Bioorthogonal Chemical Signature Enabling Amplified Visualization of Cellular Oxidative Thymines

Article abstract of DOI:10.1021/acs.analchem.1c01285

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.

Full text of DOI:10.1021/acs.analchem.1c01285

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Article
Bioorthogonal Chemical Signature Enabling Amplified Visualization
of Cellular Oxidative Thymines
Min Bai, Xiaowen Cao, Feng Chen, Jing Xue, Yue Zhao, and Yongxi Zhao*
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ABSTRACT: Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-
formyluracil (5fU), are found in the genomes of a diverse range of organisms, the
distribution of which profoundly influence biological processes and living systems.
However, the distribution of cellular oxidative thymines has not been explored because of
lacking both specific bioorthogonal labeling and sensitivity methods for single-cell
analysis. Herein, we report a bioorthogonal chemical signature enabling amplified
visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne,
an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically
linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphome-
thyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU
is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling
of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click
chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized
barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes
can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular
oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their
multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of
fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of
these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative
thymines profiles and insights into their functional role and dynamics in biology.
umerous modified bases have been identified in the
genomes of various organisms. They profoundly
DNA function disturbance.18−20 Unfortunately, the abundance
of 5hmU/5fU in the genome is very low, for example, ∼0.5−
2.5 × 10−6 dN in mESCs.8,21 The efficient analysis of low-
abundance 5hmU/5fU in single cells is a major challenge.
Especially, the structural similarity of 5fU and 5hmU hinders
the differentiated detection of these two modifications. These
factors make it extremely difficult to construct a sensitive and
specific detection method for distinguishing between 5hmU
and 5fU at the single-cell level.
So far, several methods have been developed for genomic
5hmU analysis by oxidation conversion of 5hmU to 5fU.
Balasubramanian et al. employed polymerase extension to
induce a change in the T to C bases due to the inherent ability
of the resulting 5fU to form a pair of 5fU: G bases.22 However,
even under optimized conditions, the efficiency of this
conversion is less than 40%. In addition, this method cannot
N
influence biological processes and living systems. These
modified bases are implicated in pathological associations
such as cancers,1,2 among which the most widely studied ones
are the derivatives of cytosine, including 5-methylcytosine
(5mC) and its oxidized derivatives 5-hydroxymethylcytosine
(5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine
(5caC).3−7 However, the details of thymine modifications
and their spatial distribution in cells remain elusive, especially
cellular oxidative thymines. 5-Hydroxymethyluracil (5hmU) is
an oxidation derivative of thymine determined in the genomes
of sundry organisms.8−10 In mammals, the levels of 5hmU are
different in cells and tissue types.11 Emerging evidence has
demonstrated that 5hmU mononucleoside abundance can be
used as a potential marker for the evaluation of breast cancer
risk and invasiveness.12,13 Recently, as the oxidation product of
5hmU or thymine, 5-formyluracil (5fU) was found in many
cells and tissues.8,14 5fU can be generated by exposure to
ultraviolet, reactive oxygen species, or ionizing radiation.15−17
It is well known that 5fU is mutagenic and can induce
transitional mutations. The inherent ability of strong electron-
withdrawing 5-formyl group in 5fU contributes to gene
miscoding and changes in DNA structures, which may cause
Received: March 24, 2021
Accepted: July 13, 2021
Published: July 22, 2021
https://doi.org/10.1021/acs.analchem.1c01285
© 2021 American Chemical Society
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label intracellular 5hmU with bioorthogonal tags for in situ
analysis. Another method biotinized 5fU with an aldehyder-
eactive reagent.23 However, this reagent also shows high
reactivity to other aldehyde-containing modifications consist-
ing in genomic DNA, especially 5fC. Therefore, this method
can only achieve analysis of 5hmU with 5fU-, 5hmC-, and 5fC-
absent DNA input. Recently, the enriching and sequencing of
5fU in mouse hippocampus was implemented by an azido
derivative of (2-benzimidazolyl) acetonitrile.24 Yet, simulta-
neous detection of cellular 5hmU and 5fU distribution has not
been explored.
Cell imaging in situ can observe the subcellular distribution
of biomolecules in single cells.25−28 Antibody-labeled cellular
immunofluorescence imaging has been used to detect high-
abundance DNA modifications such as 5mC and 5hmC only in
naked DNA.29,30 Some base sites in condensed chromatins or
masked by DNA-binding proteins may be sterically excluded
from antibodies with relatively large sizes. Such noncovalent,
affinity-based immunoassays have low binding efficiency and
recognition specificity and are disabled to explore 5hmU and
5fU with low abundance. The rolling circle amplification
(RCA) reaction is an isothermal signal amplification method.
This method uses circular DNA as a template to convert DNA
primer into long single-stranded DNA containing hundreds of
repetitive sequences through enzymatic catalysis.31−33 We
previously reported a method to visualize single-cell 5-
hydroxymethylpyrimidines based on the RCA reaction.34 In
this work, we have demonstrated that 5hmU DNA kinase (5-
HMUDK) uses sulfhydryl-modified ATP-γ-S substrate to
specifically phosphorylate 5hmU. Due to the large number of
sulfhydryl proteins and molecules in cells, this method cannot
be applied to the detection of 5hmU in dsDNA in the cellular
environment. Therefore, constructing cellular-specific recog-
nition of 5hmU/5fU with bioorthogonal tags and visualized
analysis strategy in single cells is still challenging.
phosphate with biological orthogonal labeling, named ATP-γ-
alkyne. After fixing the cells, ATP-γ-alkyne was used to label
intracellular 5hmU by 5-HMUDK. Then, DNA with 5-
alkynephosphomethyluracil (alkyne-5pmU) was further clicked
with azide (N3)-modified 5hmU-primer. Next, 5fU was
reduced to 5hmU by water-soluble reductant, sodium
borohydride (NaBH4). The newly generated 5hmU sites
were labeled with N3-modified 5fU-primer by the phosphor-
ylation and click reaction mentioned above. To simultaneously
visualize the low abundance of 5hmU and 5fU in single cells,
we performed RCA reaction and hybridization of fluorescent
probes. Their spatial distributions in single cells and abundance
differences in various cell types were further investigated.
Furthermore, we explored their multilevel subcellular informa-
tion and their dynamic changes during cell cycles.
EXPERIMENTAL SECTION
■
Chemicals and Materials. Propargylamine, ATP diso-
dium salt (adenosine 5′-triphosphate disodium salt), and 1-
ethyl-3-(3-dimethyllamino-propyl)carbodiimide hydrochloride
(EDC-HCl) were obtained from Sigma-Aldrich. Sodium
ascorbate, sodium borohydride, and copper(II) sulfate were
purchased from Aladdin. All of the reactions were performed in
RNase-free water and buffers. Also, all chemical reagents were
used as received without further purification. dfUTP and
dhmUTP were obtained from Trilink Biotechnologies. The
oligonucleotides of this work (Table S1) were prepared by
Sangon Biological Co. Ltd. Exonuclease I, Exonuclease III, T4
DNA ligase, and DNase I were acquired from Takara
Biotechnology Co. Ltd. 5-HMUDK, Klenow Fragment (3′
→ 5′ exo-), hSMUG1, and phi29 DNA polymerase were
acquired from New England Biolabs Ltd. Primary antibodies
used in this study: H3K4me3, γH2AX, H3K4me1, and
H3K27me3 were obtained from Cell Signaling Technology.
MitoTracker Red CMTMRos and donkey antirabbit IgG (H
+L) highly cross-adsorbed secondary antibody with Alexa fluor
488 were purchased from ThermoFisher Scientific. Antibody
dilution buffers and blocking buffer were purchased from
Beyotime Biotechnology.
Herein, we developed a bioorthogonal chemical signature
that enables amplified visualization of cellular 5hmU and 5fU
(Scheme 1). First, we have synthesized an ATP analogue of γ-
Chemical Preparation of ATP-γ-Alkyne. ATP and
propargylamine were used to prepare ATP-γ-alkyne. ATP
disodium salt (4.9 mM) and propargylamine (245 mM) were
dissolved in water.35−37 The solution pH was adjusted to 6.0
with dilute HCl. Also, then EDC-HCl (490 mM) was added to
the above solution and the mixture was stirred for 24 h at room
temperature. Then, the pH of the solution was quickly adjusted
to 8.5. After evaporating, silica column chromatography was
used to purify the light yellow viscous products and obtain the
powder product. ATP-γ-alkyne: high resolution mass spec-
trometry (HRMS) (ESI, negative mode) for C13H18N6O12P3,
[M + H]−: 543.02010 (calculated), 543.01591 (found). All of
the mass spectrometry analyses in this work were performed
on a Waters I-Class Vion IMS Qtof with electrospray
ionization. Nuclear magnetic resonance (NMR) spectra were
recorded on a Brucker AVANCE III HD 600 MHz machine.
The deuterated solvent in this work was D2O. 31P NMR
spectra are referenced to H3PO4 as an external standard.
Tetramethylsilane (TMS) was used as an external standard for
13C NMR spectra.
Scheme 1. Schematic Diagrams for a Bioorthogonal
Chemical Signature Enabling Amplified Visualization of
Cellular Oxidative Thymines
5fU Reduced to 5hmU. Freshly prepared 3.5 μg/mL
NaBH4 solutions were slowly added to 10 μg/mL 5fU
nucleobases solution. The mixture reaction was shaken at
room temperature for 1 h in the dark. To stop the reaction,
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750 mM sodium acetate (pH 5) was added the mixture until
no gas was released. The efficiency of 5fU reduction was
characterized by UV−vis absorption spectra.
mitochondria at 37 °C for 20 min before cell fixation. The
G1, S, and G2 phases of cells were synchronized with
lovastatin, hydroxylurea, and colchicine, respectively. For
immunostaining of histone, the cells were first blocked for 1
h in QuickBlock blocking buffer at room temperature. Then,
the cells were incubated with primary antibody H3K4me1
(1:800), H3K4me3 (1:400), H3K27me3 (1:1600), and
γH2AX (1:400) diluted by QuickBlock primary antibody
dilution buffer overnight at 4 °C. After washing, the fluorescent
secondary antibody was added to image the histones at room
temperature for 1 h.
All fluorescence images were acquired using laser scanning
confocal microscopy (TCS SP8 STED 3X, Leica). The cell
images in this work were captured using a 63× water objective
(NA 1.2). We randomly selected the cells to analyze. We used
Matlab 2019b to extract the spot count and fluorescence
intensity of single cells.
Reactions of 5hmU Phosphorylation by 5-HMUDK.
The double-stranded DNA substrates containing a single
5hmU:A site were synthesized by polymerization extension of
a primer-template duplex. To verify the feasibility of 5hmU
phosphorylation by 5-HMUDK, 4 μL of double-stranded DNA
substrate (50 μM), 1 μL of 5-HMUDK (20 U/μL), and 1 μL
of ATP-γ-alkyne (10 mM) was incubated in 20 μL of 1×
Cutsmart buffer for 1 h at 37 °C. NcoI-HF (0.5 μL; 20 U/μL)
was used to cut the phosphorylation products in 20 μL of 1×
Cutsmart buffer for 1 h at 37 °C. The efficiency of 5hmU
phosphorylation was characterized by melting curve analysis.
Synthesis of Circularized Padlocks. Hybridization,
ligation, and digestion are the main reaction processes in the
preparation of circularized padlocks. First, the hybridization
was operated in T4 DNA ligase reaction buffer (10 μL), which
contained 2 μL of padlock probes (50 μM) and 3 μL of
ligation linker (100 μM) for 2 h at 55 °C. Second, added 700
U T4 DNA ligase to catalyze the ligation of 3′ hydroxyl and 5′
phosphate termini of padlock probes. After reacting for 2 h at
37 °C, the mixture was inactivated at 65 °C for 40 min. Finally,
excess exonuclease was added to digest all of the linear DNA
overnight at 37 °C. Next, 80 °C for 40 min to inactivate all of
the enzymes.
RESULTS AND DISCUSSION
■
First, ATP-γ-alkyne was synthesized by a coupling reaction of
ATP with propargylamine using EDC-HCl as the activator in
water at pH 6.0. The purified reaction mixtures were analyzed
by mass spectrometry (MS). As shown in Figure 1A, the MS
spectrum peak of ATP-γ-alkyne in the negative-ion mode was
543.01591, which was consistent with its theoretical molecular
Visualizing Intracellular 5hmU and 5fU. Breast cell lines
MCF-10A, MCF-7, and MDA-MB-231 cells were cultured in
DEME with 1% antibiotic penicillin−streptomycin (100 U/
mL) and 10% FBS at 37 °C.
Then, 6000 cells were inoculated overnight on collagen-A-
coated cover glass in the poly(dimethylsiloxane) (PDMS)
chamber at 37 °C. Then, the cells were fixed with 4%
formaldehyde at room temperature for 10 min. To permeate
the cells, the cells were cultured with 0.5% Triton X-100 in
phosphate-buffered saline (PBS) for 5 min. After washing the
cells three times with PBST, we first labeled it as intercellular
5hmU. For a typical 5hmU phosphorylation reaction, the cells
were incubated with 1 μL of 5-HMUDK (20 U/μL) and 500
μM ATP-γ-alkyne in 1× Cutsmart buffer at 37 °C for 2 h.
Then, the mixed solution containing 250 nM 5hmU barcode
probe, 1 mM copper(II) sulfate, 100 mM sodium ascorbate,
and 0.5 μg/mL sperm DNA was added to perform a
copper(I)-catalyzed click reaction at room temperature for 1
h in dark. Excess probes and molecules were carefully washed
three times. Next, 5fU was reduced to 5hmU with 1 mg/mL
NaBH4 for 5 min at room temperature in dark. After washing
several times until no bubbles, the newly generated 5hmU was
labeled with a 5fU barcode probe by the phosphorylation and
click reaction mentioned above.
For the RCA reaction, two circularized padlocks (200 nM
each), 0.5 μg/mL sperm DNA, 20% formamide, and 2× SSC
formed a ring template hybrid reaction system. Then, the
hybridization mixture was reacted with the fixed cells at 37 °C
for 3 h. Next, barcoding of RCA reaction was performed in 10
μL of 1× phi29 DNA polymerase buffer containing 0.5 μL of
phi29 DNA polymerase (10 U/μL), 0.5 μL of BSA (10 mg/
mL), and 2.5 mM dNTPs at 37 °C for 2 h. After the cells were
washed three times, fluorophore-labeled DNA probes (200 nM
each) in 2× SSC buffer with 20% formamide were incubated
with the samples at 37 °C for 30 min. 4′,6-Diamidino-2-
phenylindole (DAPI) is used to stain the nucleus. In this work,
MitoTracker Red CMTMRos was used to stain the
Figure 1. Synthesis and characterization of ATP-γ-alkyne. (A)
Chemical structure and mass spectrum of ATP-γ-alkyne
(C13H18N6O12P3, [M + H]−, calculated: 543.02010, found:
543.01591). (B) 1H NMR spectrum of ATP-γ-alkyne. 1H NMR
(600 MHz,D2O): δ (ppm) = 8.50 (s, 1H, 2H), 8.17 (s, 1H, H8), 6.09
(d, J = 6.09 Hz, 1H, H1′), 4.80 (m, 1H, overlapping with HOD, H2′),
4.61−4.54 (m, 1H, H3′), 4.38 (m, 1H, H4′), 4.24 (m, 2H, H5′),
2.79−2.65 (m, 3H, H12, H14). (C) Melting temperature of model
dsDNA samples cut by restricting endonuclease NcoI-HF.
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weight. The coupling reaction with complete selectivity at the
γ-phosphate has been proved by 1H, 13C, and 31P NMR
spectroscopy (Figures 1B, S1, and S2).
Next, we studied whether 5-HMUDK used our synthesized
bioorthogonal ATP analogue to alkynephosphorylate 5hmU
site in double-stranded DNA. This double-stranded DNA
containing different modification sites and restriction endonu-
clease NcoI-HF recognition sequences was prepared as 5-
HMUDK substrates. DNA melting temperatures were used to
detect double-stranded DNA breaks; all of the double-stranded
DNA substrates were treated with ATP-γ-alkyne. As shown in
Figure 1C, only the 5hmU:A sample blocked DNA break after
the NcoI-HF excision reaction. This is because the phosphor-
ylation of 5hmU protects it from being cleaved by NcoI-HF
restriction endonuclease. Thus, ATP-γ-alkyne was proven to be
an efficient alkynephosphate donor for 5-HMUDK to
specifically mark 5hmU.
To visualize intracellular 5hmU and obtain the spatial
distribution information in single cells, we next developed a
bioorthogonal chemical-signature-encoded DNA amplification
strategy. Specifically, the resulting ATP-γ-alkyne with bio-
orthogonal tag was used to label intracellular 5hmU. To
introduce DNA amplification, 5′ azide (N3)-modified DNA
primer (5hmU-primer) reacted with the generated alkyne-
5pmU. This primer could trigger RCA reaction and produce a
tandem periodic long single DNA strand, which can capture
many molecules of fluorescence probes for signal amplification.
As shown in Figure 2A, the spot number was used to roughly
assess the number of 5hmU sites in the cells. Greater spot
count and fluorescence signal were observed in these RCA-
treated single cells compared to no RCA, which made it
possible to visualize low-abundance modified bases. Notably,
intracellular 5hmU count is single-cell heterogeneous. To
verify the specificity of our method, we performed a series of
negative experiments. In detail, we processed hSMUG1 prior
to the alkynphosphorylation reaction to prepare the 5hmU
defective sample. The other negative experiment had no 5-
HMUDK. From the cell imaging results, negligible adsorption
background fluorescence or nonspecific labeling was observed
in a series of negative samples. These results proved the
cellular genomic 5hmU-specific amplified visualization in
single-cell samples by our method. As shown in Figure 2B,
primer-probe was subsequently added. No RCA fluorescence
signal of 5fU-primer-probe was observed. This indicated that
all 5hmU modification sites were cross-linked with 5hmU-
primer after the click reaction, and there were no residual sites
to interfere with subsequent 5fU labeling. Further, the
reduction of 5fU to 5hmU was investigated. In the previous
work, the water-soluble reducing agent NaBH4 was used to
reduce 5fC in genomic DNA to 5hmC.38 NaBH4 also can
reduce 5fU to 5hmU (Figure 2C). From the above results, we
can detect 5fU by 5hmU labeling method catalyzed by 5-
HMUDK after NaBH4 reduction. These results may enable the
joint analysis of 5fU and 5hmU in the same single cells
following a proper order.
Figure 2. Bioorthogonal labeling and imaging of 5hmU in cells. (A)
Cellular genomic 5hmU-specific amplified visualization using 5-
HMUDK and ATP-γ-alkyne. Left: representative cell images (merged
picture: red, 5hmU; gray, DAPI) of single cells with different
conditions. Right: the corresponding spot count and fluorescence
intensity analysis. The cell numbers are 50. (B) Results of all
intracellular 5hmU sites were cross-linked with DNA probes (red
spot), and no residual sites were detected (green spot). (C)
Absorption spectra for 5fU reduction characterization. Green line:
absorption spectrum of 5fU; red line: absorption spectrum of
reduction product 5hmU. MCF-10A cells were used. Scale bars of
cell images are 10 μm in this work.
mentioned above. To simultaneously visualize the low
abundance of 5hmU and 5fU in single cells, we performed
RCA reaction and hybridization of fluorescent probes. Figure
3B shows representative merged images for the spatial
distributions of 5hmU and 5fU in different human breast cell
lines, including nontumorigenic MCF-10A and carcinogenic
MCF-7 and MDA-MB-231. In addition, the spot count and
fluorescence intensity of individual cells in different cell lines
were statistically analyzed (Figures 3C, S3, and S4). The
results showed that, despite significant single-cell hetero-
geneity, the mean spots/cells and fluorescence intensity/cells
of 5hmU and 5fU in MCF-7 were significantly higher than
those of MCF-10A and MDA-MB-231. Subsequently, we
investigated the colocalization of 5hmU and 5fU. The violin
chart shows Pearson’s correlation coefficient between the
5hmU signal and the 5fU signal of each cell in different cell
types (Figure 3D). The high colocalization values for these two
modifications in different cancer cell lines may imply their
potential correlation in the cells. Especially, 5fU may be
derived from 5hmU in mammalians. Overall, all these
observations revealed that the spatial distributions and the
correlation of 5hmU and 5fU in DNA differed between three
breast cell lines.
Next, we would visualize intracellular 5hmU and 5fU via the
bioorthogonal chemical-signature-encoded DNA amplification
method. Specifically, ATP-γ-alkyne was first used to label
intracellular 5hmU in single cells. The resulting alkyne-5pmU
in DNA were further clicked with 5hmU-primer. Next,
intracellular 5fU was reduced to 5hmU by NaBH4. The
newly generated 5hmU was labeled with 5fU barcode primer
(5fU-primer) by the phosphorylation and click reaction
Then, we profiled the changes of 5hmU and 5fU during cell
cycles. MDA-MB-231 cells were synchronized in G1, S, and
G2 phases, respectively. Figure 4A shows the representative
merged images of the cells. We performed a statistical analysis
of 5hmU and 5fU spot counts and signal intensities in single
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chemical modifications on the DNA in mitochondria, we
performed a colocalization analysis between 5hmU/5fU signal
and MitoTracker signal. The representative cell images of
different cell lines (MCF-10A, MCF-7, MDA-MB-231) are
shown in Figure 5A. The violin plot represents the correlation
Figure 3. Conjoint analysis of 5hmU and 5fU in three breast cell lines.
(A) Mechanism of cellular genomic 5hmU- and 5fU-amplified
visualization. (B) Representative merged cell images of three breast
cell lines (merged: gray, DAPI; red, 5hmU; green, 5fU). (C)
Statistical analysis of changes in mean 5hmU and 5fU spot counts of a
single cell in three breast cell lines. The cell numbers are 50. (D)
Pearson’s correlation coefficients between 5hmU signal and 5fU signal
in different cell types.
Figure 5. Multilevel subcellular distribution analysis of 5hmU and
5fU. (A) Representative cell images of 5hmU and 5fU labeling with
MitoTracker imaging to study mitochondrial oxidative thymines
(merged: gray, DAPI; red, 5hmU; green, 5fU; purple: mitochondria,
abbreviation as Mito). (B) Pearson’s correlation coefficients between
5hmU/5fU signal and MitoTracker signal in different cell types. The
cell numbers are 50. (C) Representative cell images of 5hmU and 5fU
labeling with four histone modifications imaging (merged: gray,
DAPI; red, 5hmU; green, 5fU; blue: histone modification). (D)
Pearson’s correlation coefficients between 5hmU/5fU signal and
histone modifications. The cell numbers are 50.
Figure 4. Exploring changes of single-cell 5hmU and 5fU during cell
cycles. (A) Representative single-cell images for G1, S, and G2 phases
of the cell cycle (merged: gray, DAPI; red, 5hmU; green, 5fU). (B)
Statistical analysis of spot count for single-cell 5hmU and 5fU during
cell cycles. The cell numbers are 50. (C) Statistical analysis of
fluorescence intensity for single-cell 5hmU and 5fU during cell cycles.
The cell numbers are 50. MDA-MB-231 cells were used here.
coefficients between 5hmU/5fU signal and MitoTracker signal
in different cell types. As shown in Figure 5B, the correlation
coefficient between 5hmU/5fU and mitochondria in carcino-
genic MCF-7 is higher than that of MCF-10A and MDA-MB-
231, especially 5fU. Thus, we also investigated the spatial
relationship between two oxidative thymines and histone
modifications such as H3K4me1, H3K4me3, H3K27me3, or
γH2AX. The representative cell images are shown in Figure
5C. We observed high fluorescence colocalization rates for
5hmU/H3K4me1, 5hmU/H3K4me3, 5hmU/H3K27me3,
5fU/H3K4me1, 5fU/H3K4me3, and 5fU/H3K27me3 despite
remarkable single-cell heterogeneity (Figure 5D). These results
indicate the potential relationship between these three histone
modifications and DNA methylation.
cells during cell cycles (Figure 4B,C). Judging from the spot
counts, as the cell cycle changes, 5hmU and 5fU do not show a
significant pattern. However, it could be seen from the signal
intensities that the intensities of 5hmU and 5fU in the G1
phase were lower than those in the S and G2 phases. This may
be related to the process of DNA or chromatin replication.
As we all know, in addition to DNA in the nucleus,
mitochondria also contain DNA. To verify whether there are
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Life Science and Technology, Xi’an Jiaotong University, Xi’an
710049 Shaanxi, P. R. China
Yue Zhao − Institute of Analytical Chemistry and Instrument
for Life Science, The Key Laboratory of Biomedical
Information Engineering of Ministry of Education, School of
Life Science and Technology, Xi’an Jiaotong University, Xi’an
710049 Shaanxi, P. R. China
CONCLUSIONS
■
In summary, we developed a bioorthogonal chemical-
signature-encoded DNA amplification method for visualization
of cellular oxidative thymines. To accomplish this, we have
synthesized a new ATP analogue with a bioorthogonal tag
modified on γ-phosphate, ATP-γ-alkyne. It was able to use as
an efficient alkynephosphate donor for 5-HMUDK to
specifically mark cellular 5hmU. Combined with NaBH4
reduction and RCA, conjoint analysis of 5hmU and 5fU in
the same single cells was realized following a proper labeling
order. Next, we revealed the cell-type-specific characteristics of
5hmU and 5fU with single-cell variants. We also found their
differences in multilevel subcellular information and dynamic
changes during cell cycles. The combination of this
bioorthogonal chemoenzymatic method can quantify and
precisely map these two oxidative thymines in the genome
and help providing insights into their function.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.1c01285
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
ASSOCIATED CONTENT
This research was supported by the China Postdoctoral
Science Foundation (Grant Numbers 2020M683448 and
2019M663658), the National Natural Science Foundation of
China (Grant Numbers 92068118 and 21874105), the Natural
Science Basic Research Program of Shaanxi (Grant Number
2020JQ-021), the Fundamental Research Funds for the
Central Universities, and “Young Talent Support Plan” of
Xi’an Jiaotong University. The authors thank Hao at the
Instrument Analysis Center of Xi’an Jiaotong University for her
assistance with fluorescence imaging analysis.
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.1c01285.
Table S1: sequence information for oligonucleotides
used in this work; Figure S1: 13C NMR spectra of ATP-
γ-alkyne; Figure S2: 31P NMR spectra of ATP-γ-alkyne;
Figure S3: statistical analysis of single-cell spot count of
5hmU and 5fU in different cell lines; Figure S4:
statistical analysis of single-cell fluorescence intensity
of 5hmU and 5fU in different cell lines; and
supplementary figures and data (PDF)
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