P-stilbazole moieties as artificial base pairs for photo-cross-linking of DNA duplex

Article abstract of DOI:10.1021/ja401835j

In this study, we report a photo-cross-linking reaction between p-stilbazole moieties. p-Stilbazoles were introduced into base-paring positions of complementary DNA strands. The [2 + 2] photocycloaddition reaction occurred rapidly upon light irradiation at 340 nm. Consequently, duplex was cross-linked and highly stabilized after 3 min irradiation. The CD spectrum of the cross-linked duplex indicated that the B-form double-helical structure was not severely distorted. NMR analysis revealed only one conformation of the duplex prior to UV irradiation, whereas two diastereomers were detected after the photo-cross-linking reaction. Before UV irradiation, p-stilbazole can adopt two different stacking modes because of rotation around the single bond between the phenyl and vinyl groups; these conformations cannot be discriminated on the NMR time scale due to rapid interconversion. However, photo-cross-linking fixed the conformation and enabled discrimination both by NMR and HPLC. The artificial base pair of p-methylstilbazolium showed almost the same reactivity as p-stilbazole, indicating that positive charge does not affect the reactivity. When a natural nucleobase was present in the complementary strand opposite p-stilbazole, the duplex was significantly destabilized relative to the duplex with paired p-stilbazole moieties and no photoreaction occurred between p-stilbazole and the nucleobase. The p-stilbazole pair has potential as a third base pair for nanomaterials due to its high stability and superb orthogonality.

Full text of DOI:10.1021/ja401835j

Article
pubs.acs.org/JACS
p‑Stilbazole Moieties As Artificial Base Pairs for Photo-Cross-Linking
of DNA Duplex
Hiromu Kashida,* Tetsuya Doi, Takumi Sakakibara, Takamitsu Hayashi, and Hiroyuki Asanuma*
Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
S
* Supporting Information
ABSTRACT: In this study, we report a photo-cross-linking
reaction between p-stilbazole moieties. p-Stilbazoles were
introduced into base-paring positions of complementary
DNA strands. The [2 + 2] photocycloaddition reaction
occurred rapidly upon light irradiation at 340 nm. Con-
sequently, duplex was cross-linked and highly stabilized after 3
min irradiation. The CD spectrum of the cross-linked duplex
indicated that the B-form double-helical structure was not
severely distorted. NMR analysis revealed only one con-
formation of the duplex prior to UV irradiation, whereas two
diastereomers were detected after the photo-cross-linking reaction. Before UV irradiation, p-stilbazole can adopt two different
stacking modes because of rotation around the single bond between the phenyl and vinyl groups; these conformations cannot be
discriminated on the NMR time scale due to rapid interconversion. However, photo-cross-linking fixed the conformation and
enabled discrimination both by NMR and HPLC. The artificial base pair of p-methylstilbazolium showed almost the same
reactivity as p-stilbazole, indicating that positive charge does not affect the reactivity. When a natural nucleobase was present in
the complementary strand opposite p-stilbazole, the duplex was significantly destabilized relative to the duplex with paired p-
stilbazole moieties and no photoreaction occurred between p-stilbazole and the nucleobase. The p-stilbazole pair has potential as
a “third base pair” for nanomaterials due to its high stability and superb orthogonality.
that planar molecules incorporated via D-threoninol linkers
serve as “artificial base pairs” to stabilize a DNA duplex.19 For
example, when azobenzene derivatives were introduced into the
base-pairing positions of both strands, the duplex was highly
stabilized relative to the unmodified DNA due to intermo-
lecular stacking interactions.20,21 Interestingly, this “base pair”
showed high orthogonality with natural base pairs. We have
also reported that base pairs of p-methylstilbazolium, which has
a positive charge, stabilize duplexes even more strongly than
natural base pairs due to electrostatic interactions between the
positive charge on p-methylstilbazolium and the phosphate
anion.22,23 In this paper, we used the photocycloaddition
reaction between stilbazole derivatives to further improve the
heat tolerance of a DNA duplex.
p-Stilbazole derivatives are known to form dimers upon UV
irradiation via a [2 + 2] photocycloaddition reaction.24−26
Therefore, if a duplex containing an artificial base pair of p-
stilbazole moieties is irradiated with UV light, the p-stilbazoles
should react with each other and the duplex should be cross-
linked (Scheme 1). If the p-stilbazole base pair shows high
orthogonality, p-stilbazoles could serve as a “third base pair” for
preparation of complex nanomaterials. Here, we introduced p-
stilbazole moieties (B in Figure 1) into DNA and investigated
their photoreactivity and effect on duplex stability. p-
INTRODUCTION
■
Watson−Crick base pairs stabilize DNA duplexes through
hydrogen-bonding and stacking interactions. Extremely high
orthogonality of base pairing is realized by strict control of
these two interactions. It has become possible to synthesize
oligodeoxyribonucleotides of essentially any sequence in an
automated fashion. This has made DNA an ideal material to
construct nanostructures and nanodevices.1,2 DNA duplexes
dissociate into single strands at high temperature. Although the
reversibility is essential for biological processes, such as
replication and transcription, the heat lability of DNA duplex
in some cases restricts applications of DNA-based nanomateri-
als. To make DNA nanomaterials more heat tolerant, it is
necessary to increase the thermal stability of DNA structures.3
Photo-cross-linking or photoligation has been widely studied
not only for its biological relevance but also for nucleic
detection and duplex stabilization.4−7 For example, Lewis et al.
reported photo-cross-linking system by introducing stilbene
units into main chain of a DNA duplex.8 Fujimoto et al.
reported several vinyldeoxyuridine and vinylcarbazole deriva-
tives that form adducts with natural nucleobases.9 Cross-linking
has been used in SNP detection, DNA computing, and
nanostructure stabilization.10−14 Furthermore, photoligation
via [4 + 4] cycloaddition reaction between anthracene moieties
has been reported by Ihara et al. and other groups.15−18
Herein, we propose a photo-cross-linking system using p-
stilbazoles as an artificial base pair. We have previously reported
Received: February 20, 2013
© XXXX American Chemical Society
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Spectroscopic Measurements. UV spectra were measured on a
JASCO model J-560 equipped with a programmed temperature
controller; 10 mm quartz cells were used. CD spectra were measured
on a JASCO model J-820 equipped with programmed temperature-
controllers using 10 mm quartz cells.
Scheme 1. Schematic Illustration of Photo-Cross-Linking via
[2 + 2] Photocycloaddition Between p-Stilbazole Moieties
Measurement of the Melting Temperature. The melting
curves were obtained with a JASCO model J-560 by measuring the
change in absorbance at 260 nm versus temperature. The melting
temperature (Tm) was determined from the maximum in the first
derivative of the melting curve. Both the heating and the cooling
curves were measured, and the calculated Tms agreed to within 2.0 °C.
The temperature ramp was 1.0 °C min−1.
UV Irradiation. A xenon light source (MAX-301, Asahi Spectra)
equipped with interference filters centered at 341.5 nm (half
bandwidth 9 nm, power density 0.15 mW/cm2) was used for photo-
cross-linking. The sample solution was added to a cuvette, and the
temperature of light irradiation was controlled using a programmable
temperature controller. Photoirradiation was conducted at 0 °C.
HPLC Analyses. A Merck LiChrospher 100 RP-18(e) column
heated at 50 °C was used for HPLC analyses. The flow rate was 0.5
mL/min. A solution of 50 mM ammonium formate (solution A) and a
mixture of 50 mM ammonium formate and acetonitrile (50/50, v/v)
(solution B) were used as mobile phases. A linear gradient of 5−35%
solution B over 30 min was employed. HPLC chromatograms were
monitored at 260 nm absorption.
Molecular Dynamic Simulation. The Insight II/Discover 98.0
program package was used for conformational energy minimization.
The duplex tethering p-stilbazole was built from canonical B-form
DNA by a graphical program. The AMBER95 force field was used for
the calculation. All the structures were energy minimized to an RMS
derivative of <0.001 kcal Å−1. Computations were carried out on a
Silicon Graphics O2+ workstation with the IRIX64 OS release 6.5.
Figure 1. Sequences of ODNs synthesized in this study. Chemical
structure of p-stilbazole monophosphate is also shown.
Methylstilbazolium (Z in Figure 1), which has a similar
structure but a positive charge, was also incorporated into DNA
to investigate the effects of positive charge on photoreactivity.
Orthogonality of stilbazole moieties with natural base pairs was
also evaluated.
RESULTS AND DISCUSSION
■
Photochemical Properties of p-Stilbazole Monophos-
phate and p-Stilbazole-Modified DNA. We first inves-
tigated the photochemical properties of p-stilbazole mono-
phosphate (Bp in Figure 1) before evaluating photochemical
properties in DNA. Figure 2a shows the UV−vis spectra of Bp
EXPERIMENTAL SECTION
■
Materials. All the conventional phosphoramidite monomers, CPG
columns, and reagents for DNA synthesis were purchased from Glen
Research. Other reagents for the synthesis of phosphoramidite
monomers were purchased from Tokyo Chemical Industry, Wako,
and Aldrich. Unmodified oligodeoxyribonucleotides (ODNs) were
purchased from Integrated DNA Technologies.
Synthesis of ODNs. All modified ODNs were synthesized on an
automated DNA synthesizer (H-8-SE, Gene World) by using
phosphoramidite monomers bearing B or Z. Syntheses of phosphor-
amidite monomers B and Z were reported previously.22 p-Stilbazole
monophosphate was synthesized as described in the Supporting
Information. Sample handling was conducted under dark conditions.
After the workup, ODNs were purified by reversed phase HPLC and
characterized using a MALDI-TOF MS (Autoflex II, Bruker
Daltonics). Matrix-assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS) data for the synthesized
ODNs: Ba: obsd 3072 (calcd for [Ba + H+]: 3073). Bb: obsd 3112
(calcd for [Bb + H+]: 3113). Bc: obsd 2206 (calcd for [Bc + H+]:
2206). Bd: obsd 2126 (calcd for [Bd + H+]: 2126). Za: obsd 3088
(calcd for [Za + H+]: 3088). Zb: obsd 3128 (calcd for [Zb + H+]:
3128).
Figure 2. UV−vis spectra of (a) stilbazole monophosphate Bp and (b)
Ba/N before and after UV irradiation (340 nm). Tm of Ba/N was 43.2
°C. Solution conditions were 5.0 μM Bp, 5.0 μM DNA, 100 mM
NaCl, 10 mM phosphate buffer (pH 7.0), 0 °C.
NMR Measurements. NMR samples were prepared by dissolving
lyophilized DNA in an H2O/D2O 9:1 solution containing 10 mM
sodium phosphate (pH 7.0) to give a duplex concentration of 1.0 mM.
NaCl was added to give a final sodium concentration of 200 mM.
NMR spectra were measured with a Varian INOVA spectrometer (700
MHz) equipped for triple resonance at a probe temperature of 275 K.
Resonances were assigned by standard methods using a combination
of 1D, totally correlated spectroscopy (TOCSY) (60 ms of mixing
time), and nuclear Overhauser effect spectroscopy (NOESY) (150 ms
of mixing time) experiments. All spectra in the H2O/D2O 9:1 solution
were recorded using the 3−9−19 WATERGATE pulse sequence for
water suppression.27
before and after photoirradiation. Before photoirradiation, Bp
showed peaks at 303 and 312 nm arising from the ππ*
transition of trans p-stilbazole (black line). When the sample
was irradiated with 340 nm UV light for 1 min, these peaks
immediately disappeared, and a new broad peak appeared at
297 nm due to photoisomerization to the cis form (purple
line).28 Thus, the photoisomerization rate of free p-stilbazole is
intrinsically very rapid. Further photoirradiation at 340 nm for
480 min resulted in a decrease in absorbance at around 300 nm
and appearance of a peak at 247 nm (red line). These spectral
B
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shifts are due to cyclization and subsequent oxidation to a
benzoquinoline derivative as shown in Scheme 2a.29 The
Scheme 2. Photochemical Reaction Observed with (a) p-
Stilbazole Monophosphate and (b) p-Stilbazole Moiety in
DNA Duplex
Figure 3. UV−vis spectra of Ba/Bb before and after photoirradiation.
Solution conditions were 5.0 μM DNA, 100 mM NaCl, 10 mM
phosphate buffer (pH 7.0), 0 °C.
The photo-cross-linked product of Ba/Bb was further
analyzed by reversed-phase HPLC (Figure 4). Before photo-
cyclized product was detected by ESI-MS (see Supporting
Information). The peak at around 300 nm did not disappear
even after 480 min irradiation, indicating that the [2 + 2]
photocycloaddition reaction did not occur under these
conditions. These results show that photoirradiation of p-
stilbazole potentially causes several side reactions but not the
desired [2 + 2] photocycloaddition (Scheme 2a).
In striking contrast, incorporation of p-stilbazole into DNA
drastically suppressed these side reactions. The Ba/N duplex
with a stilbazole moiety in one strand of the DNA duplex
showed absorption maximum (λmax) at 327 nm (black line in
Figure 2b). The peak did not change after irradiation at 340 nm
for 5 min. Although the peak slightly decreased after 30 min
irradiation, these results clearly demonstrated that introduction
of p-stilbazole moiety into DNA strongly suppressed photo-
isomerization to cis form (Scheme 2b). This is in sharp contrast
to azobenzene, which can isomerize even within DNA duplex.30
This difference in isomerization is attributable to differences in
the isomerization mechanisms: Isomerization of the stilbene
derivative proceeds exclusively through rotation, whereas that
of azobenzene occurs via both rotation and inversion.31
Consistently, photoisomerization of stilbene derivatives is
suppressed in viscous solution, whereas isomerization of
azobenzene is not affected significantly by viscosity.32 Within
a DNA duplex, isomerization of p-stilbazole is presumably
suppressed by stacking interactions with natural base pairs.
Photo-Cross-Linking of p-Stilbazole in DNA Duplex.
The photochemical properties of the duplex Ba/Bb, which has
B residues in complementary positions on the two strands,
were investigated in order to evaluate the photo-cross-linking of
p-stilbazole within a duplex.33 Before photoirradiation, the
UV−vis spectrum of the Ba/Bb duplex had a peak at 323 nm
(Figure 3). A slight hypsochromic shift compared with the
single-stranded Ba (λmax 325 nm) indicated that B residues
formed an H dimer (Figure S1).34 Upon irradiation at 340 nm
for 3 min, the peak at 323 nm had completely disappeared. This
disappearance of the π−π* absorption band assigned to the p-
stilbazole moieties strongly suggested that a [2 + 2]
photocycloaddition reaction between two p-stilbazole moieties
had occurred. The λmax of Ba/Bb did not shift upon
photoirradiation, indicating that trans-to-cis isomerization did
not occur and that neither p-stilbazoles were in the cis form.
Instead, the trans forms reacted. Note that isomerization to the
cis form causes a blue-shift in the absorption spectrum as shown
in Figure 2A.
Figure 4. HPLC chromatograms of Ba/Bb before and after indicated
times of photoirradiation.
irradiation, two peaks corresponding to the single-stranded
forms of Ba and Bb were observed. After 3 min photo-
irradiation, however, the two peaks disappeared and a new peak
appeared at a shorter retention time. MALDI-TOF MS analysis
of this new peak proved it to be a photo-cross-linked duplex
(Figure S2). From these results, we conclude that a [2 + 2]
photocycloaddition reaction occurred between p-stilbazole
moieties in the DNA duplex upon UV irradiation at 340 nm.
Structure of Photodimer of p-stilbazole. Photocycload-
dition of stilbene derivatives usually produces several isomers.24
When the stilbene derivatives are within a DNA scaffold, the
orientations of p-stilbazoles should be restricted, and thus the
stereochemistry of photodimerized product should be con-
trolled. In order to investigate the structure of photo-cross-
linked product, the Bc/Bd duplex was analyzed by NMR. The
imino region of the 1D spectrum of Bc/Bd had six peaks
corresponding to imino protons from the six natural base pairs
(Figure 5a). These sharp peaks indicated that hydrogen
bonding of natural base pairs was not disturbed by the
presence of the p-stilbazole moieties. After irradiation at 340
nm, these peaks disappeared, and eight new peaks appeared.
Although several peaks overlap, the appearance of more than six
peaks indicates that there are at least two species in the photo-
cross-linked product.
The aromatic regions of the 1D NMR spectra in D2O of Bc/
Bd before and after irradiation are shown in Figure 5b. In the
nonirradiated sample, 11 peaks corresponded to 4 vinyl and 8
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Figure 6. Presumed structures of diastereoisomers produced upon
photocycloaddition.
that the p-stilbazoles in Ba/Bb may adopt either of the possible
conformations without disturbing the overall B-form structure.
Thermal Stability of Photo-Cross-Linked Duplex. We
next evaluated thermal stability of the cross-linked duplex.
Melting curves of Ba/Bb before and after photoirradiation are
shown in Figure 7a. Before photoirradiation, the Tm of Ba/Bb
Figure 5. 1H NMR spectra of Bc/Bd in (a) imino regions measured in
H2O/D2O and (b) aromatic regions in D2O. White and black arrows
in (b) indicate vinyl and phenyl protons of the p-stilbazole moiety,
respectively.
phenyl protons of 2 p-stilbazole moieties.35 In the spectrum of
the irradiated sample, 4 peaks assigned to vinyl protons
disappeared as expected, and 12 new, partially overlapping
peaks appeared. If the photo-cross-linked product were a single
species, only eight peaks should be observed. Hence, the
appearance of 12 peaks clearly shows that more than one
species was produced upon photoirradiation. Furthermore,
TOCSY in D2O gave eight cross-peaks in the aromatic region
after photoirradiation, whereas four cross-peaks were observed
before photoirradiation (Figure S3). These results undoubtedly
show that there are two diastereomers of the photo-cross-linked
product. The existence of two diastereomers was further
supported by the appearance of two peaks in the HPLC
chromatogram of Bc/Bd after irradiation (Figure S4).36
Previously, we prepared DNA duplexes containing hetero-
dimers of azo compounds. NMR structural analyses revealed
that the dyes were stacked in an antiparallel manner and located
at 3′ side of the complementary strand.21 Thus, p-stilbazole
moieties in this study are expected to adopt similar stacking
structures. A trans-to-cis isomerization within the duplex could
be ruled out based on the fact that the λmax of Ba/Bb did not
shift during photoirradiation (Figure 3). Furthermore, NMR
and HPLC analyses revealed that equal amounts of
diastereomers were produced. Thus, we concluded that the
two observed products are diastereomers (Figure 6, right
panel). The very fast rotation around the single bond between
phenyl and vinyl groups of p-stilbazole results in the production
of the two diastereomers upon irradiation. Before photo-
irradiation, p-stilbazole can adopt either of two stable stacking
modes (Figure 6, left panel). Since interconversion between the
two conformers is rapid, NMR does not distinguish these two
conformers. Photo-cross-linking prevents interconversion and
fixes the conformation. As a result, duplexes containing each of
these two “fixed” conformers are observed both by NMR and
HPLC. We have also conducted molecular dynamics
simulations of Ba/Bb (Figure S5). These simulations indicate
Figure 7. (a) Melting curves and (b) CD spectra of Ba/Bb before
(dotted line) and after photoirradiation (solid line). Solution
conditions were 5.0 μM DNA, 100 mM NaCl, 10 mM phosphate
buffer (pH 7.0), 0 °C.
Table 1. Comparison of Thermal Stabilities and
Photoreactivities of Duplexes Containing p-Stilbazole and p-
Methylstilbazolium
a
Tm/°C
b
c
sequence
before UV irradiation
after UV irradiation
kobs/min−1
Ba/Bb
Za/Zb
Ba/Zb
46.4
51.5
49.7
>80
>80
>80
1.1
1.5
0.9
a
Conditions: 5.0 μM DNA, 100 mM NaCl, 10 mM phosphate buffer
b
(pH 7.0), 20 °C. UV light at 340 nm was irradiated for 30 (Ba/Zb),
60 (Ba/Bb) or 120 min (Za/Zb). Tm of native duplex without
stilbazole was 38.2 °C. Observed rate constant determined from
c
initial slope of Figure 9.
was 46.4 °C (Figure 7a and Table 1), which was 8.2 °C higher
than that of a native duplex without B residues (38.2 °C). The
Tm increased to more than 80 °C after photoirradiation. Thus,
thermal stability of duplex was dramatically improved through
photodimerization of the p-stilbazole moieties. The overall
structure of photo-cross-linked duplex was analyzed by CD
spectroscopy (Figure 7b). Before photoirradiation, positive
exciton couplet was observed at around 260 nm, showing that
D
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natural nucleobases adopted B-form duplex. An induced CD
was observed at 320 nm, indicating that the p-stilbazoles are
located in the chiral environment of DNA duplex. This induced
CD band disappeared upon light irradiation because π−π*
absorption of p-stilbazole at 325 nm disappears upon
photocycloaddition. In the cross-linked species, the CD couplet
at 260 nm remained almost unchanged to that prior to
irradiation. These results indicated that overall B-form double-
helical structure remained intact in spite of nonplanar structure
of photodimerized p-stilbazoles.24
Effect of Positive Charge on Photo-Cross-Linking
Properties. We then investigated photo-cross-linking of p-
methylstilbazolium, a stilbazole derivative containing a positive
charge (Z in Scheme 1). The UV−vis spectra of a duplex
tethering these moieties, Za/Zb, are shown in Figure 8. Before
Figure 9. Reaction kinetics of Ba/Bb (red line), Za/Zb (blue line),
and Ba/Zb (green line) determined from UV−vis spectra.
Absorbances of all duplexes were adjusted to be almost the same
(Ba/Bb: 0.186, Ba/Zb: 0.196, Za/Zb: 0.187).
important roles in both yield and stereoselectivity.24,39
Although the positive charge does affect the association process
of stilbazoles, the “intrinsic” reactivity should be almost the
same between p-stilbazole and p-methylstilbazolium. In other
words, intrinsic photoreactivity of molecules can be evaluated
by using DNA as a scaffold. Use of a DNA scaffold also allows
reactivity of hetero combinations to be investigated while
excluding the corresponding homo combinations.
Orthogonality with Natural Nucleobases. In order to
serve as artificial base pairs, orthogonality is one of the most
important prerequisites. Thus, we investigated orthogonality of
B-B or Z-Z pairs with natural base pairs. Melting temperatures
of Xa/Xb duplexes (where X was A, T, G, C, B, or Z) before
photoirradiation are shown in Table 2. The B residue more
Figure 8. UV−vis spectra of (a) Za/Zb and (b) Ba/Zb before and
after photoirradiation. Solution conditions were 5.0 μM DNA, 100
mM NaCl, 10 mM phosphate buffer (pH 7.0), 0 °C.
photoirradiation, an absorption maximum was observed at 340
nm. This peak completely disappeared after photoirradiation
for 3 min, unambiguously demonstrating that the photo-
cycloaddition reaction between Z moieties occurred. The
photo-cross-linking between Z moieties was further confirmed
by HPLC and MALDI-TOF MS analyses (Figure S6). The Tm
of Za/Zb before photoirradiation was 51.5 °C (Table 1 and
Figure S7), about 5.1 °C higher than that of Ba/Bb (46.4 °C).
The high stability of the Z-Z pair is due to electrostatic
interactions between phosphate anions and cations of Z
residues as we reported previously.22 In addition, cation−π
interaction should also contribute to the high stability. The Tm
of the Za/Zb increased to over 80 °C after photoirradiation at
340 nm, demonstrating significant stabilization upon photo-
cross-linking of the Z-Z pair. We also investigated photo-
cycloaddition of the hetero combination between B and Z
residues (Figure 8b). Absorption maxima of B and Z residues at
about 320 and 350 nm, respectively, simultaneously decreased
upon irradiation. Thus, the B−Z pair can be photo-cross-linked
in the context of DNA duplexes.
The observed rate constant of the reaction between p-
methylstilbazolium residues was also determined from UV−vis
spectra (Table 1 and Figure 9). The observed rate constants
measured under the conditions of the same absorbance and
light intensity are a measure of the relative quantum yields of
reaction.37 The observed rate constant of Za/Zb was 1.5 min−1,
which was almost the same order as that of the p-stilbazole
duplex (1.1 min−1).38 The observed rate constant of hetero B-Z
pair was also similar (0.9 min−1). Thus, the cationic charge on
the Z moiety did not significantly affect photoreactivity. In
photodimerization reactions of p-stilbazole and p-methylstilba-
zolium not conjugated with DNA, the positive charge played
Table 2. Orthogonality of B or Z Moiety with Natural
Nucleobases before Photoirradiation
a
Tm/°C
Aa
Ta
Ga
Ca
Ba
Za
Ab
Tb
Gb
Cb
Bb
Zb
25.7
42.8
30.7
27.3
33.7
32.5
40.3
28.7
31.9
26.8
37.4
34.4
30.6
33.6
29.8
48.8
36.3
35.1
24.4
26.4
45.9
23.8
36.0
34.7
31.6
34.0
33.9
35.1
46.4
49.7
30.4
31.8
32.4
32.2
50.9
51.5
a
Conditions: 5.0 μM DNA, 100 mM NaCl, 10 mM phosphate buffer
(pH 7.0), 20 °C.
strongly hybridizes when the complementary residue is B than
when it is a natural nucleobase. For example, Ba/Bb had a Tm
of 46.4 °C, whereas duplexes of Ba with Ab, Tb, Gb, and Cb
had much lower Tms (31.6, 34.0, 33.9, and 35.1 °C,
respectively). These Tms were also significantly lower than
those of duplexes with natural A-T or G-C pairs: The Tm of
Aa/Tb (42.8 °C) was much higher than that of Ab/Bb (33.7
°C). The same tendency was observed with Z-Z pairs (Table
2). These results clearly demonstrated that B-B and Z-Z pairs
show excellent orthogonality with natural nucleobases. In other
words, the B-B or Z-Z pair could be used as “third base pair”
for construction of DNA-based nanomaterials. The instability
of a natural base paired with either B or Z is attributable to the
sizes of the B and Z moieties; these stilbazoles are much larger
than nucleobases, and intercalation of these moieties should
E
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Notes
cause steric hindrance with D-ribose. In contrast, when B and Z
moieties are paired, steric hindrance is likely lessened by flexible
D-threoninol linkers. In addition, strong intermolecular stacking
interactions between B and Z moieties should contribute to
high stabilities of the duplexes containing two of these residues.
Next, orthogonality in the photo-cross-linking reaction was
evaluated. When a B or Z moiety was located counter to A, T,
G, or C, the UV−vis spectra did not change even after
photoirradiation for 30 min (Figure S9).40 Spectra of Ba/N
(Figure 2b) and Za/N (Figure S10) did not change upon
photoirradiation of the sample. This indicates that neither B
nor Z react with natural nucleobases, whereas fast reactions
occurred between B-B, Z-Z, and B-Z pairs. It has been reported
that natural nucleobases, especially pyrimidine bases, may cause
[2 + 2] photocycloaddition reactions. For example, a thymine
dimer is formed upon UV irradiation that may induce point
mutations.41 Photo-cross-linking agents, such as psoralen, form
interstrand cross-link with thymine via photocycloaddition
reactions.42,43 In our design, the vinyl group of p-stilbazole was
located near the center of the natural base pair (Figure S11),
but the reaction sites of nucleobases (e.g., C5 and C6 of
pyrimidine bases) are toward the outside of the DNA duplex.
Separation of vinyl group from pyrimidine inhibited cyclo-
addition reaction between p-stilbazole and natural nucleobases.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a Grant-in-Aid for Scientific
Research (A) (21241031) and a Grant-in-Aid for Scientific
Research on Innovative Areas “Molecular Robotics” (no.
24104005) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan. Partial support by The Asahi
Glass Foundation (for H.A.) is also acknowledged.
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In conclusion, we have successfully developed artificial base
pairs that can be photo-cross-linked in the context of a DNA
duplex. When a duplex with a pair of p-stilbazoles located on
opposite strands was irradiated with UV light, a [2 + 2]
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that photo-cross-linking reaction was complete within 3 min.
NMR analyses indicated that two diastereomers are produced
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showed high orthogonality with natural nucleobases in terms of
both stability and photo-cross-linking reaction.
DNA has recently been used for construction of nanoma-
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natural base pairs, A-T and G-C, are not necessarily optimal in
these materials. Furthermore, it is not necessary to mimic
natural nucleobases when DNA is chemically synthesized and
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ASSOCIATED CONTENT
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(21) Fujii, T.; Kashida, H.; Asanuma, H. Chem.Eur. J. 2009, 15,
S
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* Supporting Information
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Synthetic routes of p-stilbazole monophosphate, UV−vis,
NMR, HPLC charts of duplexes tethering p-stibazole and p-
methylstilbazolium, energy-minimized structures are available.
These materials are available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
(27) Liu, M.; Mao, X.-a.; Ye, C.; Huang, H.; Nicholson, J. K.; Lindon,
kashida@nubio.nagoya-u.ac.jp; asanuma@nubio.nagoya-u.ac.jp
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(33) Here, we did not introduce spacers at the counter positions of p-
stilbazole because incorporation of spacers rather destabilize duplex.
See ref 20.
(34) B residues should be stacked in antiparallel manner in DNA. See
also ref 21.
(35) Because peaks of p-stilbazole in cis form were not observed, all
the p-stilbazole took trans form in DNA.
(36) On the other hand, only single peak was observed with Ba/Bb
after photoirradiation as shown in Figure 4. Longer DNA portion of
Ba/Bb would lessen the effect of structural difference of diastereomers
compared with Bc/Bd.
(37) It is known that rate constant of photoreaction is affected by
light intensity, absorbance, and quantum yield. Here, absorbances of
Ba/Bb, Ba/Zb, and Za/Zb were adjusted to about 0.19. In addition,
reaction kinetics of all duplexes was measured within several hours to
avoid change of light intensity during the experiments. As a result,
initial slope of Figure 9 (observed rate constant in Table 1) should be
proportional to quantum yields.
(38) We also investigated effects of flanking sequence and mismatch
on reaction rate constant of B-B combination. As a result, flanking AT
pairs strongly facilitated photocrosslinking of p-stilbazole. Incorpo-
ration of mismatches decreased the rate constant. See Figure S8 for
actual data.
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decreased probably due to photodegradation, the reaction was much
slower than photocrosslinking reaction.
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dx.doi.org/10.1021/ja401835j | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX