Discrimination of N6-methyl adenine in a specific DNA sequence

Article abstract of DOI:10.1039/c0cc00172d

We have developed a novel chemistry-based method to detect adenine methylation. A DNA probe functionalized with a formyl group discriminates between adenine and N6-methyl adenine by the formation of a selective interstrand cross-link. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0cc00172d

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Discrimination of N6-methyl adenine in a specific DNA sequencew
Chikara Dohno, Tomonori Shibata and Kazuhiko Nakatani*
Received 1st March 2010, Accepted 26th April 2010
First published as an Advance Article on the web 11th May 2010
DOI: 10.1039/c0cc00172d
We have developed a novel chemistry-based method to detect
adenine methylation. A DNA probe functionalized with a formyl
group discriminates between adenine and N6-methyl adenine by
the formation of a selective interstrand cross-link.
Methylated nucleobases are widely used in many organisms
to add extra information to the DNA. Three methylated
nucleobases, 5-methylcytosine (5mC), N4-methylcytosine,
and N6-methyladenine (6mA), are natural components of
genomic DNA.1–3 Patterns of C5-cytosine methylation in the
genomic DNA determine epigenetic gene regulation and
cellular differentiation in eukaryotes, and are associated
with many human tumors.2 6mA is mostly found in bacterial
genomes and some archaeal and eukaryotic genomes.
The roles of adenine methylation include genome defense,
replication, mismatch repair, and control of gene expression.3
Since 5mC plays a significant role in mammalian cells, many
methods for analyzing the distribution of 5mC have been
developed over the past decade.4 In contrast, few methodo-
logies have been developed for the analysis of other
methylated nucleobases. For the detection of adenine
methylation, the few methods that have been developed rely
on specific enzymatic reactions.5 Here, we demonstrate a novel
chemical approach to distinguish adenine methylation in
specific target sequences.
Fig. 1 Schematic illustration of the discrimination between adenine
and 6mA based on the selective interstrand cross-link (ICL)
formation.
f
Synthesis of G-containing DNA was achieved by post-
synthetic modification of the DNA containing a vicinal diol
moiety with sodium periodate (NaIO4; Fig. S1, ESIw).10 The
reaction between the fG-containing DNA and its comple-
mentary DNA was analyzed by denaturing polyacrylamide
gel electrophoresis (PAGE; Fig. 2a). DNA without any ICLs
should be electrophoresed as a single-stranded form under
denaturing conditions. The precursor DNA containing a
vicinal diol was mixed with its complement and NaIO4, and
For discrimination of the methylated states of adenine, we
have focused on the reactions involving an exocyclic amino
group of adenine-N6. The base amino groups can react with
naturally occurring and artificially introduced formyl groups
in DNA, leading to an interstrand cross-link (ICL) via imine
or carbinolamine formation.6–9 Such a nucleophilic addition
will be sensitive to the presence and absence of the methyl
group at the N6 position. To discriminate between the
methylation states of adenine with the reaction involving the
base amino group, we synthesized the modified nucleobase,
fG, which possessed an electrophilic formyl group at the O6
position of guanine (Fig. 1). The covalently attached formyl
group was located in the major groove of the DNA duplex,
and allowed the ICL formation by reacting with the neighboring
adenine base. The presence of a methyl group at the N6
position prevented the formyl group from approaching the
amino group, resulting in methylation-dependent suppression
of the ICL formation.
Fig. 2 (a) Sequence-selective ICL formation with fG-containing
DNAs. A series of DNA duplexes was incubated at 30 1C for 24 h,
and then subjected to denaturing PAGE analysis. 2A represents
2-aminopurine (lane 6). (b) HPLC profiles for the ICL formation in
The Institute of Scientific and Industrial Research (ISIR),
Osaka University, Ibaraki 567-0047, Japan.
E-mail: nakatani@sanken.osaka-u.ac.jp; Fax: +81 6-6879-8459;
Tel: +81 6-6879-8457
w Electronic supplementary information (ESI) available: Synthetic
details and the DNA properties. See DOI: 10.1039/c0cc00172d
the 50-TfGT-30/50-ACA-30 sequence before (top) and after 26 h
ꢀ
incubation (bottom). dT was added as an internal standard. (c) Time
course for the ICL formation of the duplex containing 50-TfGT-30/
50-ACA-30 at pH 6 (filled circle) and 7 (open square).
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the duplex possessing the in situ generated fG was incubated in
sodium cacodylate buffer (pH 6) and 100 mM NaCl for 24 h.
Bands that migrated more slowly than the 13-mer single-
stranded DNAs corresponded to DNA containing the ICL
and appeared in a sequence-dependent manner (Fig. 2a). The
ICLs were observed only for the sequence containing an
f
adenine opposite the 30-adjacent base of G, i.e., 50-fGT-30/
50-AC-30 (lanes 1 and 2). In contrast, the adenine bases at
ꢀ
other positions were inert toward the ICL formation. The
observed specificity was very similar to that previously
reported for ICLs between adenine-N6 and the aldehyde of
a C40-oxidized abasic site.7 The 2-aminopurine (lane 6) and the
guanine (Fig. S3, ESIw), which had an amino group in the
minor groove of the double-stranded DNA, were not active
Fig.
3
Discrimination between methylated and unmethylated
adenine by fG-containing DNA. (a) The ICL formation for the target
adenine and 6mA changing the opposite base of fG (N). (b) Discrimi-
nation between adenine and 6mA in regulatory sequence of the E. coli
f
pap operon. 1 eq. and 4 eq. of the G DNA probe were used for the
f
toward G. A weak ICL band was observed for the cytosine
f
single stranded and double stranded target DNA, respectively.
base opposite the 30-adjacent base of G (5% yield, Fig. S3,
ESIw). These results indicated that the ICL formation
was highly sensitive to the spatial orientation between the
electrophilic formyl group and the nucleophilic sites of
nucleobases.6–9
fG DNA probe, followed by incubation for 24 h in the
presence of NaIO4. ICL formation was clearly observed for
nonmethylated adenine in both single- and double-stranded
targets, whereas no ICL was detected for the methylated
counterpart (Fig. 3b). Methylated and nonmethylated adenine
in the predetermined sequence were clearly distinguished by
the fG DNA probe, although the method needs to be
improved in the ICL yield for higher sensitivity.
The cross-link reaction with the adenine base in the
sequence 50-TfGT-30/50-ACA-30 was monitored by HPLC
ꢀ
(Fig. 2b). The amount of ICL almost reached a plateau after
20 h incubation (27% yield). Saturation of the reaction
suggests the reversible nature of the ICL formation. In fact,
the ICLs could be reverted to the original single-stranded
DNAs after heat denaturation of the duplex, for which
subsequent incubation reproduced the ICL (Fig. S4, ESIw).
Comparison of the time-course of product yields at pH 6 and 7
showed that a slightly faster reaction occurred at pH 6
(Fig. 2c). The observed pH dependency and reversibility in
ICL formation suggested that ICLs could be attributed to the
formation of carbinolamine8 and/or a Schiff base9 between the
In conclusion, we have demonstrated a chemistry-based
methodology to discriminate between methylated and
nonmethylated adenine. Since the adenine methylation plays
a significant role in the prokaryotic methylome and remains
mostly unknown in eukaryotes,3 the new chemical methodo-
logy presented here will be useful to study the biological roles
of adenine methylation.
This work was supported by Grant in Aid for Scientific
Research (S) (18105006) and Grant in Aid for Young
Scientists (B) (21750171) from the Japan Society for the
Promotion of Science (JSPS).
f
formyl group of G and the N6 amino group of adenine.11
Having established that fG undergoes a sequence-dependent
cross-linking with adenine bases, we exploited these reactions
to detect the methylation states of adenine. To discriminate
between adenine and 6mA, we prepared a series of oligo-
nucleotides containing the core sequence, 50-fGT-30/
50-(6mA/A)N-30, where the target adenine was placed at the 50
Notes and references
1 M. M. Suzuki and A. Bird, Nat. Rev. Genet., 2008, 9, 465–476;
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adjacent to the opposite base of fG (N). The relative intensities
of the ICL bands investigated by denaturing PAGE with
regard to the methylation states of adenines are summarized
2 K. D. Robertson and A. P. Wolffe, Nat. Rev. Genet., 2000, 1,
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f
in Fig. 3a. Regardless of the base opposite G, an ICL band
was undetectable for 6mA, whereas ICL bands were
unambiguously observed for nonmethylated adenines.12 These
results clearly showed that electrophilic attack by the formyl
group on the N6 of adenine is sensitive to the presence and
absence of a methyl group. The observed weak sequence-
dependency in ICL formation is favorable for the discrimina-
tion of 6mA in any sequence context.
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We next conducted preliminary experiments to discriminate
between methylated and nonmethylated bases using the
regulatory sequence of the E. coli pap operon.13 Methylation
of the adenine in the GATC site of the regulatory sequence is
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responsible for transcription of the pap operon and the
resulting epigenetic phase variation of bacteria.13 The target
20-mer DNA containing a single methylation site in single- or
double-stranded form was annealed with the complementary
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10 H. Sugiyama, S. Matsuda, K. Kino, Q. M. Zhang, S. Yonei
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11 MALDI-tof-MS measurements of the isolated ICL showed peaks
only for each single stranded DNA even after NaBH3CN or
NaBH4 treatment. A very slow or no reduction reaction with
NaBH3CN has been reported for ICLs containing an imine or a
carbinolamine.7,8 Resistance to reducing agents was again
observed at nucleoside level in the enzymatic digestion experiments
(Fig. S7, ESIw).
f
12 When the opposite base of G is purines, the efficiency of the ICL
was lower than the pyrimidines. Since O6-alkylguanosine can
adopt syn glycosidic torsion angle by pairing with purines, this
local conformational change may cause unfavorable positioning of
the formyl group for the ICL.
13 M. van der Woude, B. Braaten and D. Low, Trends Microbiol.,
1996, 4, 5–9; A. Hernday, M. Krabbe, B. Braaten and D. Low,
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5532 | Chem. Commun., 2010, 46, 5530–5532