4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA

Article abstract of DOI:10.1093/nar/gkt197

The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a nee

Full text of DOI:10.1093/nar/gkt197

6774–6781 Nucleic Acids Research, 2013, Vol. 41, No. 13
doi:10.1093/nar/gkt197
Published online 18 June 2013
4-vinyl-substituted pyrimidine nucleosides exhibit
the efficient and selective formation of interstrand
cross-links with RNA and duplex DNA
Atsushi Nishimoto1, Daichi Jitsuzaki1, Kazumitsu Onizuka1, Yosuke Taniguchi1,2
Fumi Nagatsugi2,3 and Shigeki Sasaki1,2,
,
*
2
1Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan, CREST, Japan
3
Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan and Institute of Multidisciplinary
Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan
Received January 29, 2013; Revised February 27, 2013; Accepted March 1, 2013
such as mitomycin C, exert their effects by the alkylation
of DNA (4). On UV irradiation, psoralen forms DNA
adducts with both duplex strands to form an interstrand
cross-link. This compound is widely used for therapy (5)
and mechanistic studies of processes, such as DNA repair
(6). The enhanced inhibition of translation by antisense
oligodeoxynucleotides (ODNs) has been demonstrated
by the cross-link formation between psoralen and target
RNA (7,8). Cross-link formation is also used to maintain
3D nucleic acid structures (9–11). A variety of functional
groups have also been developed to enable interstrand
cross-linking, including disulfide bonds (12), benzoph-
enone derivatives (13), carbazoles (14), quinone methides
(15,16), phenylselenyl derivatives of pyrimidines (17) and
furan derivatives (18). To further advance these studies, an
efficient cross-linking method is still desired.
ABSTRACT
The formation of interstrand cross-links in nucleic
acids can have a strong impact on biological
function of nucleic acids; therefore, many cross-
linking agents have been developed for biological ap-
plications. Despite numerous studies, there remains
a need for cross-linking agents that exhibit both ef-
ficiency and selectivity. In this study,
a
4-vinyl-substituted analog of thymidine (T-vinyl de-
rivative) was designed as a new cross-linking
agent, in which the vinyl group is oriented towards
the Watson–Crick face to react with the amino group
of an adenine base. The interstrand cross-link
formed rapidly and selectively with a uridine on the
RNA substrate at the site opposite to the T-vinyl de-
rivative. A detailed analysis of cross-link formation
while varying the flanking bases of the RNA sub-
strates indicated that interstrand cross-link forma-
tion is preferential for the adenine base on the
50-side of the opposing uridine. In the absence of a
50-adenine, a uridine at the opposite position under-
went cross-linking. The oligodeoxynucleotides probe
incorporating the T-vinyl derivative efficiently formed
interstrand cross-links in parallel-type triplex DNA
with high selectivity for dA in the homopurine
strand. The efficiency and selectivity of the T-vinyl
derivative illustrate its potential use as a unique
tool in biological and materials research.
To address the need for an efficient cross-linking agent,
we previously developed a 2-amino-6-vinylpurine deriva-
tive (1) based on a hybridization-assisted strategy. This
compound exhibited efficient and selective cross-linking
to cytosine bases (19–25). The close proximity of the
vinyl group to the cytosine base in the hybridized
complex contributed to the high reactivity of
1
(Figure 1). An ODN incorporating a sulfide-protected de-
rivative of 2-amino-6-vinylpurine was shown to be useful
for inhibiting and modulating intracellular gene expres-
sion (26). A characteristic feature of this 2-amino-
6-vinylpurine is that the vinyl group is directed towards
the Watson–Crick base pairing face to react with the
4-amino group of cytosine. Generally, the reactive group
for cross-link formation is not located near the Watson–
Crick face. The efficient cross-link formation by 1 suggests
that partial base pairing and/or shape complementarity
can benefit from a proximity effect. Further efforts have
continued towards the biological applications and im-
provement of the 2-amino-6-vinylpurine unit (27–30). In
this study, newly designed 4-vinylpyrimidine-2-one nu-
cleoside analogs (T-vinyl 2 and U-vinyl 3) have been
INTRODUCTION
Many chemical entities, of either exogenous or endogen-
ous origins, cause the alkylation of or damage to DNA
and RNA; thus, they have a strong impact on biological
functions of nucleic acids (1–3). Chemotherapeutic agents,
*To whom correspondence should be addressed. Tel: +81 92 642 6615; Fax: +81 92 642 6615; Email: sasaki@phar.kyushu-u.ac.jp
ß The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acids Research, 2013, Vol. 41, No. 13 6775
H2N
N
R
NH2
N
N
N
N
N
N
RNA
N
O
N
O
O
N
NH
2 O
N
RNA
N
O
O
1
O
2: R=CH3
3: R=H
O
Figure 1. The 2-amino-6-vinylpurine derivative (1) for cross-linking with cytosine and newly designed 4-vinyl substituted pyrimidine derivatives,
T-vinyl (2) and U-vinyl (3).
shown to exhibit fast and selective cross-link formation
with either adenine or uracil bases, depending on the
target sequence. Herein, we describe in detail the synthesis
of nucleoside analogs 2 and 3, evaluation of their
cross-linking reactivity, analysis of the cross-linked
products derived from 2 and highlight its potential appli-
cation for cross-linking in triplex DNA.
treated with 0.5 M NaOH solution to generate the vinyl
group, thus providing the cross-linking ODNs (3,4 and 5)
in good yields. The vinyl group of ODN was hydrated
with a half-life of ꢀ4 h at 25ꢁC and pH 7.0.
RESULTS AND DISCUSSION
Evaluation of the cross-linking reaction
MATERIALS AND METHODS
The vinyl-ODNs (3 and 4) obtained were used for the
cross-linking reaction with the target RNA1 having differ-
ent nucleoside residues at the complementary positions
(Figure 2). Surprisingly, the cross-linking reaction of
ODN3 (R = CH3) to the RNA1 with U at the opposite
position (X = U) was complete within 15 min (Figure
2A). The RNA1 with an opposing G or A (X = G or A)
showed lower reactivity, whereas the RNA1 with an
opposing C (X = C) did not form any appreciable
cross-link product. Interestingly, ODN4 (R = H) contain-
ing U-vinyl 3 showed much slower reaction rates while re-
taining the selectivity for U (Figure 2B). Using Arrhenius
plots, kinetic parameters for the cross-linking reactions
were obtained, which indicated that T-vinyl (2) is superior
to U-vinyl (3), as indicated by the smaller negative value
of activation entropy (Supplementary Figure S6 and
Supplementary Table S1). It is reasonable to interpret
from this result that the vinyl group of the T-vinyl
(R = CH3) requires less conformational change than
that of the U-vinyl (R = H) for cross-link formation to
occur. This interpretation is supported by molecular
orbital calculations (B3LYP/6-31G*); a syn-conformation
(15), in which the vinyl group is directed towards the
Watson–Crick face, is 2.57 kcal/mol more stable than the
anti-conformation (16) of T-vinyl (R = CH3), whereas this
conformation is only 0.52 kcal/mol more stable in the
U-vinyl (R = H) (Figure 3 and Supplementary Figure
S6C and D). The 5-methyl group on the T-vinyl (2) plays
a role in directing the vinyl group to the Watson–Crick face
by steric repulsion.
The 4-vinyl-(1H)-5-methylpyrimidine-2-one derivative (2)
was designed for cross-link formation with the 6-amino
group of adenine, based on the expectation that the two
bases would exhibit shape-complementarity resembling a
T–A base pair. A similar derivative lacking the 5-methyl
group (3) was also synthesized for comparison. The syn-
thesis of the nucleoside units and their incorporation into
ODNs are shown in Scheme 1. The syntheses described
herein are an extension of earlier reports from this group
concerning the introduction of a vinyl group via Suzuki–
Miyaura
coupling.
The
2,4,6-triisopropylbenzen-
sulfonyl derivative of tert-butyldimethylsilyl (TBDMS)-
protected thymidine (7) or 20-deoxyuridine (8) was
treated with 2,4,6-trivinylcyclotriboroxane pyridine
complex in the presence of Pd(PPh3)4, LiBr and K2CO3
in H2O-dioxane. As the resulting vinylated products (9
and 10) were not sufficiently stable for isolation, they
were isolated after protection with octanethiol (11 and
12) (31). They were then converted to the corresponding
phosphoramidite precursors (13 and 14) using conven-
tional methods and were then used in a DNA/RNA auto-
mated synthesizer to incorporate 2 or 3 into ODN1 and
ODN2 with flanking bases (M5 and M3). Initially, vinyl
derivative (9) was protected with methanethiol. However,
the methylsulfide protecting group was cleaved during the
reaction with DMTrCl and resulted in a complex mixture.
This problem was overcome by protecting the vinyl group
with octanethiol. Additionally, thioanisole was required as
a scavenger for the DMTr cation during protection with
DMTrCl. The ODNs thus synthesized were cleaved from
the resin by treatment with a solution of K2CO3 in dry
methanol in the presence of octanethiol. After HPLC puri-
fication, the DMTr group was deprotected in 5% aqueous
AcOH solution, and the deprotected ODNs were again
purified using HPLC to produce the octylsulfide-protected
ODNs (1 and 2). The octylsulfide group was oxidized with
magnesium metaperoxyphthalate (MMPP) in carbonate
buffer, and the resulting sulfoxide derivatives were
Although this new cross-linking agent was designed for
reaction with an adenine base, both T–(2) and U-vinyl (3)
showed selectivity for target RNAs having U at the com-
plementary site. To examine the effect of duplex stability
on selectivity, Tm values were measured for each duplex.
For this purpose, the vinyl group of ODN3 (R = CH3)
was reduced with NaBH4 to give a non-reactive ODN
that had an ethyl group instead of a vinyl group
(Supplementary Figure S4). The Tm values for the

6776 Nucleic Acids Research, 2013, Vol. 41, No. 13
Scheme 1. Synthesis of 4-vinyl-(1H)-pyrimidine-2-one derivatives (2 and 3)a. (a) (i) TBDMSCl, imidazole, N,N-dimethylformamide, 94% and
(ii) 2,4,6-triisopropylbenzensulfonyl chloride, Et3N, N,N-dimethyl-4-aminopyridine, CH2Cl2, 93%; (b) 2,4,6-trivinylcyclotriboroxane pyridine
complex, Pd(PPh3)4, LiBr, K2CO3, H2O:1,4-dioxane = 1:3 solution; (c) C8H17SH, CH3CN, 94% (2 steps); (d) (i) tetrabutylammonium fluoride,
tetrahydrofuran, 79%, (ii) DMTrCl, thioanisole, N,N-Diisopropylethylamine (DIPEA), CH2Cl2, 88% and (iii) 2-cyanoethyl N,N-diisopropylchlor-
ophosphoramidite, DIPEA, CH2Cl2, 72%; (e) (i) DNA/RNA synthesizer, (ii) K2CO3 in dry methanol in the presence of octanethiol and (iii) 5%
aqueous AcOH; and (f) (i) MMPP carbonate buffer, and (ii) 0.5 M NaOH.
duplexes formed between reduced ODN3 and RNA1
(X = U, G, A and C) were determined to be 42.2ꢁC
(X = U), 43.5ꢁC (X = A), 49.4ꢁC (X = G) and 41.5ꢁC
(X = C), indicating that the cross-linking reaction took
place at temperatures lower than the Tm values, and that
the selectivity was not reflective of the Tm value
(Supplementary Figure S5). To gain insight into the
cross-linking reaction, it was monitored using HPLC.
The reaction mixture at pH 7 gave two major peaks at
ꢀ12 min, both of which were confirmed to be the
cross-linked products by Matrix Assisted Laser
Desorption Ionization-Time of Flight (MALDI-TOF)/
MS (Figure 4 and Supplementary Table S2). The faster
peak became predominant after the reaction at pH 5,
whereas the slower peak dominated at pH 9. The
cross-link peaks were isolated and subjected to enzym-
atic hydrolysis using bacterial alkaline phosphatase,
venom phosphodiesterase and P1 nuclease for the HPLC
analysis of the product structure. Interestingly, the faster
peak produced an adenosine adduct [electrospray
ionization (ESI)-MS m/e calcd 520.22 (M+H)+, found
520.07]; in contrast, the slower peak gave a uridine
adduct (ESI-MS m/e calcd 497.19 [M+H]+, found
497.30). As DNA substrates showed base selectivity and
the pH-dependence of the corresponding HPLC profile
similar to those of RNA substrates (Supplementary
Figure S7), DNA substrates were used for more de-
tailed analysis. The authentic adducts with dA and
T were synthesized by adding tert-butyldimethylsilyl
(TBS)-protected dA or T during the synthesis of 9
(Supplementary Schemes S2 and S3). Their structures
were confirmed by 1H–1H correlation spectroscopy for
1
17 (Supplementary Figure S1) or H–13C hetero-nuclear
multiple-bond connectivity for 18 (Supplementary Figure
S2). An HPLC comparison with authentic 17 confirmed
that the cross-link was predominantly formed with the
6-amino group of dA at acidic pH (Figure 5A).
Conversely, Figure 5B clearly shows that the cross-link

Nucleic Acids Research, 2013, Vol. 41, No. 13 6777
R
N
3
ODN :R=CH3
ODN4: R=H
N
O
5'd-CTTT
TTCTCCTTTCT
1
3'r-GAAA -X- AAGAGGAAAGA-(FAM)
RNA
Yield (%) with ODN3
Yield (%) with ODN4
A
B
100
100
80
60
40
20
0
U
80
60
40
20
0
U
G
A
A
C
G
C
0
15
30
45
60
0
15
30
45
60
Time (min)
Time (min)
C
X=U
X=G
X=A
X=C
Cross-link
RNA1
0
5
15 30 60
0
5
15 30 60
0
5
15 30 60
0
5
15 30 60
Time (min)
Figure 2. Cross-linking reaction with ODN3,4 and RNA1. Cross-linking conditions: [ODN3 or 4] = 10 mM, RNA1 = 1 mM, 50 mM MES buffer,
100 mM NaCl, 37ꢁC, pH 7.0. The reaction was followed by electrophoresis using 15% denatured polyacrylamide gel. The fast- and slow-moving
bands represent RNA1 and the cross-linked products, respectively. The cross-link yield was obtained by quantification of the bands by FAM
fluorescence (ꢀem = 518 nm, ꢀex = 494 nm), and it was plotted against time. (A) Yield obtained with ODN3 (R = CH3). (B) Yield obtained with
ODN4 (R = H). (C) An example of gel-shift analysis of the reaction with ODN3 (R = CH3).
A
B
Nu
R
R
N
N
N
O
N
O
dR
15 sy n
dR
16 anti
Figure 3. A syn- (15) and an anti-conformation (16) of the vinyl-substituted derivative.
was formed at the N3 atom of T (18) at alkaline pH
(Figure 5B). These results suggest that the reactivity of
the vinyl group of 2 is affected by the pH of the reaction
medium.
an HPLC analysis of the reaction and the enzymatic
hydrolyzates of the isolated products (Supplementary
Figure S8). It should be noted that cross-link formation
predominantly occurred with an adenine residue at the 50
position relative to U (striped columns in lanes 1–4). The
adenosine residue at the 30 position did not undergo
cross-linking (lanes 5, 9 and 13). Cross-link formation
with the opposing U was slower than with A at the
50-position (white columns in lanes 5–16); however,
yields ꢀ80% were obtained after 60 min (Figure 6B).
Because having a U residue opposite to T-vinyl 2 is
important for efficient cross-link formation with a 50-A,
it may be postulated that this uracil base plays a role in
maintaining the duplex structure. As the distance between
Determination of the cross-linked products
As two adenosine residues flank the opposing uridine in
the target RNA1, it is necessary to determine which of
these adenosine residues is responsible for cross-link for-
mation. Accordingly, cross-link formation was analyzed
using all 16 possible combinations of RNA2 (N3-U-N5)
with various 30-, 50-flanking nucleotide relative to U and
the complementary ODN5 (M5-2-M3) (Figure 6). The
yields of the A- and U-adducts were determined using

6778 Nucleic Acids Research, 2013, Vol. 41, No. 13
A-Crosslink
MMPP RNA1(X=U)
N
pH 5
N
O
pH 7
5'd-CTTM5
M3TCTCCTTTCT
ODN5
RNA2
3'r-GAAN3 U N5AGAGGAAAGA
U-Crosslink
pH 9
0
10 11 12 13 14 15 16
8 9
U C
G A
Lane
N5’
1
A
A
2
3
4
5
6
U
U
7
U
C
A
U
A
A U
G A
C
U
C
C
C
G A
G G G G
2
4
6
8
10
12
N3’
C
U
C
G
Time(min)
5 min
A 100
Figure 4. HPLC trace of the cross-linking reaction using ODN3 and
non-labeled RNA1 (X = U). Cross-linking conditions:
80
60
40
20
0
A-crosslink
U-crosslink
[ODN3] = 15 mM, RNA1 = 10 mM, 50 mM MES buffer, 100 mM
NaCl, 37ꢁC, pH 5 or pH 7. The reaction at pH9 was performed in
50 mM carbonate buffer. HPLC conditions: column SHISEIDO C18,
4.6 ꢂ 250 mm; solvent A 0.1 M TEAA buffer, solvent B CH3CN, B 10–
20% /20 min 20–100%/25 min, linear gradient; flow rate 1.0 ml/min; UV
254 nm. Each peak was assigned to the A- or U-cross-link, as described
in the text. MMPP used for preparation of ODN3 appeared in HPLC
chart.
60 min
B
100
80
60
40
20
0
NH
O
N
N
N
N
N
Figure 6. The ratio of A- to U-cross-linking at 5 and 60 min. The ratio
was determined using HPLC. The cross-linking conditions were
[ODN5] = 15 mM, [RNA2] = 10 mM, 50 mM MES buffer, 100 mM
NaCl, pH 7.0, 37ꢁC. Flanking nucleosides M3 and M5 of ODN5 are
complementary to N5 and N3, respectively.
N
N
O
N
O
N
N
O
HO
OH
HO
OH
O
O
O
O
OH
OH
OH
OH
18
17
A
T
A
B
A
G
T
G
Cross-linked adduct
Cross-link formation with an opposing U decreased at
pH 5 (Figure 4), probably because of low reactivity of
its keto form as a major component. The enolate form
may also contribute to the selective cross-link formation
with an opposing U at pH 9 (Figure 4).
C
Cross-linked adduct
C
Co-injection with 18
Co-injection with 17
Cross-link formation within triplex DNA
5
10
15
5
10
15
We next investigated cross-link formation in triplex DNA.
ODN3 contains T-vinyl 2 in the homopyrimidine strand
and can, therefore, form triplex DNA with a homopurine–
homopyrimidine duplex. ODN6 contains 5-methylcytidine
(dmC) instead of dC, which is used for triplex formation
under weakly acidic conditions. The target duplex was
prepared using DNA1 and DNA2, either of which was
labeled with 6-carboxyfluorescein-aminohexyl (FAM) to
clarify the cross-linked strand. Figure 7A illustrates gel
analysis of the reaction between ODN3 and the duplex
formed with FAM-labeled DNA1 and non-labeled
DNA2. Slow mobility bands were formed only in the
reaction with the duplex containing XY = AT.
Efficiency and selectivity were clearly demonstrated by
examining the time course of the reaction (Figure 8B).
When the duplex was formed with non-labeled DNA1
and FAM-labeled DNA2, no slow mobility bands were
observed (Figure 8C). In addition, FAM–DNA1 alone
did not produce the slow mobility bands, clearly
indicating that cross-link formation took place with
DNA1 in the triplex. This was also confirmed by an
HPLC analysis of the cross-linking reaction medium
using non-labeled DNA1 and DNA2. The ODN3 and
DNA1 peaks disappeared, whereas the DNA2 peak
remained and the cross-linked product appeared at
Time (min)
Time (min)
Figure 5. HPLC analysis of the enzymatic hydrolyzates of the
cross-linked products obtained at pH 5 (A) and those obtained at
pH9 (B). The peaks appearing at ꢀ15 min in each chart showed iden-
tical retention times and MSs with those corresponding to the authentic
samples (17 and 18).
the T-vinyl derivative and U is too far for direct H-bond
formation, we speculate that a bridging water molecule
may be important for this H-bonding interaction, as
shown in 19 (Figure 7). Such bridging water molecules
were shown to contribute to the formation of a U–C
base pair in a crystal structure, thus highlighting this inter-
action (32,33). Molecular modeling (Supplementary
Figure S9) indicates that the 6-amino group of the
adenine residue at the 50 side is in van der Waals contact
with the vinyl group of 2, as shown schematically in 20
(Figure 7). The fact that the cross-link reaction was faster
at pH 5 might imply that protonation at the N3 atom of 2
increases the reactivity of the vinyl group towards the
6-amino group of 50-A, which is in close proximity.
RNA2 lacking a 50-A formed a cross-link with the N3
atom of the opposite U (Figure 6B), most likely by
reaction with its enol or enolate form (Figure 7, 21).

Nucleic Acids Research, 2013, Vol. 41, No. 13 6779
N
O
O
N
O
O
N
RNA
H2N
H N
N
H
N
N
N
N
RNA
N
N
O
N
H
H
H
O
N
O
H
O
H O
RNA
O
RNA
N
N
N
5'
H
O
H
ODN O
ODN
ODN
21
19
20
Figure 7. Speculated hydrogen bonds between T-vinyl 2 and a uracil base. A bridging water molecule was assumed in 19. A close proximity between
6-NH2 of 50-A is shown schematically in 20. The cross-link formed with the N3 of the opposite U likely by the reaction with its enol or enolate form
as shown in 21.
3:
ODN
C=dC
2
5'-CTTT- -TTCTCC TTT CT
HPLC of the cross-linking reaction
A
m
6
ODN : C=d C
DNA1 DNA2 ODN3
(FAM)- 5' TTTGAAA -X- AAGAGGAAAGA
3' AAACTTT -Y- TTCTCCTTT CT -5' (FAM)
DNA1
DNA2
0 min
MMPP
Gel-analysis with ODN3
A
AT
GC
CG
TA
XY =
Time (min)
DNA2 Cross-linked adduct
60
0
60
0
60 0
60
0
Cross-linked
adduct
60 min
FAM-DNA1
0
5
10
15
20
Yield (%) with FAM-DNA1(X=)
Yield (%) with FAM-DNA2(Y=)
B
C
Time (min)
TFO
O
TFO
60
40
60
B
A
N
N
H
N
H
N
H
O
N
H
40
20
0
H
H
O
N
N
H
H
N
N
H
O
N
O
N
N
20
0
G
T
C
N
N
A,G,C,T
N
O
N
H
DNA
DNA
N
N
DNA
N
N
H
DNA
0
20
40
60
0
20
40
60
H
22
23
Time(min)
Time(min)
Figure 9. (A) HPLC analysis of cross-link formation in the triplex
DNA. The reaction was done using ODN3 (30 mM) and DNA1 and
2 (10 mM each) in 50 mM MES buffer (pH 5.0), including 100 mM
NaCl and 10 mM MgCl2 at 25ꢁC for 1 h (B) Expected complex struc-
tures in the triplex DNA between a protonated C and a GC pair (22)
and that between a protonated 2 and an AT pair (23). MMPP used for
preparation of ODN3 appeared in HPLC chart.
Figure 8. Evaluation of triplex cross-linking. Cross-linking conditions,
[ODN3 or ODN6] = 25 mM, [DNA1/DNA2] = 5 mM, 50 mM MES
buffer, 100 mM NaCl, 10 mM MgCl2, 25ꢁC, pH 5.0 (C = dC) or pH
6.1 (C = dmC). (A) Electrophoresis, 15% denatured polyacrylamide gel.
The reaction was checked at 0, 5, 15, 30 and 60 min. (B) The yield of
the cross-linked product was obtained using FAM-labeled DNA1. (C)
The yield was obtained using FAM-labeled DNA2.
dmC is protonated at weakly acidic pH, these results
also support that cross-link formation takes place in the
triplex DNA. The reactivity of the vinyl group is enhanced
at acidic pH because of protonation at N3; therefore, such
a protonated form might contribute to the access of the
vinyl group to 6-NH2 of the adenosine residue in the
homopurine strand as depicted in 23 (Figure 9B).
ꢀ15 min in the HPLC profile (Figure 9A). As the reaction
was performed at acidic conditions, the hydration reaction
of the vinyl group of ODN3 took place rapidly to produce
side products observed between the peaks of DNA1 and 2.
The cross-linked product was subjected to enzymatic hy-
drolysis and HPLC analysis to demonstrate that the
cross-link was formed with the 6-amino group of
adenine (Figure 5, 17). ODN6, containing dmC instead
of dC, produced the cross-link at weakly acidic pH,
whereas no adduct was formed with ODN3 under the
same conditions (Supplementary Figure S10). As a
protonated dC is responsible for the formation of triplex
DNA containing CGC base triplets (Figure 9, 22), and
CONCLUSION
Analogues of thymidine (T-vinyl, 2) and 20-deoxyuridine
(U-vinyl, 3) containing 4-vinyl-sustituents were designed
as new cross-linking agents. In these compounds, the vinyl

6780 Nucleic Acids Research, 2013, Vol. 41, No. 13
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group is oriented towards the Watson–Crick face to facili-
tate a reaction with the amino group of the adenine base.
ODN containing 2 or 3 exhibited rapid cross-link forma-
tion with RNA substrates containing U at the position
opposite to 2 or 3. In the case of RNA1 (X = U) contain-
ing A at the 50 side of U, a cross-link formed with the
6-amino group of the 50-A. RNA1 (X = U) lacking 50-A
formed a cross-link with the N3 atom of a U at the
opposing position. T-vinyl (2) showed more efficient re-
activity than U-vinyl (3), most likely because the direction
of the vinyl group of T-vinyl is pre-organized towards the
Watson–Crick face by the steric bulkiness of the 5-methyl
group. The ODN probe incorporating 2 efficiently formed
interstrand cross-links in parallel-type triplex DNA with
high selectivity for dA in the homopurine strand. Thus,
the efficiency of the cross-linking reaction of 2 has been
demonstrated for DNA/RNA, DNA/DNA duplexes and
triplex DNA. The efficiency and selectivity suggest that
T-vinyl 2 may become a unique tool in biological and
materials research.
16. Rokita,S.E. (2009) Reversible alkylation of DNA by quinone
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SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Tables 1 and 2, Supplementary Figures
1–10 and Supplementary Schemes 1–3.
18. Op de Beeck,M. and Madder,A. (2012) Sequence specific DNA
cross-linking triggered by visible light. J. Am. Chem. Soc., 134,
10737–10740.
FUNDING
Grant-in-Aid for Scientific Research (S) [21229002] from
the Japan Society for the Promotion of Science (JSPS);
CREST from the Japan Science and Technology
Agency. Funding for open access charge: Grant-in-Aid
for Scientific Research (S) [21229002] from the Japan
Society for the Promotion of Science (JSPS).
19. Nagatsugi,F., Kawasaki,T., Usi,D., Maeda,M. and Sasaki,S.
(1999) Highly efficient and selective cross-linking to cytidine based
on the new strategy for auto-activation within duplex. J. Am.
Chem. Soc., 121, 6753–6754.
20. Nagatsugi,F., Tokuda,N., Maeda,M. and Sasaki,S. (2001) A new
reactive nucleoside analogue for highly reactive and selective
cross-linking reaction to cytidine under neutral conditions. Bioorg.
Med. Chem. Lett., 11, 2577–2579.
21. Nagatsugi,F., Usui,D., Kawasaki,T., Maeda,M. and Sasaki,S.
(2001) Selective reaction to a flipping cytidine of the duplex DNA
mediated by triple helix formation. Bioorg. Med. Chem. Lett., 11,
343–345.
Conflict of interest statement. None declared.
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