Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5-Methylcytosine

Article abstract of DOI:10.1002/anie.201511520

5-Methyl-2′-deoxycytosine, the most common epigenetic marker of DNA in eukaryotic cells, plays a key role in gene regulation and affects various cellular processes such as development and carcinogenesis. Therefore, the detection of 5mC can serve as an important biomarker for diagnostics. Here we describe that modified dGTP analogues as well as modified primers are able to sense the presence or absence of a single methylation of C, even though this modification does not interfere directly with Watson-Crick nucleobase pairing. By screening several modified nucleotide scaffolds, O⁶-modified 2′-deoxyguanosine analogues were identified as discriminating between C and 5mC. These modified nucleotides might find application in site-specific 5mC detection, for example, through real-time PCR approaches.

Full text of DOI:10.1002/anie.201511520

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International Edition: DOI: 10.1002/anie.201511520
German Edition: DOI: 10.1002/ange.201511520
Epigenetics
Modified Nucleotides for Discrimination between Cytosine and the
Epigenetic Marker 5-Methylcytosine
Janina von Watzdorf+, Kim Leitner+, and Andreas Marx*
Abstract: 5-Methyl-2’-deoxycytosine, the most common epi-
genetic marker of DNA in eukaryotic cells, plays a key role in
gene regulation and affects various cellular processes such as
development and carcinogenesis. Therefore, the detection of
5mC can serve as an important biomarker for diagnostics. Here
we describe that modified dGTP analogues as well as modified
primers are able to sense the presence or absence of a single
methylation of C, even though this modification does not
interfere directly with Watson–Crick nucleobase pairing. By
screening several modified nucleotide scaffolds, O6-modified
2’-deoxyguanosine analogues were identified as discriminating
between C and 5mC. These modified nucleotides might find
application in site-specific 5mC detection, for example,
through real-time PCR approaches.
based on the selective bisulfite-mediated deamination of C to
uracil (U) in the presence of 5mC, which remains unaffected
as a result of slower deamination.[14] The sites of epigenetic
markers can be revealed by comparison of the output of
conventional sequencing methods before and after bisulfite
treatment, as C will be sequenced as thymine (T), and 5mC as
C.[15]
Although bisulfite sequencing can be used for the
genome-wide detection of 5mC, it possesses several draw-
backs. Since many steps are required and two sequencing runs
are needed for comparison, the method is time consuming
and prone to contaminations.[16] In addition, the conditions
used for the bisulfite treatment are harsh and destroy
approximately 95% of the genomic DNA, and thus a large
amount of DNA is required.[17] Furthermore, deamination of
C and 5mC after bisulfite treatment is incomplete, thereby
leading to an error-prone output.[18]
Nucleotides that are able to discriminate between C and
5mC would be highly interesting tools for the development of
new approaches for the site-specific detection of 5mC.
However, since the methylation of cytosines at C5 does not
directly affect Watson–Crick base pairing, attempts along
these lines are challenging and have not yet been described.
Here we present a class of modified nucleotides that can be
used for discrimination between C and 5mC in reactions
catalyzed by DNA polymerases. For this purpose, we
screened and investigated several purine-based 2’-deoxynu-
cleotides for their ability to sense 5mC as a result of diverging
incorporation efficiencies opposite a template containing C or
5mC by different DNA polymerases (Figure 2). We found
that several O6-modified 2’-deoxyguanosine derivatives (Fig-
ure 3a) are incorporated opposite 5mC and extended from
5mC with significantly different efficiencies compared to the
unmodified counterparts.
First, we screened a variety of different modified nucle-
otides (Figure 2) in combination with the thermostable DNA
polymerases KlenTaq and KOD exoÀ[19] in primer extension
experiments, followed by analysis through denaturing poly-
acrylamide gel electrophoresis (PAGE) and visualization by
autoradiography. Both DNA polymerases were able to
incorporate differently modified nucleotides opposite C or
5mC, although with decreased incorporation efficiencies
compared to the unmodified dGTP (1; Figure 2). KOD exoÀ
showed the highest potency for the desired application, with
the most pronounced differences in the incorporation effi-
ciencies being observed during the processing of the nucle-
otides O6-methyl-dGTP (3), dATP (10), and 5-nitro-1-indolyl-
2’-deoxyribose-5’-triphosphate (21). Since modified dATP
analogues (nucleotides 11–16) showed decreased incorpora-
tion efficiencies as well as decreased discrimination between
E
pigenetic modifications, caused by
methylation of cytosine residues (5-
methyl-2’-deoxycytosine, 5mC;
Figure 1), have been proven to have
an impact on a variety of cellular
processes that affect development[1,2]
and gene expression[3] as well as the
development of various diseases.[4]
Genes are frequently found to be
Figure 1. Chemical
structure of C (left)
and 5mC (right).
silenced[3] if CpG dinucleotides in the corresponding pro-
moters exhibit significant levels of 5mC; thus, 5mC is known
to regulate gene transcription and thereby affect tumori-
genesis.[5] The level of epigenetic methylation has to be
precisely regulated in eukaryotic genomes, since changes of
the methylation pattern lead to severe genetic malfunctions.[6]
Therefore, the detection of the occurrence and distribu-
tion of 5mC in the genome holds the potential to serve as an
important biomarker for diagnosis as well as disease ther-
apy.[7] This requires efficient strategies for the detection of
5mC. Different concepts for discriminating between cytosine
(C) and 5mC have been described which rely on endonuclease
digestion,[8] affinity enrichment,[9] nanopore sequencing,[10]
different chemical behavior concerning redox reactivity,[11]
or selective deamination of C using sodium bisulfite.[12]
Bisulfite sequencing has become routine for the detection
of 5mC with single-nucleotide resolution.[13] This method is
[*] J. von Watzdorf,[+] K. Leitner,[+] Prof. Dr. A. Marx
Fachbereich Chemie, Graduiertenschule Chemische
Biologie Konstanz, Universität Konstanz
Universitätsstrasse 10, 78457 Konstanz (Deutschland)
E-mail: andreas.marx@uni-konstanz.de
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201511520.
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solutions. The obtained nucleosides 23a–c were converted
into the corresponding 2’-deoxynucleoside-5’-triphosphates
(Figure S2).[21,22]
The potency of O6-methyl-, O6-ethyl-, O6-propyl-, and O6-
isopropyl-dGTP towards diverging incorporation efficiencies
opposite C and 5mC was further investigated. Both DNA
polymerases were able to incorporate all four nucleotide
analogues opposite C and 5mC, although the incorporation
efficiency decreased with increased steric demand of the
introduced modifications (Figure 3b and see Scheme S4).
KOD exoÀ showed the highest potency for the desired
application, with the most pronounced differences in incor-
poration efficiencies being observed during the processing of
the nucleotides dGTP, O6-methyl-, O6-ethyl-, O6-propyl-, and
O6-isopropyl-dGTP opposite C compared to 5mC (Fig-
ure 3b). Although the processing efficiencies of those modi-
fied nucleotides were decreased compared to natural dGTP,
the discrimination between the two templates was increased
markedly (Figure 3b). Of note, as reported before, O6-
methyl-dGTP was also processed opposite T[23] and we
could show that the same holds true for the other O6-alkyl-
dGTP derivatives (Figure S5). In contrast, KlenTaq DNA
polymerase again showed no significant difference in incor-
poration efficiencies for either dGTP or the modified
nucleotides (Figure S4).
To further investigate this observation, we determined the
steady-state kinetics[24] for the processing of the nucleotides
dGTP, O6-methyl-, O6-ethyl-, O6-propyl-, and O6-isopropyl-
dGTP by KOD exoÀ opposite C and 5mC (Table 1; see also
Scheme S2 and Figure S7). Comparison of the catalytic
efficiencies (kcat/Km) observed during processing of dGTP
and the modified nucleotides opposite C or 5mC in the
template strand confirms all the tendencies observed in the
qualitative primer extension reactions (Table 1 and see
Scheme S2). The ratio of the catalytic efficiency observed
Figure 2. Structures of modified dNTP analogues, including the per-
centage incorporation opposite C or 5mC on employing KODexoÀ in
primer extension by incorporation of a single nucleotide. 50 mm dGTP
or dN*TP and 5 nm KODexoÀ were used; the reactions were stopped
after 20 min. Discrimination ratios were determined by calculating the
quotient of the % incorporation opposite C and % incorporation
opposite 5mC.
both templates, and the synthesis of derivatives of nucleotide
21 seems to be tedious and challenging, we decided to focus
on O6-methyl-dGTP (3) for further derivatization. KlenTaq
DNA polymerase showed no significant difference in incor-
poration efficiencies for either dGTP or the modified
nucleotides (see Figure S1 in the Supporting Information).
Next, we focused on optimizing the system by synthesis
and functional evaluation of O6-alkylated dGTPs. O6-Alky-
lated dGTP derivatives were synthesized starting from
commercially available 2’-deoxyguanosine, which was acety-
lated and subsequently chlorinated at the 6-position by known
procedures (see Figure S2 in the Supporting Information).[20]
The different alkoxy groups were introduced in position 6 by
the reaction of 22 with the respective sodium alkoxide
Figure 3. a) Partial primer/template sequence used. b) PAGE analysis
of primer extension experiments on incorporation of a single nucleo-
tide of dGTP, O6-methyl-, O6-ethyl-, O6-propyl-, and O6-isopropyl-dGTP
opposite a template containing C, in comparison to a template
containing 5mC (with use of KODexoÀ). 50 mm dGTP or dG*TP and
5 nm KODexoÀ were used; reactions were stopped after the indicated
time points.
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Table 1: Steady-state kinetic analysis of primer extension on incorpo-
ration of a single nucleotide opposite C or 5mC by KODexoÀ DNA
polymerase.[a]
(3’-)Nucleotide
Template
Kcat/Km [sÀ1 mmÀ1
]
Ratio
1.36
dGTP
C
5mC
C
5mC
C
5mC
C
5mC
C
5mC
C
5mC
C
5mC
C
1.5Æ0.1
1.1Æ0.6
O6-methyl-dGTP
O6-ethyl-dGTP
O6-propyl-dGTP
O6- isopropyl-dGTP
G
0.16Æ0.02
2.58
4.24
1.08
1.48
3.48
6.72
3.53
0.062Æ0.010
0.14Æ0.02
0.033Æ0.007
0.105Æ0.015
0.097Æ0.011
0.031Æ0.005
0.021Æ0.004
8.7Æ1.3
Figure 4. a) Sequence context of the primer/template complex used in
the described studies. b) PAGE analysis of the primer extension
experiments for incorporation after C or 5mC paired with the 3’-
terminally unmodified G or modified O6-methyl-G and O6-ethyl-G
primers (by using KlenTaq). 50 mm dCTP and 0.1 nm KlenTaq were
used for G, 10 nm KlenTaq was used for O6-methyl-G and O6-ethyl-G;
reactions were stopped after the indicated time points.
30.3Æ3.4
O6-methyl-G
O6-ethyl-G
0.0043Æ0.0008
0.0289Æ0.0079
0.0066Æ0.0002
0.0233Æ0.0080
5mC
nation with the unmodified primer and the two modified
primers paired with both templates (Table S3, Figure S8).
These experiments confirm that both modified primers are
elongated with lower efficiency than the unmodified primer
and that elongation of the primers from 5mC is more efficient.
We found that the primer bearing O6-methyl-G showed the
best discrimination between C and 5mC (Table 1 and
Scheme S3). The catalytic efficiencies (kcat/Km) for the two
reactions differ by a factor of 6.7 (Table 1).
The different behavior of the DNA polymerases KlenTaq
and KOD exoÀ when processing the modified nucleotides in
the context of C and 5mC is intriguing. Structural data of both
enzymes bound to a primer and a template reveals signifi-
cantly different interaction patterns of the respective protein
with its substrate.[26] This difference might be the origin of the
observed effects.
Next we investigated whether we were able to exploit
these findings in polymerase chain reaction (PCR) experi-
ments. We used KlenTaq DNA polymerase and a primer
modified at the 3’-terminus. The modified nucleotide forms
a base pair with a methylated or unmethylated cytosine in
human genomic DNA (gDNA; Figure 5). We designed
a forward primer that should lead to a 155 bp PCR product
and analyzed a specific CpG site in the promotor region of
NANOG in gDNA.[1,27] We used gDNA purified from HeLa
cells because it was shown that this CpG site is unmethyla-
ted.[27b] However, an enzymatically methylated HeLa gDNA
was used as a fully methylated control. We found in real-time
PCR experiments that discrimination between both un- and
methylated cytosine in gDNA was improved by using the
primer bearing O6-methyl-G at the 3’-end relative to the
unmodified primer (Figure 5). The use of the HeLa gDNA
leads to a delayed amplification of 2.95 Æ 0.80 (mean Æ stan-
dard deviation (SD), n = 5) compared to the fully methylated
gDNA when paired with the primer modified at the 3’-
terminus. In contrast, the employment of the unmodified
primer leads to a delayed amplification of 0.8 Æ 0.23 (mean Æ
SD, n = 5) in the case of the wild-type HeLa gDNA (Figure 5).
In summary, we found that O6-modified 2’-deoxyguano-
sine analogues are able to sense the presence or absence of
a single methylation in C in the template strand, despite this
[a] Ratio: kcat/Km (C) to kcat/Km (5mC) (nucleotide incorporation) or
kcat/Km (5mC) to kcat/Km (C) (modified primer extension).
during incorporation of the respective nucleotide opposite C
compared to 5mC varied. The best discrimination was
observed for the processing of O6-ethyl-dGTP, with the
catalytic efficiencies kcat/Km for incorporation opposite C
and 5mC differing by a factor of 4.2 (Table 1 and see
Scheme S2).
Next, we aimed at investigating the effect of the modifi-
cations when they are embedded at the 3’-terminus of primer
strands and placed directly opposite C or 5mC.[25] Thus, the
modified nucleosides were converted into phosphoramidites,
which were employed in DNA solid-phase synthesis of the
different primer strands (see Figure S3 in the Supporting
Information). The methyl and ethyl modifications in O6-
methyl-dGTP and O6-ethyl-dGTP showed the most promis-
ing results, and so we focused on these modifications
(Figure 3, Table S1).
After their synthesis, the primer strands were first
employed in primer extension studies (Figure 4b). Again,
both modifications were well tolerated by both DNA
polymerases tested, with all three primers elongated,
although the modified primers to a smaller extent than the
unmodified primer (Figure 4b and see Figure S6). Interest-
ingly, no significant discrimination between C and 5mC was
observed with KOD exoÀ, either when the unmodified or
when the modified primer strands were employed (Fig-
ure S6). In contrast, significant discrimination was observed
with the KlenTaq DNA polymerase for both modified primers
(Figure 4b). A marginal difference in the incorporation
efficiencies between extension from C or 5mC was observed
when the unmodified primer was extended with dCTP. In
contrast, elongation of the modified primers bearing either
O6-methyl-G or O6-ethyl-G at the 3’-end showed extensive
discrimination between both templates (Figure 4b).
Interestingly, in these primer extension reactions, elonga-
tion from the modified primer is more efficient when paired
with 5mC. To confirm these results, we determined steady-
state kinetics[24] using KlenTaq DNA polymerase in combi-
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Acknowledgements
We gratefully acknowledge the European Research Council
(ERC Advanced Grant 339834). We also acknowledge the
Konstanz Research School Chemical Biology for support.
Keywords: 5-methylcytosine · DNA methylation ·
DNA polymerase · epigenetics · polymerase chain reaction
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Received: December 11, 2015
Published online: February 2, 2016
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