
lator base pairs.
Spectroscopic measurements: Fluorescence spectra were measured on a
JASCO model FP-6500 with a microcell. The excitation wavelength was
500 nm. The sample solutions were as follows: [NaCl]=100 mm, pH 7.0
(10 mm phosphate buffer), [DNA]=1.0 mm. Quantum yield were deter-
mined from the quantum yield of Rhodamine 6G in ethanol (0.94) as a
reference.
Measurement of the melting temperature: The melting curve of duplex
DNA was obtained with a Shimadzu UV-1800 by measurement of the
change in absorbance at 260 nm versus temperature. The melting temper-
ature (Tm) was determined from the maximum in the first derivative of
the melting curve. Both the heating and the cooling curves were mea-
sured, and the calculated Tms agreed to within 2.08C. The temperature
ramp was 0.58C minÀ1. The sample solutions were as follows: [NaCl]=
100 mm, pH 7.0 (10 mm phosphate buffer), [DNA]=5.0 mm.
Acknowledgements
This work was supported by Core Research for Evolution Science and
Technology (CREST), the Japan Science and Technology Agency (JST).
Partial support by a Grant-in-Aid for Scientific Research from the Minis-
try of Education, Culture, Sports, Science and Technology, Japan (for
H.A.) is also acknowledged.
In conclusion, the quantum yield of PDI dramatically in-
creased following the introduction of “insulator base pairs”
with cyclohexane moieties. The quantum yield of PDI was
increased from <0.001 to as high as 0.59 when six H–H
pairs were introduced between PDI and nucleobases. Thus,
this duplex has the potential to be used for labeling of DNA
or RNA. Because even PDI, whose fluorescence is com-
pletely quenched by natural nucleobases, showed high quan-
tum yield, the “insulator base pairs” could be utilized for
the enhancement of quantum yield of other fluorophores.
We recently reported the assembly of fluorophores in DNA
duplexes, in which the fluorophores and the natural base
pairs were alternately introduced.[22] The “insulator base
pairs” can also be utilized to assemble fluorophores without
decreasing the quantum yield.
Keywords: cyclohexane
insulators · perylenediimide
· DNA · fluorescent probes ·
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Experimental Section
Synthesis of the DNA modified with perylenediimide and insulators: The
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Received: June 10, 2010
Published online: August 26, 2010
Chem. Eur. J. 2010, 16, 11554 – 11557
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