Ultrafast reversible photo-cross-linking reaction: Toward in situ DNA manipulation

Article abstract of DOI:10.1021/ol801112j

(Figure Presented) We describe a novel ultrafast reversible DNA interstrand photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside ( ^CNVK). Oligodeoxynucleotide (ODN) containing ^CNVK can be photo-cross-linked by irradiation at 366 nm for 1 s, and the photo-cross-linked ODN can be split by irradiation at 312 nm for 60 s.

Full text of DOI:10.1021/ol801112j

ORGANIC
LETTERS
2008
Vol. 10, No. 15
3227-3230
Ultrafast Reversible Photo-Cross-Linking
Reaction: Toward in Situ DNA
Manipulation
Yoshinaga Yoshimura† and Kenzo Fujimoto*,†,‡
School of Materials Science, Japan AdVanced Institute of Science and Technology,
1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan, and InnoVation Plaza Ishikawa, Japan
Science and Technology Agency, 2-13, Asahidai, Nomi, Ishikawa 923-1211, Japan
kenzo@jaist.ac.jp
Received May 14, 2008
ABSTRACT
We describe a novel ultrafast reversible DNA interstrand photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside (CNVK).
Oligodeoxynucleotide (ODN) containing CNVK can be photo-cross-linked by irradiation at 366 nm for 1 s, and the photo-cross-linked ODN can
be split by irradiation at 312 nm for 60 s.
The cross-linking reaction has widely been used to stabilize
complexes with DNA by a covalent-bond formation. Bio-
technological approaches based on the cross-linking reactions
have been also used in inhibiting gene expression,1 ap-
preciating DNA damage,2 and investigating RNA structures.3
Psoralen and its analogues have enjoyed remarkable suc-
cesses as T-selective photo-cross-linkers, applicable to a wide
variety of in vitro as well as in vivo studies.4 However, the
photo-cross-linking reaction from the use of psoralens limits
the choice of the target DNA, as an efficient cross-linking
reaction requires a TpA step.5 Additionally, the photo-cross-
linked DNAs, via a [2 + 2] cycloaddition between psoralen
and thymine base, were regenerated to the parent DNA by
254 nm irradiation resulting in fatal damage to normal DNA,
due to the formation of pyrimidine dimer.6 To overcome such
sequence dependence and DNA damage, researchers have
been seeking alternative types of photo-cross-linkers with
high reactivity. We have also been studying artificial DNA
bases as a tool for the photochemical DNA cross-linking
method.7 By using the carbazole nucleoside incorporated into
DNA, we have observed the repair of a thymine dimer and
have used the same as a tool for photochemical DNA
† Japan Science and Technology Agency.
‡ Japan Advanced Institute of Science and Technology.
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Broughton-Head, V. J.; Peng, G.; Powers, V. E. C.; Ovens, M. J.; Fox,
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10.1021/ol801112j CCC: $40.75
Published on Web 06/27/2008
2008 American Chemical Society

manipulation.8 Herein, we report the development of a novel
ultrafast interstrand photo-cross-linking reaction via 3-cy-
anovinylcarbazole nucleoside (CNVK) in duplex DNA. We
have demonstrated that the modified oligodeoxynucleotide
(ODN) containing CNVK was reversibly photo-cross-linked
with an adjacent pyrimidine base via UV irradiation at two
different wavelengths.
to ODN(ACNVK-GT) in 97% yield along with the disappear-
ance of ODN(ACNVK) and ODN(GT) peaks (Figure 2).10
The synthesis of the phosphoramidite of CNVK is outlined
in Scheme 1. Compound 1 was prepared according to a
Scheme 1. Synthesis of the Phosphoramidite of
3-Cyanovinylcarbazole Nucleoside (4, CNVK)
Figure 2.
HPLC analysis of the irradiated ODN(ACNVK) in the
presence of ODN(GT). 2′-Deoxyuridine (dU) was used as an
internal standard.
MALDI-TOF-MS indicates that the isolated ODN(ACNVK-
GT) obtained from HPLC purification was a photo-cross-
linked product of ODN(ACNVK) and ODN(GT) (calcd
5546.76 for [M + H]+, found 5546.69). The enzymatic
digestion of isolated ODN(ACNVK-GT) showed the formation
of dCyd, dGuo, dThd, and dAdo in a ratio of 5:6:2:3, together
with CNVK<>T photoadduct, which was confirmed by
MALDI-TOF-MS (calcd 599.2118 for [M + Na]+, found
599.2033). On the other hand, when ODN(GCNVK) (5′-
d(TGCGCNVKCCGT)-3′) and ODN(GC) (5′-d(ACGGGCG-
CA)-3′) were used in photo-cross-linking, the cytosine base
reacted with photoexcited CNVK to produce a photo-cross-
linked product ODN(GCNVK-GC) efficiently.11 As shown in
Figure 3, the photo-cross-linking reaction between ODN
(ACNVK) and ODN(GT) was finished by 366 nm irradiation
for only 1 s.
To examine the effect of sequence contexts around the
photo-cross-linking site, we constructed 64 sequences of
ODNs, ODN(Y′ZX′) (X′, Y′, Z ) A, G, C, or T), in the
opposite strand. The photo-cross-linking yield obtained by
photoirradiation at 366 nm for 1 s was determined by HPLC
and UPLC analysis. Among the various combinations of base
pairs between CNVK and natural bases Z (Z ) A, G, C, or
T), we observed that ODNs containing CNVK produced a
method reported in literature.9 Compound 2 was synthesized
from 1 and acylonitrile, while compound 3 was synthesized
from 2 and Hoffer’s R-chlorosugar. The deprotection of 3
with sodium methoxide afforded 4 (CNVK). Compound 4 was
dimethoxytritylated and converted into nucleoside phos-
phoramidite 5. The various modified ODNs, ODN(XCNVKY)
(X, Y ) A, G, C, or T), were prepared, according to standard
phosphoramidite chemistry, on a DNA synthesizer using
phosphoramidite 5. ODNs containing CNVK were character-
ized by MALDI-TOF-MS.
We determined the feasibility of the interstrand photo-
cross-linking via ODN containing CNVK as shown in Figure
1. When ODN(ACNVK) (5′-d(TGCACNVKCCGT)-3′) and
(8) (a) Yoshimura, Y.; Fujimoto, K. Chem. Lett. 2006, 35, 386–387.
(b) Ogasawara, S.; Kyoi, Y.; Fujimoto, K. ChemBioChem 2007, 8, 1520–
1525. (c) Fujimoto, K.; Yoshino, H.; Ami, T.; Yoshimura, Y.; Saito, I. Org.
Lett. 2008, 10, 397–400.
Figure 1. Schematic illustration of photo-cross-linking of ODNs
with CNVK. X, X′, Y, Y′, or Z ) A, G, C, or T.
(9) Bonesi, S. M.; Erra-Balsells, R. J. Heterocycl. Chem. 2001, 38, 77–
87.
(10) The reaction mixture (total volume 30 µL) containing ODN(ACNVK)
and ODN(GT) (each 20 µM, strand concn) in 50 mM sodium cacodylate
buffer (pH 7.0) and 100 mM sodium chloride was irradiated with a UV-
LED (366 ( 15 nm light at 1600 mW/cm2) at a distance of 1.5 cm at 0 °C
for 1 s. After irradiation, the progress of the photoreaction was monitored
by HPLC. The yield was calculated on the basis of ODN(GT). Quantum
yield of the formation of photo-cross-linked product was measured at 366
nm, based on the disappearance of ODN(GT) by employing valerophenone
as an actinometer. The formation of ODN(ACNVK-GT): Φ ) 0.251.
(11) MALDI-TOF-MS: calcd 5547.74 for ODN(GCNVK-GC) [(M + H)+],
found 5547.88; calcd 584.2121 for CNVK<>C photoadduct [(M + Na)+], found
584.2102. The yield was calculated on the basis of ODN(GC).
ODN(GT) (5′-d(ACGGGTGCA)-3′) were irradiated at 366
nm for 1 s, HPLC showed the appearance of a peak relating
(7) (a) Yoshimura, Y.; Ito, Y.; Fujimoto, K. Bioorg. Med. Chem. Lett.
2005, 15, 1299–1301. (b) Ogino, M.; Fujimoto, K. Angew. Chem., Int. Ed.
2006, 45, 7223–7226. (c) Ogasawara, S.; Yoshimura, Y.; Hayashi, M.; Saito,
I.; Fujimoto, K. Bull. Chem. Soc. Jpn. 2007, 80, 2124–2130. (d) Ami, T.;
Ito, K.; Yoshimura, Y.; Fujimoto, K Org. Biomol. Chem. 2007, 5, 2583–
2586.
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Org. Lett., Vol. 10, No. 15, 2008

ature of 36.4 °C, whereas ODN(ACNVK-GT) melted at 67.9
°C (see Supporting Information). An example of this
behavior has been seen for photo-cross-linked ODNs by the
ODN-containing p-carbamoylvinyl phenol nucleoside.7 The
duplex ODN(GCNVK)/ODN(GC) showed a melting temper-
ature of 40.4 °C, whereas ODN(GCNVK-GC) melted at 75.8
°C. Thus, photo-cross-linking increased Tm of ODN, a
dramatic stabilization of the duplex form.
To confirm the photoreversibility of the photo-cross-linked
product, irradiation of the photo-cross-linked ODN(ACNVK-
GT) at 312 nm was examined. The rapid disappearance of
ODN(ACNVK-GT) was observed by 312 nm irradiation for
60 s to revert to two ODNs (Figure 5),12 while the reverse
Figure 3.
Time-course of photo-cross-linking reaction with ODN(AC-
NVK) (red) and ODN(GCNVK) (blue).
photo-cross-linked product efficiently in each base pair
(Figure 4A, 4B). Importantly, adenine and guanine bases on
the photo-cross-linking site are inactive toward photo-cross-
linking reaction with ODNs containing CNVK (see Supporting
Information). Therefore, we can determine the difference
between the pyrimidine and purine bases of the target DNAs
using a photo-cross-linking reaction.
Figure 5. Time-course of the photosplitting reaction with ODN
(ACNVK-GT) (red) and ODN(GCNVK-GC) (blue).
photoreaction produced only ODN(ACNVK) and ODN(GT)
without any byproducts. When the photo-cross-linked ODN
(GCNVK-GC) was used in reverse photoreaction, the rapid
disappearance of ODN(GCNVK-GC) was observed by 312
nm irradiation for 3 min to revert to two ODNs. Therefore,
we succeeded in the reverse reaction by irradiation at 312
nm, resulting in no damage to normal DNA.
We examined molecular modeling studies of the duplexes
of ODN(ACNVK) and ODN(GT). The vinyl group of CNVK
is stacked on the C5-C6 double bond of the T base of
ODN(GT) (see Supporting Information). The molecular
weight of ODN(ACNVK-GT) was equal to the sum of the
molecular weights of ODN(ACNVK) and ODN(GT). As
judged from the molecular modeling, the photoreversibility,
and UV spectrum of CNVK<>T photoadduct, there is a strong
suggestion that the photo-cross-linking proceeded via [2 +
2] cycloaddition between the double bond of CNVK and the
C5-C6 double bond of thymine, giving rise to the formation
of a cyclobutane structure.13
In conclusion, we have demonstrated that a modified
ODN-containing CNVK can be cross-linked by irradiating at
366 nm with an adjacent pyrimidine base in a [2 + 2]
manner. The photo-cross-linked ODNs were reverted to the
Figure 4. Photo-cross-linking yield obtained by photoirradiation
at 366 nm for 1 s by using ODNs containing CNVK. Y, Y′, Z ) A,
G, C, or T. (A) Photoreaction with T base, (B) photoreaction with
C base.
(12) A solution (total volume 60 mL) containing ODN(ACNVK-GT) (20
µM, strand concn) in CH3CN/H2O (1:1) containing urea (2 M) was irradiated
with 15 W transilluminator (312 nm) at 25 °C for 60 s. After irradiation,
the progress of the photoreaction was monitored by HPLC. The yield was
calculated on the basis of ODN(ACNVK-GT).
(13) For reports for the photochemical reaction of acrylonitrile, see: (a)
He´le`ne, C.; Brun, F. Photochem. Photobiol. 1970, 11, 77–84. (b) Chang,
C. K. J. Chem. Soc., Chem. Commun. 1977, 800–801. (c) Mangion, D.;
Frizzle, M.; Arnold, D. R.; Cameron, T. S. Synthesis 2001, 8, 1215–1222.
To examine the influence of the photo-cross-linking
reaction on thermal stability, the melting temperature (Tm)
of the duplex ODN(ACNVK)/ODN(GT) or ODN(ACNVK-GT)
was determined by UV-monitored thermal denaturation. The
duplex ODN(ACNVK)/ODN(GT) showed a melting temper-
Org. Lett., Vol. 10, No. 15, 2008
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original ODN by 312 nm irradiation. The photo-cross-linking
reaction of ODN containing CNVK is ultrafast (with an
irradiation time of only 1 s) and clean. The photo-cross-
linking reaction would require no added reagents to carry
out the reaction and would be controllable with space and
time by the choice of proper irradiation methods. The
reactivity, selectivity, and reversibility of the novel photo-
cross-linker will be beneficial to the in situ DNA manipula-
tion applicable to the inhibition of gene expression and
fluorescent labeling of DNA.
Acknowledgment. This work was supported by a Grant-
in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science, and Technology, Japan.
Supporting Information Available: Synthetic procedures
for CNVK-containing ODN and detailed experimental data of
the reversible photo-cross-linking reaction. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL801112J
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