A new strategy for site-specific alkylation of DNA using oligonucleotides containing an abasic site and alkylating probes

Article abstract of DOI:10.1039/c5cc03915k

Selective chemical reactions with DNA, such as its labelling, are very useful in many applications. In this paper, we discuss a new strategy for the selective alkylation of DNA using an oligonucleotide containing an abasic site and alkylating probes. We d

Full text of DOI:10.1039/c5cc03915k

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ARTICLE TYPE
A New Strategy for Site-Specific Alkylation of DNA using
Oligonucleotides Containing an Abasic site and Alkylating Probes
Norihiro Sato, Genichiro Tsuji, Yoshihiro Sasaki, Akira Usami, Takuma Moki, Kazumitsu Onizuka, Ken
Yamada and Fumi Nagatsugi*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Selective chemical reactions with DNA, such as its labelling,
moiety into close proximity and inducing selective alkylation. As
are very useful in many applications. In this paper, we discuss 50 a hydrophobic pocket, an AP site in duplex DNA has been
1
0
a new strategy for the selective alkylation of DNA using an
oligonucleotide containing an abasic site and alkylating
probes. We designed three probes consisting of 2-AVP as a
reactive moiety and three kinds of binding moiety with high
configured in our study, because it has been reported that a
variety of ligands can recognize the nucleobase opposite the AP
site through hydrogen bonding even in the aqueous media.
In this study, we have designed the new probes for reactions
17-22
affinity to duplex DNA. Among these probes, Hoechst-AVP 55 involving an AP site in duplex DNA. These probes consist of 2-
1
5
probe exhibited high selectivity and efficient reactivity to
thymine bases at the site opposite an abasic site in DNA. Our
method is potentially useful for inducing site-directed
reactions aimed at inhibiting polymerase reactions.
AVP as a reactive moiety and penta-arginine (2: (Arg) ), acridine
(3), or Hoechst (4) as a binding moiety with high affinity to
duplex DNA (Fig.1.).
5
Alkylating agents react with DNA bases in the generation of a
variety of covalent adducts and can cause cancer by inducing
mutagenic modifications of the constitutive nucleic acid bases in
6
0
2
2
3
3
0
5
0
5
2-amino-6-vinyl
purine (1: AVP)
Target DNA
DNA strand
1
DNA. However, certain alkylating agents are commonly used as
strand
2
chemotherapeutic drugs in cancer treatment. In light of the
Abasic
site
double-edged properties of alkylating agents, methods for the
site-specific alkylation of DNA with sequence selectivity offer
new potential strategies for the development of selective
chemotherapeutic drugs. In addition, selective chemical
modification of DNA and RNA can significantly impact their
respective functions. Reports exist on the development of small
T
+
2
3
4
: R=(Arg)5
: R=Acridine
: R=Hoechst
Fig. 1 Schematic representation of the strategy for site-specific alkylation to
3,4
5,6
probes for selective reactions with DNA or RNA. Aryl
nitrogen mustard conjugated to various functional groups has
DNA
6
5
7-11
12
Penta-arginine binds to DNA with high affinity by a non-specific,
electrostatic interaction. The acridine moiety binds to the duplex
demonstrated selective alkylation with DNA
and RNA.
Rhodium-catalyzed carbene transfer was achieved in the selective
alkylation of cytosine and adenine in the unpaired DNA or RNA
23-24
DNA as an intercalator.
Hoechst is a representative minor
13
grove binder with high affinity and high specificity to sequences
sequences. The combination of catalytic RNA and electrophilic
probes was developed as a tool for selective, irreversible RNA
2
5-26
7
0
rich in adenine and thymine.
These binding moieties are
14
postulated to be capable of promoting access of 2-AVP to the AP
site. The 2-AVP derivatives are expected to form hydrogen bonds
and to react selectively with the target base opposite the AP site.
modification.
Here, we propose a strategy for the site-specific alkylation of
DNA using oligonucleotides containing an abasic (AP) site and
the alkylating probes. We previously reported that a 2’-OMe
oligonucleotide containing 2-amino-6-vinylpurine (1: AVP)
efficiently forms a covalent linkage with the complementary
sequence of mRNA at the uridine residue across from the AVP.
4
0
Scheme 1.
7
5
15,16
The efficient reactivity observed with AVP was attributed to
5
6
4
5
the proximity effect enabled by the formation of hydrogen bonds
with the target uridine. Based on these results, we anticipated that
(
a) BrCH
2
CO
2
t-Bu, t-BuOK, DMSO, 75 % (b) 1) CH
2 3 3 2 2 2
=CHSnBu ,(Ph P) Pd Cl ,
dioxane 2) MeSNa, CH
3
CN, 54 % for 2 steps 3) TFA, 98 %
2
-AVP derivatives might also form hydrogen bonds with target
The reactive base portion (6) was synthesized as shown in
Scheme 1. The N9 position of chloropurine was alkylated with t-
nucleobases located in a hydrophobic pocket, forcing the reactive
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butyl bromoacetate to yield the t-butyl ester (5) in 75% yield. The
cross-coupling reaction of (5) with nBu SnCHCH
gave the 35 terminal position. Subsequently, the stable precursor of the 2-
resin was coupled with the 2-AVP derivative (6) at the N-
3
2
AVP probe conjugated with the penta-arginine (7) was cleaved
from the resin by treatment with TFA and triisopropylsilane. The
acridine derivative (8) was obtained from 9-acridinecarboxylic
acid and coupled with 6 to produce the stable precursor of the 2-
40 AVP probe conjugated to the acridine derivative (9). The Hoechst
derivative (12) was synthesized through a modification of a
purine derivative, which was, without purification, reacted with
methanethiol to give the thiol protected purine derivative (54%
for 2 steps). The removal of t-butyl group was performed with
5
TFA to afford 6.
Scheme 2.
Synthesis of the (Arg)
5
probe (7)
27,28
method described in the literature
and conjugated with 6 to
1
R =
afford the stable precursor of the 2-AVP probe (13). The
sulphide-protected precursors of the 2-AVP probes (7, 9, 13)
45 were converted to the sulphoxides by oxidation with magnesium
monoperphtalate (MMPP), followed by elimination under
alkaline conditions to yield the AVP probes (2, 3, 4). The
reactivities of synthesized probes were evaluated using duplex
DNA containing an AP site and fully-matched DNA. In our
HBTU, HOBT
DIPEA, NMP
6
1
1
2
0
TFA,
triisopropylsilane
5
0
experiments, tetrahydrofuranyl residue (dSpacer) was used as an
AP site. The duplexes were formed with 5’ fluorescein labelled
DNA2, which was complementary to DNA1, containing thymine
2
R =
7
(
T), cytosine (C), adenine (A) and guanine (G) bases opposite the
(
Arg)5
Synthesis of the acridine probe (9)
AP site. Each probe (2, 3, 4) was added to both duplex DNA and
single-stranded DNA. After 24 h, each reaction mixture was
analysed by gel electrophoresis with 20% denaturing gel (Fig.
5
5
2
A).
EDC, HOBT
DMF
DNA1:
DNA2: 5’6-FAM-d(CAG CGC N AA TTC GCG AGT)-3’ (N=T, C, A, G)
(A)
3’-d(GTC GCG APTT AAG CGC TCA)-5’
AP
5
8
9
Acridine
6
0
(
Arg)
5
-AVP (2)
Acridine-AVP (3)
Hochst-AVP (4)
1
2
2 2
) TFA/CH Cl
N=
T
C
A
G
F
SS
N=
T
C
A
G
F
SS
)
HBTU, HOBT
DIPEA, NMP
6
adduct
ssDNA
adduct
ssDNA
Synthesis of the Hoechst probe (13)
6
5
(B)
(
Arg)
5
-AVP (2)
Acridine-AVP (3)
Hochst-AVP (4)
70
70
60
50
40
30
20
10
0
70
60
50
40
30
20
N=T
60
N=T
N=C
5
0
0
DIAD, Ph
3
P, THF
SS
4
1
0
30
20
0
1) SOCl
2
1) Pd/C, H
2
, EtOH
N=T
N=G,C
10
10
SS, F
N=G,A
N=A,
F
SS, F
2
)
MeNHOMe▪HCl
Pyridine, CH Cl
2
)
10, Na
2
S O , H
2 5 2
O
O
N=A,G,C 0
0
0
20
40
60
80
3) LiAlH , THF, Et
4
2
0
20
40
60
80
2
2
0
20
40
60
80
Reaction time (h)
7
0
Reaction time (h)
Reaction time (h)
Fig. 2 Evaluations of the reactivities of the probes by the denaturing PAGE
The reaction was performed with 5 uM target DNA and 100 uM (Arg) -AVP (2), 100
5
uM Acridine-AVP (3), or 25 uM Hoechst-AVP (4) in 100 mM NaCl, 50 mM MES pH 7
1
1
2
1
o
at 37 C. (A) Gel electrophoresis analysis performed on the products of the
)
1-methylpyperadine
CO , DMF
Pd/C, H , EtOH
alkylation reaction after 24 h, SS: single strand (DNA2, N=T) F: full match (DNA1,
AP=A, DNA2, N=T) (B) The time course for alkylation reactions.
K
2
3
)
2
The higher-molecular-weight band detected in reactions between
2 2 5 2
3) 11, Na S O , H O
1
2
75 the probes (2, 3, 4) and duplex DNA contained thymine opposite
an AP site. The adduct was purified from each higher molecular-
weight band and measured by MALDI TOF mass spectra. Each
isolated species was shown to have a molecular weight consistent
with a probe-DNA1 (N=T) adduct (supporting information). No
1
2
)
TFA, CH
2
Cl2, quant
2
5
) 6, HBTU, HOBT
1
3
DIPEA, NMP
Hoechst
8
8
9
0
5
0
adducts were observed in reactions between the probes (2, 3, 4)
and fully matched DNA duplexes. The yields of the alkylated
products (Y-axis) were determined by quantification of the
fluorescent bands in comparison to the bands corresponding to
non-reacted target DNA. Fig. 2B shows the time-dependent
changes observed in the alkylation yields with the three probes.
The (Arg) -AVP (2) probe exhibited efficient reactivity with the
5
duplex DNA containing thymine opposite the AP site, but similar
reactivity to single-stranded DNA. These results indicated that
Synthesis of the reactive probes (2~4)
MMPP
aqNaOH
7
9
: R=(Arg)5
: R=acridine
2: R=(Arg)5
3: R=acridine
4: R=Hoechst
1
3: R=Hoechst
3
0
Each probe was synthesized as shown in Scheme 2. The 2-AVP
probe conjugated to penta-arginine was prepared by (Fmoc)-
based solid-phase synthesis. The protected penta-arginine on the
5
the (Arg) -AVP (2) may also bind to single-stranded DNA
through electrostatic interactions, arranging itself in sufficiently
2
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close proximity to react the target base. The acridine-AVP (3)
DNA as a template (lane 8~12). These results suggested that the
alkylation by Hoechst-AVP (4) took place at the thymine
opposite the AP site, stopping the primer extension at that site.
and Hoechst-AVP (4) probes did not produce adducts with
single-stranded DNA. These results suggested that acridine and
Hoechst do not interact with single-stranded DNA sufficiently to
enable selective reactivity with duplex DNA containing an AP
site. The reactivity of 4 to duplex DNA containing a thymine
opposite the AP site was higher than that of 3. This more efficient
reactivity might be due to a higher binding affinity between
duplex DNA and Hoechst-AVP (4) than shown by acridine-AVP
(3).
1
8
11
9
(A)
5
0
5
0
5
0
5
0
5
0
5
0
AAG CGC TCA- FAM -5’
5
’-CAG CGC N AA TTC GCG AGT-3
Non-modified DNA
(N=T)
Alkylated DNA
Control
-
T
C
A
G
All
-
T C A G All
dNTP:
1
1
8 mer:
1 mer:
1
1
2
2
3
3
4
4
5
5
6
The alkylation of duplex DNA containing an AP site using
Hoechst-AVP (4) was monitored by HPLC (Fig. S7). After the
newly observed peak was purified, the isolated species was
confirmed by MALDI TOF mass spectrometry to be DNA
alkylated with Hoechst-AVP (4). We tried enzymatic hydrolysis
of the purified alkylated DNA to determine the structure;
however, we could not isolate the hydrolyzed product derived
from the alkylated nucleoside. The potential alkylated sites with
Hoechst-AVP (4) are O4, O2 and N3 of thymine (Fig. 3)
9 mer:
Lane
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
1
8
11
(
B)
TT AAG CGC TCA- FAM -5’
’-CAG CGC X AA TTC GCG AGT-3
5
Non-modified DNA
(N=T)
Alkylated DNA
Control
dNTP:
-
T
C
A
G
All
-
T
C
A G All
1
8 mer:
1
1 mer:
Lane
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Fig.4. Gel analysis of primer extension assay using alkylated DNA by Hoechst-
AVP or non-modified DNA as the template. The template DNA (X=T or T-
HoechstAVP) was annealed with the primer (5’ FAM labeled 9 mer DNA (A) or 11
mer DNA (B). The primer extension reactions were catalyzed by Klenow
fragment in the presence of dTTP (Lane 2 and 8), dCTP (Lane 3 and 9), dATP
(Lane 4 and 10), dGTP (Lane 5 and 11) and four dNTP (Lane 6 and 12). Lane
O4-adduct
O2-adduct
N3-adduct
R=Hoechst
Fig.3. Proposed structures for the adduct to thymine with Hoechst-AVP (4)
For the considerations on the adduct structure, we examined the
stability of the purified adduct. This adduct was highly stable
under acidic (pH 5.0) or basic conditions (pH 9.0) for 24 hr, and
heating conditions (for 10 min, 90 °C) (Fig. S8A~D). In addition,
treatment of the adduct with 20 % acetic acid (pH 1.9) at 50 ºC
for 30 min did not induced the remarkable degradation product
1
3~15: 5’FAM labeled DNA as a size marker.
6
5
Next, we investigated the influence of the distance between the
AP site and the Hoechst binding site (AATT) in the target DNA
on Hoechst-AVP (4) reactivity.
(
A)
AP0bp
Rev.AP
3’
(
Fig. S8E). Under these conditions, O2-alkylthymidine is cleaved
3’
5
5
-
d(GTCGCG X TTAA GCGCTCA)- ’
FAM-d(CAGCGC T AATT CGCGAGA)-
- d(GTCGCG TTAA X GCGCTCA)- ’
FAM-d(CAGCGC AATT T CGCGAGA)-
5
’
3’
5’
3’
of the glycosyl bond so rapidly and O4-alkylthymidine is induced
29
AP1bp
3’
W/O AATT
dealkylation. N3-alkyl derivative is stable under the strong
5
3’
5
30
- d(GTCGCG X G TTAA GCGCTCA)- ’
FAM-d(CAGCGC T C AATT CGCGAGA)-
- d(GACTCG X TCGTCAGTCAG)- ’
FAM-d(CAGCGC T AGCAGTCAGTC)-
acidic conditions. Based on these reports and our results, we
propose that the major adduct structure might be the N3-adduct.
Next, primer extension reaction assays were used to confirm that
the alkylation took place at the thymine opposite the AP site.
Either purified DNA alkylated by Hoechst-AVP (4) or non-
modified DNA was annealed to the fluorescein labelled primer,
and each mixture was subjected to the primer extension reaction
using Klenow DNA polymerase. The DNA fragments extended
by nucleotide incorporation were visualized by fluorescence
through sequencing polyacrylamide gel (PAGE) analysis. Fig. 4A
shows the denatured PAGE bands obtained through the
elongation of the fluorescein-labelled 9 mer primer annealed with
DNA alkylated by Hoechst-AVP (4) or non-modified DNA. Two
base elongations were observed using unmodified DNA as a
template with TTP to produce 11 mer DNA (lane 2). Complete
elongation to the full-length product was detected in the presence
of four dNTP in the reference experiment (lane 6). In contrast,
when using alkylated DNA as a template, 11 mer DNA was
produced in the presence of dTTP (lane 8) and four dNTP (lane
12). In Fig. 4B, single nucleotide incorporations were examined
using the fluorescein-labelled 11mer primer. When un-modified
DNA was used as a template, elongation by one or two bases was
found in the presence of dATP (Lane 4) and full length
elongation in the presence of four dNTP (Lane 6) was observed.
No elongation was observed with any dNTP using the alkylated
5’
3’ 5’
3’
AP2bp
3’
X: AP
5
- d(GTCGCG X GG TTAA GCGCTCA)- ’
FAM-d(CAGCGC T CC AATT CGCGAGA)-
5’
3’
AP3bp
3’
5
-
d(GTCGCG X GGC TTAA GCGCTCA)- ’
FAM-d(CAGCGC T CCG AATT CGCGAGA)-
5’
3’
(
B) 80
70
4
60
50
40
30
2
4 hr
4
7
8 hr
2 hr
2
0
0
0
1
AP
bp
AP
1bp
AP
2bp
AP
3bp
Rev.
AP
w/o
AATT
0
Fig.5. (A) Sequences of the duplex DNA using in this study. (B) Comparison of
the alkylation yields with 16 to target duplex DNA contained the different
distances between AATT and an AP site.
Fig. 5A shows the sequences of duplexes in this experiment.
The reactivity of 4 with duplex DNA decreased in order of
AP1bp≥AP2bp>AP0bp>>AP3bp, showing a dependence on the
distance between AATT site and the AP site. The duplex DNA
7
0
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without an AATT site (w/o AATT) gave only a modest yield of
the alkylated product.
Promotion of Science (JSPS). This work was also supported in
55 part by the Management Expenses Grants National Universities
Corporations from the Ministry of Education, Science, Sports and
Culture of Japan (MEXT).
14
5
0
5
0
5
(A)
DNA concentration
0 M
(B)800
700
600
500
400
300
200
2
Notes and references
700
600
500
400
300
200
a
60 Institute of Multidisciplinary Research for Advanced Materials, Tohoku
University, 2-1-1 Katahira, Aoba-ku, Sendai-shi, 980-8577, Japan. Tel:
+81-22-217-5633; E-mail: nagatugi@tagen.tohoku.ac.jp
0 M
1
1
2
2
100
0
100
0
†
Electronic Supplementary Information (ESI) available: [details of any
360
400
440
480
0
0.5
1
1.5
2
65 supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
Wavelength(nm)
DNA concentration (M)
Fig.6. (A) Changes in fluorescence spectra of 14 (0.1M) in 50 mM MES (pH 7.0),
00 mM NaCl with increasing duplex DNA (AP0bp) concentration. (B) Changes in
1
fluorescence intensity at 450 nm are plotted versus the DNA concentration.
1. D. Fu, J. A. Calvo and L. D. Samson, Nature Reviews Cancer, 2012,
1
2, 104.
Interestingly, the alkylated product was nearly absent in the
duplex DNA containing the Hoechst binding site (AATT) at the
' side of the target base in the fluorescein-labelled strand
7
7
8
8
9
0
5
0
5
0
2. K. W. Wellington, Rsc Advances, 2015, 5, 20309.
3
.
M. P. McCrane, M. A. Hutchinson, O. Ad and S. E. Rokita, Chem.
Res. Toxicol., 2014, 27, 1282
3
(
Rev.AP). For the non-reactive Hoechst-AVP derivative (14) that
4. D. Verga, M. Nadai, F. Doria, C. Percivalle, M. Di Antonio, M.
Palumbo, S. N. Richter and M. Freccero, J. Am. Chem. Soc., 2010,
132, 14625.
was prepared by a route described in the supporting information,
the binding constants to duplex DNA targets were determined
by fluorescence titrations. Fig. 6 shows the change in the
fluorescence spectra of 14 with increasing concentrations of
duplex AP-site-containing DNA. The binding constants were
obtained by analysis of the titration curve (Table 1).
5
.
R. C. Spitale, P. Crisalli, R. A. Flynn, E. A. Torre, E. T. Kool and H.
Y. Chang, Nature Chem. Biol., 2013, 9, 18.
S. Kellner, L. B. Kollar, A. Ochel, M. Ghate and M. Helm, Plos One,
2013, 8.
6
.
7. R. K. Singh, D. N. Prasad and T. R. Bhardwaj, Med. Chem. Res.,
015, 24, 1534
8
2
Table 1 Binding constants of 14 to duplex DNA
.
K. M. Johnson, Z. D. Parsons, C. L. Barnes and K. S. Gates, J. Org.
Chem., 2014, 79, 7520
8
-1
8
-1
DNA
Ks 10 M
DNA
AP3bp
Ks 10 M
AP0bp
3.0
2.3
2.1
3.0
3.3
9. W. B. Chen, Y. Y. Han and X. H. Peng, Chem. Eur. J., 2014, 20, 741
10. J. Wu, R. Huang, T. L. Wang, X. Zhao, W. Y. Zhang, X. C. Weng, T.
Tian and X. Zhou, Org. Biomol. Chem., 2013, 11, 2365
AP1bp
AP2bp
Rev.AP
w/oAATT
weak
1
1. Y. Y. Kuang, K. Baakrishnan, V. Gandhi and X. H. Peng, J. Am.
Chem. Soc. 2011, 133, 19278.
Values were obtained by a non-linear least squares data analysis.
The non-reactive Hoechst-AVP derivative (14) exhibited the
similar binding constants for duplex DNA, AP0~3bp and Rev.AP,
indicating that the binding affinity of 14 did not depend on the
location of the Hoechst binding site (AATT) in the target DNA.
Taken together, the efficient reactivity of the Hoechst-AVP
derivative (4) required appropriate positioning of the AVP moiety
for interaction with the thymine opposite the AP site.
12. L. Guan and M. D. Disney, Angew. Chem. Int. Ed., 2013, 52, 10010.
13. K. Tishinov, K. Schmidt, D. Haussinger and D. G. Gillingham,
Angew. Chem. Int. Ed., 2012, 51, 12000.
1
4. R. I. McDonald, J. P. Guilinger, S. Mukherji, E. A. Curtis, W. I. Lee
3
0
and D. R. Liu, Nature Chem. Biol., 2014, 10, 1049.
15. S. Hagihara, S. Kusano, W. C. Lin, X. G. Chao, T. Hori, S. Imoto and
95
F. Nagatsugi, Bioorg. Med. Chem. Lett., 2012, 22, 3870.
6. S. Hagihara, W. C. Lin, S. Kusano, X. G. Chao, T. Hori, S. Imoto and
F. Nagatsugi, Chembiochem, 2013, 14, 1427
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1
1
Conclusions
1
1
1
1
00 18. M. Li, Y. Sato, S. Nishizawa, T. Seino, K. Nakamura and N. Teramae,
J. Am. Chem. Soc. , 2009, 131, 2448
19. Y. Sato, S. Nishizawa, K. Yoshimoto, T. Seino, T. Ichihashi, K.
Morita and N. Teramae, Nucl. Acids Res., 2009, 37, 1411
3
4
4
5
5
0
5
0
In conclusion, we have developed a strategy for the selective
alkylation of DNA using single-stranded DNA containing an
abasic site and an alkylating probe. The Hoechst-AVP probe
exhibited high selectivity and efficient reactivity to thymine bases
at the site opposite an abasic site in DNA. In addition, the primer
extension reaction with polymerase was stopped at the alkylated
site by the Hoechst-AVP probe. These results suggested that
Hoechst-AVP probe may exert inhibitory effects on DNA
replication. Our studies provide the proof-of concept that AVP
derivatives conjugated with DNA binding molecules might form
hydrogen bonds with target nucleobases located in a hydrophobic
pocket and induce the selective alkylation. Based on the concept,
the investigations of the probes for inducing selective alkylation
in other hydrophobic pockets are now in progress.
2
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This work was supported by a Grant-in-Aid for Scientific
Research on Innovative Areas (“Chemical Biology of Natural 120
Products: Target ID and Regulation of Bioactivity”) and a Grant-
in-Aid for Scientific Research (B) from the Japan Society for the
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