Selective detection of 5-formyl-2′-deoxycytidine in DNA using a fluorogenic hydroxylamine reagent

Article abstract of DOI:10.1021/ol401290d

Fluorogenic hydroxylamine reagents were used for detecting 5-fC through a labeling pathway. Chemical synthesis, HPLC, denaturing PAGE, and DNA MS were applied to testify that the probe reacted with 5-fC with oligodeoxynucleotide selectivity to achieve 5-fC detection conveniently and quantificationally with the method of fluorescence. The feasibility of fluorescently detecting 5-fC in a genome was also investigated.

Full text of DOI:10.1021/ol401290d

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Selective Detection of
5‑Formyl-20-deoxycytidine in DNA Using a
Fluorogenic Hydroxylamine Reagent
Pu Guo,† Shengyong Yan,† Jianlin Hu,† Xiwen Xing,† Changcheng Wang,† Xiaowei Xu,†
Xiaoyu Qiu,† Wen Ma,† Chunjiang Lu,† Xiaocheng Weng,† and Xiang Zhou*,†,‡
College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers
of Ministry of Education, Wuhan University, Hubei, Wuhan, 430072, P. R. of China, and
State Key Laboratory of Natural and Biomimetic Drugs, Beijing University, Beijing,
100191, P. R. China
xzhou@whu.edu.cn
Received May 9, 2013; Revised Manuscript Received June 11, 2013
ABSTRACT
Fluorogenic hydroxylamine reagents were used for detecting 5-fC through a labeling pathway. Chemical synthesis, HPLC, denaturing PAGE, and
DNA MS were applied to testify that the probe reacted with 5-fC with oligodeoxynucleotide selectivity to achieve 5-fC detection conveniently and
quantificationally with the method of fluorescence. The feasibility of fluorescently detecting 5-fC in a genome was also investigated.
The epigenetic base 5-methylcytosine (5-mC) in mam-
malian genomes plays essential roles in maintaining cellular
function and genomic stability and is involved in biological
processes including chromosome inactivation, genomic
imprinting, and transposon silencing.1 Recently, studies2
on an active oxidative demethylation process for cytidine
derivatives were reported. The demethylation mechanism
involvesthe iterativeoxidation of5-methylcytosine(5-mC)
to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine
(5-fC), and 5-carboxycytosine (5-caC) by the 10-11 trans-
location (TET) enzymes, followed by base excision repair
by thymine DNA glycosylase (TDG).3 As the main oxidative
product, 5-hmC has been implicated in stem cell biology4 and
cancer diagnosis using several detection methods.5 It has been
reported that 5-formylcytosine (5-fC) is present in mouse
embryonic stem (ES) cells and the mouse cerebral cortex
through LC-MS and deep sequencing.3b,6 It has been con-
firmed that Activation-induced deaminase (AID)/APOBEC
family cytosine deaminases cannot convert 5-hmC into
(5-hydroxymethyluracil) 5-hmU to complete the DNA
demethylation process,7 whereas 5-fC, as an oxidation
† Wuhan University.
‡ Beijing University.
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10.1021/ol401290d
XXXX American Chemical Society

intermediate of TET enzymes, remains detectable.3b As the
major product after the TET-mediated oxidation of 5-mC,
5-fC is situated at a pivotal step in the DNA demethylation
process. In addition, a recent report indicated that 5-fC
plays potential roles in differentiation.6b
DNA containing 5-fC with hydroxylamine probes could
occur successfully in ammonium acetate buffer (pH = 4.5)
when catalyzed by p-anisidine.
In view of the importance of 5-fC in the genome,
developing a sensitive, selective, and convenient detection
method for 5-fC has become a challenge. The most recent
breakthrough in this field came with the development of
three new methods allowing the measurement of 5-fC.3b,6
The methods include LC-MS and deep sequencing tech-
nology. Our group just reported that a primary amino
could be used to detect 5-fC; however, scant proof and
interference of abasic sites limit its application in the
detection of 5-fC in a genome.6c Herein, considering the
highcost and complicated nature ofthe abovemethods, we
developed a simple and effective approach to qualitatively
and quantitatively detect 5-fC in DNA. Furthermore, we
deduced that our method will hopefully be applied in the
detection of 5-fC in a genome.
In this paper, we report a new approach for the simple
detection of 5-fC in DNA using a fluorogenic hydroxyla-
mine reagent. Hydroxylamine, an active carbonyl-reactive
group, is widely used in fluorescence labeling applications.
Therefore, we designed fluorescent probes for 5-fC based
on the hydroxylamine group. The oxime ligation of the
hydroxylamine and carbonyl groups is a popular reaction
in chemical biology.8 Oxime ligations can be significantly
accelerated by using aniline and its derivatives as nucleophilic
catalysts under acidic conditions.9 Because of the high
reactivity of hydroxylamine on benzyl, the reaction of
Scheme 1. Structure of Synthetic Hydroxylamine Probes
First, we designed and synthesized the probes BODIPY-
B (1), BODIPY-L(2), and Coumarin-HA (3) (see Scheme 1).
BODIPY-B (1) was obtained in two steps. Amino-BODIPY
was subjected to a condensation reaction and produced
BODIPY-L (2) to increase the length of the carbon chain.
In addition, Coumarin-HA (3) was synthesized from
4-bromomethyl-7-hydroxy-chromen-2-one through base-
promoted HBr-elimination and hydrazinolysis. Reagents 1
and 2 were treated with hydrazine hydrate to release the
free hydroxylamine group just before exposure to 5-fC.8b,10
We first tested the activity of the hydroxylamine probes
using an organic reaction (see Scheme 2). Compound 10,
derived from the hydrazinolysis of BODIPY-B (1), and
probe 3 were added separately to MeOH containing 5-fC,
followed by the addition of several drops of acetic acid.
The reaction occurred in the presence of p-anisidine as the
catalyst for 12 h at 50 °C, giving fluorescent product 4 in a
95% yield and 5 in an 83% yield.11 The satisfactory yields
support the use of hydroxylamine probes in the detection
of 5-fC. Furthermore, we measured the fluorescence spec-
tra of compound 5 to facilitate the detection of 5-fC in
DNA (see Figure S1). (We added the fluorescence spectra
with excitation and emission wavelengths of compound 5
in the Supporting Information (Figure S1).)
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Next, we chose a 21-mer oligodeoxyribonucleotide con-
taining 5-fC (ODN-1)12 and incubated it with probe 3
under the optimized conditions (Figure 1a). A solution of
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Org. Lett., Vol. XX, No. XX, XXXX

Scheme 2. Reaction of 5-fC with Hydroxylamine Probes
was incubated at rt for 12 h to give the ODN-1-CHA-
containing compound 5 in > 97% yield. After probe 3 was
added, the reaction mixture was monitored by HPLC, and
the chromatograms are displayed in Figure 1b. After the
reaction proceeded for 2 h, an obvious new peak was
observed, and with increasing time, this new peak in-
creased in intensity as ODN-1 disappeared. As shown in
Figure 1c, the new peak in the HPLC chromatogram was
collected, and its identity was confirmed by MALDI-TOF
analysis (see Figures 1c, S2). The MS analysis indicated the
DNA product was the desired ODN-1-CHA. Probes 1 and
2were also used to detect 5-fC in ODN-1 (see Figures S3, S4).
The products of ODN-1 that reacted with probes 1 and 2 had
higher fluorescence intensities than the product of ODN-1
reacted with probe 3. Even at the concentration of 5 μM, we
could see an unambiguous green color with the naked eye
under UV light (see Figure S5).
In view of the fact that there are many kinds of bases in a
genome besides normal ones, and the reaction of abasic
sites either apurinic or apyrimidinic (AP) with compounds
including a long carbon chain primary amine has been
reported,13 experiments focused on selectivity were neces-
sary. Because a fluorophore was introduced into ODN-1,
the reaction mixture was also analyzed by denaturing
PAGE followed by fluorescence detection, and the results
supported our previous conclusion.
ODN-2, ODN-3, ODN-4, and ODN-5wereindividually
incubated with probe 3 under the same reaction condi-
tions. The fluorescence images of the denaturing PAGE gel
revealed favorable selectivity for 5-fC among the tested
oligodeoxynucleotides (see Figure 2). As shown in the
upper image of Figure 2a and 2b, fluorescent probe 3
reacted with ODN-1, which contains 5-fC, but not with
ODN-2, ODN-3, ODN-4, or ODN-5. In view of the fact
thatAP has a similar level orgreater comparedwith5-fC in
genome, ODN-9 (containing AP and 5-fC) and excess
ODN-5 were used to serve as controls (see Figure 2b).
15-mer ODN-8 (5-GACTCAA5-fCAGCCGTA-30) and
Figure 1. Conversion of ODN-1 (50-CCCGAGGTG5-fC
GTGAGTACCGG-30) into ODN-1-CHA with probe 3. (a)
Reaction scheme with the sequences of ODN-1 and product
ODN-1-CHA. (b) HPLC chromatogram of ODN-1 (starting
DNA) and ODN-1-CHA (product DNA after probe 3 added at
0, 2, 6, and 12 h). (c) MALDI-TOF analysis of ODN-1 and
ODN-1-CHA.
ODN-10 (50-GACTCAA-AP-AGCCGTA-30) were also
checked as shown in Figure S6. The results still revealed
excellent selectivity for 5-fC. The proposedreasonsare that
the steric hindrance of probe 3 and adjacent bases of AP
hinder the possible reaction. The lower images of Figure 2a
and 2b taken under 254 nm UV light indicate that un-
reacted ODN-2, -3, and -4 were present in lanes 2, 3, and 4
in Figure 2a and unreacted ODN-5 was present in lanes 2
(0.2 nmol) and 4 (0.6 nmol) in Figure 2b respectively. The
relative positions are consistent with their molecular
weights. Consistent with the upper image, the image of
lane 1 in Figure 2a and lanes 1 and 3 in Figure 2b contained
an unambiguous blue-fluorescent ODN band. We also
interrupted the reaction at 3 h and performed denaturing
PAGE analysis to assess the intermediate state in which
ODN-1 and ODN-1-CHA coexist to observe the different
locations of ODN-1 and ODN-1-CHA in the same lane
(see Figure S7). To facilitate the fluorescence experiment,
different concentrations of ODN-1 were incubated in the
presence of equal amounts of probe 3, and the fluorescence
image of the denaturing PAGE gel indicated an apparent
difference with increasing concentrations of ODN-1 (see
Figure S8).
Finally, the fluorescence intensity of the reaction prod-
uct was measured in ddH2O after the removal of excess
probe 3 and p-anisidine by extraction from the reaction
mixture with ethyl acetate (AcOEt). As shown in Figure 3a,
we used fluorescence-based methods to demonstrate
that probe 3 can distinguish ODN containing 5-fC from
ODN containing 5-hmC, 5-mC, C, and AP. It is obvious
that the fluorescence intensity of ODN-1-CHA is much
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Figure 2. Denaturing PAGE probed fluorescent image analysis.
(a) Lane 1: 0.2 nmol of ODN-1 (50-CCCGAGGTG5-fCGT-
GAGTACCGG-30). Lane 2: 0.2 nmol of ODN-2 (50-CCC-
GAGGTG5-hmCGTGAGTACCGG-30). Lane 3: 0.2 nmol of
ODN-3 (50-CCCGAGGTG5-mCGTGAGTACCGG-30). Lane
4: 0.2 nmol of ODN-4 (50-CCCGAGGTGdCGTGAGTAC-
CGG-30). (b) Lane 1: 0.2 nmol of ODN-1. Lane 2: 0.2 nmol of
ODN-5 (50-CCCGAGGTG-AP-GTGAGTACCGG-30). Lane 3:
0.2 nmol of ODN-9 (50-CCCGAGGTG5-fCGTGAGTA-AP-
CGG-30). Lane 4: 0.6 nmol of ODN-5 reacted with probe 3.
stronger than those of ODN-2, -3, -4, and -5 after incuba-
tion with probe 3. We observed a linear correlation
between the concentration of ODN-1 and the fluorescence
intensity over the range 50ꢀ1000 nmol (Figure 3b). We
could calculate the content of 5-fC based on the observed
linear correlation with the fluorescence intensity. In order
to investigate the feasibility of our method applied in the
detection of 5-fC in genome, we chose DNA collected from
mouse embryonic stem cells as practical samples. After
reaction with probe 3 for 24 h at 37 °C, the genome was
hydrolyzed with S1 nuclease, alkaline phosphatase, and
snake venom phosphodiesterase I. Through collecting frac-
tions at the corresponding retention time of compound 5
several times, we detected the presence of 5 in the enzymatic
hydrolysate of the genome by LC-MS (see Figure S9). This
result supported that our probes could be applied in the
detection of 5-fC in genomes. (The limit of detection is a
little high for being applied to the analysis in a genome.
However, we could achieve this purpose through DNA
enrichment or choosing a new probe with hydroxylamine.
Additionally, we also proved the possibility in our manu-
script (Figure S9).)
In summary, we have established a new method for the
detection of 5-fC in DNA that is selective, convenient,
inexpensive, and quantitative. Our method does not re-
quire complicated steps or separation processes before
measuring the fluorescence. With the help of fluorescence,
we are able to observe the presence of 5-fC in oligonucleo-
tides and calculate the amount of 5-fC (this site is the same
as that noted at the end of the previous paragraph). We
also estimated the feasibility of our method applied in the
detection of 5-fC in the genome of mouse embryonic stem
Figure 3. (a) Fluorescence intensity of 1 μM ODN-1, -2, -3, -4, -5
(containing 5-fC, 5-hmC, 5-mC, dC, AP) in H2O reacted with
10 μM probe 3 after removal of excess compounds. (b) Fluor-
escence intensity (at 479 nm) of the reaction mixture containing
various amounts of the ODN derived from ODN-1 in ddH2O.
Fluorescence measurements were performed at 25 °C with
excitation at 325 nm. The limit of detetion is 42 nM. (Through
the linearity of concentrations of DNA and fluorescence inten-
sity, we could report the limit of detection for 5-fC is 42 nM.)
cells. Finding a new probe to decrease the limit of detection
and using a fluorescence-based approach for the detection
of 5-fC in various genomic DNA from cancer cells will be
the focus of our future work.
Acknowledgment. This work was supported by the
National Basic Research Program of China (973 Program)
(2012CB720600, 2012CB720603), the National Science
Foundation of China (Nos. 91213302, 21272181,
21072115). Supported by Program for Changjiang
Scholars and Innovative Research Team in University
(IRT1030), the National Grand Program on Key Infec-
tious Disease (2012ZX10003002-014). Supported by “the
Fundamental Research Funds for the central Universities”
(2012203020212).
Supporting Information Available. Full experimental
details, compound characterization, HPLC, denaturing
PAGE, MALDI-TOF and fluorescence data. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
The authors declare no competing financial interest.
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