Template-directed DNA photoligation via α-5-cyanovinyldeoxyuridine

Article abstract of DOI:10.1021/ol050709g

(Chemical Equation Presented) We describe an efficient template-directed photoligation of oligodeoxynucleotides (ODNs) using α-5- cyanovinyldeoxyuridine (α^cU). An efficient photoligation was produced by photoirradiation of an ODN containing thymine at the 5′ end with an ODN containing thymine at the 5′ end in the presence of a template ODN. This photoligation method is a new and efficient way to synthesize branched ODNs.

Full text of DOI:10.1021/ol050709g

ORGANIC
LETTERS
2005
Vol. 7, No. 14
2853-2856
Template-Directed DNA Photoligation via
-5-Cyanovinyldeoxyuridine
r
Masayuki Ogino,† Yoshinaga Yoshimura,‡ Akio Nakazawa,§ Isao Saito,§ and
Kenzo Fujimoto*,†,‡
The School of Materials Science, Japan AdVanced Institute of Science and Technology,
Ishikawa 923-1292, Japan, PRESTO, Japan Science and Technology Agency, Ishikawa
923-1292, Japan, and Department of Synthetic Chemistry and Biological Chemistry,
Faculty of Engineering, Kyoto UniVersity, Kyoto 606-8501, Japan
kenzo@jaist.ac.jp
Received April 3, 2005
ABSTRACT
We describe an efficient template-directed photoligation of oligodeoxynucleotides (ODNs) using
r
-5-cyanovinyldeoxyuridine (
r
CU). An efficient
end in the
photoligation was produced by photoirradiation of an ODN containing end with an ODN containing thymine at the 5′
r
CU at the 3
′
presence of a template ODN. This photoligation method is a new and efficient way to synthesize branched ODNs.
Nonenzymatic template-directed DNA has recently attracted
considerable interest because of its potential application in
genetic regulation and use as a tool for molecular biology
research, as well as for its role in prebiotic nucleic acid
synthesis.1 Many template-directed chemical ligation ap-
proaches have been reported that join the ends of two DNA
strands by a native phosphodiester bond or by nonnatural
linkages.2 Although Fujimoto et al. reported the template-
directed reversible photoligation of DNA oligomer via
5-vinyldeoxyuridine derivative at the 5′ end, they did not
observe photoligation via 5-vinyldeoxyuridine at the 3′ end
(Figure 1).3 We used molecular modeling to study the
Figure 1. Contrasting photoreactions of template-directed DNA
photoligation (a) for an ODN containing âCU at the 5′ end and (b)
for an ODN containing âCU at the 3′ end (X, modified base).
duplexes of oligodeoxynucleotides (ODNs) containing â-5-
† Japan Advanced Institute of Science and Technology.
‡ Japan Science and Technology Agency.
§ Kyoto University.
cyanovinyldeoxyuridine (âCU) (Figure 2) as usual at the 3′
end. Our molecular modeling study suggested that there is
less stacking between the cyanovinyl group and the C5-C6
double bond of the 5′-terminal dT (Figure 3a). We examined
an R-anomer as an alternative probe. A molecular modeling
study suggested base stacking between the cyanovinyl group
in R-5-cyanovinyldeoxyuridine (RCU) (Figure 2) and the
C5-C6 double bond. Our molecular modeling study pre-
dicted that the cyanovinyl group of RCU is effectively stacked
(1) Rex, X. F. R.; Narayan, C. C.; Pamela, L, P.; Rumney, S., IV; Kool,
E. T. J. Am. Chem. Soc. 1996, 118, 7671-7678.
(2) (a) Jiamin, G.; Christoph, S.; Deborah, T.; Kool, E. T. J. Am. Chem.
Soc. 2002, 124, 11590-11591. (b) Liu, J.; Taylor, J. S. Nucleic Acids Res.
1998, 26, 3300-3304.
(3) (a) Fujimoto, K.; Matsuda, S.; Takahashi, N.; Saito, I. J. Am. Chem.
Soc. 2000, 122, 5646-5647. (b) Fujimoto, K.; Matsuda, S.; Ogawa, N.;
Hayashi, M.; Saito, I. Tetrahedron Lett. 2000, 41, 6451-6454. (c) Fujimoto,
K.; Matsuda, S.; Hayashi, M.; Saito, I. Tetrahedron Lett. 2000, 41, 7897-
7900. (d) Fujimoto, K.; Ogawa, N.; Matsuda, S.; Hayashi, M.; Saito, I.
Tetrahedron Lett. 2000, 41, 9437-9440. (e) Yoshimura, Y.; Ito, Y.;
Fujimoto, K. Bioorg. Med. Chem. Lett. 2005, 15, 1299-1301.
10.1021/ol050709g CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/14/2005

Scheme 1
Figure 2. Structures of â-5-cyanovinyldeoxyuridine (âCU) and R-5-
cyanovinyldeoxyuridine (RCU).
Table 1. ODNs Used in This Study
Figure 3. Molecular modeling of the stacking geometry in B-form
DNA. The model was optimized by AMBER* force field in water
using MacroModel version 8.1. Blue, red, green, and water blue
molecules are âCU, RCU, dA, and dT, respectively. The vinyl group
and C5-C6 double bond of the 5′-terminal dT are shown in
yellow: (a) 3′-terminal âCU and 5′-terminal dT, (b) 3′-terminal RCU
and 5′-terminal dT.
sequences
ODN 1
ODN 2
ODN 3
ODN 4
ODN 5
ODN 6
ODN 7
5′-d(TGTGCRCUp)-3′
5′-d(TGCGTG)-3′
5′-d(CACGCAAGCACA)-3′
5′-d(CACGCAGGCACA)-3′
5′-d(CACGCACGCACA)-3′
5′-d(CACGCATGCACA)-3′
5′-d(AAAAAATGCGTG)-3′
on the C5-C6 double bond of the 5′-terminal dT (Figure
3b). We here report our development of a novel, nonenzy-
matic, template-directed photoligation of ODN via the RCU
contained at the 3′ end. We used melting temperature (Tm)
experiments to study the hybridization affinity of the ODN
for complementary and mismatched ODNs in detail.
Scheme 2
Chlorosugar 3 was coupled with 5-iodouracil in the
presence of CuI. The two resulting isomers were deprotected
with concentrated aqueous ammonia to yield 5-iodouridine
5. Compound 5 was transferred to 2 by the Heck reaction.
R,â-Anomers were isolated by silica gel column chroma-
tography. 2 was dimethoxytritylated and converted to the
corresponding cyanoethyl phosphoramidite using a conven-
tional method (Scheme 1). The RCU-containing ODN, 5′-
d(TGTGC RCUp)-3′ (ODN 1), was synthesized on an ABI
3400 DNA synthesizer using 3′-phosphate CPG.4 Incorpora-
tion of RCU into the ODN was confirmed by enzymatic
digestion and matrix-assisted laser desorption ionization-
time-of-flight mass spectrometry (MALDI-TOF MS).5 The
ODNs used in this study are summarized in Table 1.
at 0 °C in the presence of template ODN 3.6 Capillary gel
electrophoresis analysis of a mixture of ODN 1 and ODN 2
photoirradiated with template ODN 3 indicated a clean and
efficient formation of ligated ODN 8 and the concomitant
disappearance of ODN 1 and ODN 2 (Figure 4). MALDI-
TOF MS indicated that ODN 8 was a ligated product of ODN
1 and ODN 2.7 Enzymatic digestion of isolated ODN 8
showed the formation of dC, dG, and dT in a ratio of 2:5:3,
together with a new product. Spectroscopic data, including
MALDI-TOF MS, indicated that this new product was the
We next determined the feasibility of photoligation of the
RCU-containing ODN (Scheme 2). ODN 1 and 5′-d(T-
GCGTG)-3′ (ODN 2) were irradiated at 366 nm for 30 min
(6) When an ODN with the phosphate group of ODN 1 at the 3′ end
removed and ODN 2 were irradiated at 366 nm for 1 h at 0 °C in the
presence of template ODN 3, the product was obtained in 10% yield.
(7) MALDI-TOF MS: calculated for ODN 8 (C120H151N42O76P11) [M
- H]- 3735.6277, found 3735.8486.
(4) Walton, T. A.; Lyttle, M. H.; Dick, D. J.; Cook, R. M. Bioconjugate
Chem. 2002, 13, 1155-1158.
(5) MALDI-TOF MS: calculated for ODN 1 (C61H76N20O40P6) [M -
H]- 1913.2876, found 1913.5459.
2854
Org. Lett., Vol. 7, No. 14, 2005

of a double helix, which resulted from the cooperative
dissociation of ODN 8 from ODN 3. The Tm value (40.5
°C) of ODN 8 and ODN 3 was higher than that of two 6-mers
ODN 1 and ODN 2 and template ODN 3 (Tm ) 18.2 °C)
(Figure 5). These results indicated that ligated ODN 8 can
Figure 4. Capillary gel electrophoresis of photoreaction of ODN
1 (30 µM) and ODN 2 (30 µM) in the presence of template ODN
3 (33 µM) (a) before photoirradiation, (b) after irradiation at 366
nm for 5 min at 0 °C (42% yield), and (c) after irradiation at 366
nm for 30 min at 0 °C (95% yield).
Figure 5. UV melting curves (a) before photoirradiation (Tm
)
18.2 °C) and (b) after irradiation at 366 nm for 30 min (Tm ) 40.5
°C) in 5.0 µM each strand, 100 mM NaCl, and 50 mM Na
cacodylate buffer (pH 7.0).
form a stable duplex with the complementary ODN 3.
We measured the thermal stability of the double helix
formed between modified ODNs and complementary ODNs.
We applied the same method to measure the Tm values of
the duplexes of ODN 1‚ODN 2 and mismatched comple-
mentary strands ODN 4, ODN 5, and ODN 6. As shown in
Table 3, the Tm value of the ODN 1‚ODN 2 and comple-
Table 2. Template-Dependent Conversion of ODN 1 and ODN
2 by Photoirradiation
conversion (%)
ODN 1
ODN 2
ODN 3 (Y ) A)
ODN 4 (Y ) G)
ODN 5 (Y ) C)
ODN 6 (Y ) T)
95
11
9
95
15
12
13
14
Table 3. Tm Values for Duplex to Single-Strand Conversions
of ODN 1 and ODN 2 in the Presence of ODNs 3-6
d(RCU-T) photoadduct.8 The molecular weight of ODN 8
was equal to the sum of the molecular weight of ODN 1
and ODN 2. Judging from the molecular modeling (Figure
3b), it is strongly suggested that the photoligation reaction
proceeded via [2 + 2] cycloaddition between the double bond
of RCU side chain and the C5-C6 double bond of thymine,
giving rise to the formation of a cyclobutane structure as
observed for CVU.3d
Tm (°C)a
DTm (°C)
ODN 3 (Y ) A)
ODN 4 (Y ) G)
ODN 5 (Y ) C)
ODN 6 (Y ) T)
18.2
14.6
16.4
15.9
-3.6
-1.8
-2.3
a Conditions: 100 mM NaCl, 50 mM Na cacodylate buffer (pH 7.0),
5.0 µM each strand.
We carried out a test of selectivity of RCU against the
opposite-site base (Scheme 2). ODN 1 and ODN 2 were
irradiated at 366 nm for 30 min at 0 °C in the presence of
ODN 4, ODN 5, and ODN 6, respectively. Results showed
that photoligation proceeded with low conversion. Conver-
sion after 30 min was estimated at 9-15% in the singly
mismatched case (Table 2). These results suggested that this
photoligation depends on sequence specificity and that RCU
has the ability to recognize the opposite-site dA.
mentary ODN 3 was the greatest of all combinations, and
when another ODN was used, a decrease in the Tm value of
1.8-3.6 °C was observed. As a result, we considered that
the photoligation reaction is promoted by thermal stability
and that the cyanovinyl group and C5-C6 double bond are
fixed by the A-RCU base pair.9
There is a great potential for using DNA as a generic
material instead of a genetic material. Branched DNA
molecules have various uses in signal amplification technol-
We performed UV melting studies using ODN 8 and the
12-mer template ODN 3. The thermal melting behavior
exhibited a monophasic helix-coil transition characteristic
(9) We examined molecular modeling of ODNs 1-3. As a result, We
expected a weak hydrogen bond between the lone pair of 1N in dA and
3NH of RCU. When ODNs 4-6 were used as the template ODN, the
interaction was considered to be not effective on the basis of the result of
molecular modeling.
(8) MALDI-TOF MS: calculated for d(RCU-T) (C22H27N5O10) [M +
H]+ 522.1836, found 522.1866.
Org. Lett., Vol. 7, No. 14, 2005
2855

ogy,10 nanotechnology applications such as DNA comput-
ing,11 DNA nanostructures using self-assembled branched
units,12 DNA sensors,13 and nanoelectronic devices.14 The
past 10 years have seen remarkable success in the construc-
tion of DNA nanoarchitectures. For example, one- and two-
dimensional DNA lattices15 and dendrimerlike DNA16 have
been constructed from a rich set of branched DNA. Various
autonomous DNA walker devices based on DNA cleavage
and the ligation of branched DNA were explored experi-
mentally. Various template-directed enzymes and the intro-
duction of a branch structure via RCU have also been
explored theoretically (Scheme 3). Figure 6 shows the results
Scheme 3
Figure 6. Capillary gel electrophoresis of photoreaction of ODN
1 (30 µM) and ODN 7 (30 µM) in the presence of template ODN
3 (33 µM) (a) before photoirradiation, (b) after irradiation at 366
nm for 2 h at 0 °C (30% yield), and (c) after irradiation at 366 nm
for 6 h at 0 °C (82% yield).
of capillary gel electrophoresis of the photoirradiated mixture
of 5′-d(AAAAAATGCGTG)-3′ (ODN 7) and ODN 1 in the
presence of template ODN 3, with the clean and efficient
formation of the expected ligated 18-mer ODN 9 and the
disappearance of ODN 7 and ODN 1. MALDI-TOF MS
indicated that ODN 9, purified by HPLC, was the ligated
product of ODN 7 and ODN 1.17 Enzymatic digestion of
isolated ODN 9 showed the formation of dA, dG, dT, and
dC in a ratio of 6:5:3:2, together with a d(RCU-T) photo-
adduct. We observed no formation of ODN 9 after photoir-
radiation of ODN 7 and ODN 1 in the absence of template
ODN 3. These results clearly indicated that ODN 9 was a
branched ODN formed by template-directed cross-linking
between the thymine of ODN 7 and the RCU of ODN 1.
In conclusion, we have demonstrated that an ODN
containing RCU at the 3′ end can be employed in the
photoligation of DNA by irradiation at 366 nm in the
presence of a template DNA, with no side reaction. This
method has the same photosensitivity as the usual method
using âCU. Using this novel photoligation method, we have
demonstrated a convenient and versatile method of generating
branched oligonucleotides, which should be particularly
useful in DNA nanotechnology.
(10) (a) Urdea, M. S. Bio/Technology 1994, 12, 926-928. (b) Colling,
M. L.; Fine, E.; Zayati, C.; Horn, T.; Ahle, D.; Detmer, L. P.; Shen,;
Kolberg, J.; Bushnell, S.; Urdea, M. S.; Ho, D. D. Nucleic Acids Res. 1997,
25, 2979-2984.
(11) Aldyen, P.; Jonoska, N.; Seeman, N. C. J. Am. Chem. Soc. 2004,
126, 6648-6658.
(12) Scheffler, M.; Dorenbeck, A.; Jordan, S.; Wustefeld, G.; Kiedrowski,
G. Angew. Chem., Int. Ed. 1999, 38, 3312-3315.
(13) Nakamura, F.; Ito, E.; Sakou, Y.; Ueno, N.; Gatuna, I. N.; Ohuchi,
F. S; Hara, M. Nano Lett. 2003, 3, 1083-1086.
(14) Becerril, H. A.; Stoltenberg, R. M.; Wheeler, D. R.; Davis, R. C.;
Hard, J. N.; Woolley, A. T. J. Am. Chem. Soc. 2005, 127, 2828-2829.
(15) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Nature 1998,
394, 539-544.
(16) (a) Shchepinov, M. S.; Udalova, I. A.; Bridgman, A. J.; Southern,
E. M. Nucleic Acid. Res. 1997, 25, 4447-4454. (b) Shi, J.; Bergstrom, D.
E. Angew. Chem., Int. Ed. Engl. 1997, 36, 111-113.
(17) MALDI-TOF MS: calculated for ODN 9 (C180H223N72O106P17) [M
+ H]+ 5618.7373, found 5618.7555.
Supporting Information Available: Experimental pro-
cedures for the synthesis, purification, and analysis of ODNs
2-7, oligonucleotide digestion, and melting studies. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL050709G
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Org. Lett., Vol. 7, No. 14, 2005