Aqueous Wittig reaction-mediated fast fluorogenic identification and single-base resolution analysis of 5-formylcytosine in DNA

Article abstract of DOI:10.1039/d0cc04950f

A highly reactive ylide^tBuAwas introduced, which could react rapidly with the 5-formyl and 4-amino groups of 5-formylcytosine (5fC) under mild conditions without any co-solvent or catalyst in a manner of Wittig olefination and intramolecular nucleophilic substitution to yield a cyclized fluorescent adduct, which meets the demands of both single-base resolution sequencing and fluorescence switch-on detection of 5fC in DNA.

Full text of DOI:10.1039/d0cc04950f

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Aqueous Wittig Reaction-mediated Fast Fluorogenic Identification
and Single-base Resolution Analysis of 5-Formylcytosine in DNA
Qian Zhou,a Kun Li,*a Kang-Kang Yu,a Na Li,a Lei Shi,a Hao Chen,b Shan-Yong Chena and Xiao-Qi Yu*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A highly reactive ylide BuA was introduced, which could react Höbartner et al. reported trimethylindole derivatives5 to react
rapidly with the 5-formyl and 4-amino groups of 5-formylcytosine with 5fC based on Knoevenagel condensation, enabling
(5fC) under mild conditions without any co-solvent or catalyst in a quantification of 5fC and making up for the instability of C=N.
manner of Wittig olefination and intramolecular nucleophilic Nevertheless, the above two kinds of reagents have poor
substitution to yield a cyclized fluorescent adduct, which meets the selectivity for 5fC, because they fail to rule out the interference
demands of both single-base resolution sequencing and from 5-formyluracil (5fU),6 a major oxidative lesion of thymine
t
fluorescence switch-on detection of 5fC in DNA.
(T) in DNA. Furthermore, such fluorescence-based assays only
realize qualitative and quantitative analysis of 5fC at globe-wide
level, while the specific location of 5fC in the genome remains
unknown.
5fC, now considered to be the 7th base of the genetic DNA, is widely
present in mammalian cells, tissues and all major organs at a level of
0.02% to 0.002% of cytosines. As one of sequential oxidation
products of 5-methylcytosine (5mC) catalysed by TET dioxygenases,
5fC can return to canonical cytosine (C) through thymine DNA
glycosylase (TDG)-assisted base excision repair (BER) or direct
deformylation. 1 Therefore, one proposed role of 5fC is to serve as an
important intermediate in active DNA demethylation. More recently,
5fC was thought to be a semi-permanent DNA modification as TDG
only removes 50% 5fC within specific genomic regions.2 5fC increases
DNA flexibility, alters the structure of DNA double helix, and forms
imine-cross-links with lysine residues of histone proteins, whereby
affects nucleosomes organization, chromatin remodelling and
transcription regulation.3 All above suggest 5fC has independent
epigenetic functions in DNA. However, due to the limited abundance
of 5fC and the lack of sensitive and selective detection methods, we
still know very little about this derivative.
Chemistry-based methodological innovations have
significantly advanced this field (Scheme 1). Chemosensors
bearing a formyl-binding site to covalently attach 5fC have
received a lot of attention and become one of the most
powerful tools for 5fC quantification. Hydrazine, amine and
hydroxylamine derivatives4 were firstly developed to
fluorogenic labelling 5fC via a Schiff base-type reaction
mechanism. However, these reagents are inherently unstable
and the newly formed C=N is prone to hydrolysis over time.
To achieve single-base resolution sequencing of 5fC, which is
critical for studying its regulatory functions, He and Balasubramanian
et al. developed fCAB-seq7 and redBS-seq,8 relying on either EtONH2-
based protection of 5fC or NaBH4-based reduction of 5fC to 5hmC,
respectively. Then, inspired by Friedländer synthesis, Yi and co-
workers invented fC-CET with 1,3-indandione, although its poor
solubility is worrying.9 Most recently, using malononitrile, a modified
version termed “CLEVER-seq” was reported.10 Next, Zhou et al.
presented another cyano reagent that can also locate 5fC in DNA.11
However, the above three “-CH2-” reagents suffer from poor
reactivity to -CHO as they take a long time (10-24 h) to completely
modify 5fC. In addition, up to date, only amino, indole, and active -
CH2- derivatives were applied to label 5fC-DNA. Hence, developing
new kinds of aldehyde-reagents and methods to selectively and
rapidly analyze 5fC both at global-wide level and single-base
resolution are highly needed.
The Wittig reaction is a principal methodology for carbon-
carbon double bond construction. Not long ago, we have developed
a Wittig-initiated triple domino reaction for fluorescence detection
of 5fC using PPh3=CN.12 However, PPh3=CN has many defects when
labelling 5fC. First, it is so sensitive to water that the derivatization
must be performed in organic solvent; Second, the generated
nucleoside adduct only emits strong fluorescence in water, thus a
solvent replacement step is necessary before sample test; Third, the
introduction of photocatalysis increases the complexity of operation
and the high-energy UV causes additional oxidative damage; Forth,
without enzymatic hydrolysis, 5fC in DNA cannot be detected; even
worse, it is no longer possible to obtain the sequence information
sequence information of 5fC. To circumvent these limitations, we
expanded our scope of Wittig reagents screening. One stable and
a. Key Laboratory of Green Chemistry and Technology (Ministry of Education),
College of Chemistry, Sichuan University, Chengdu 610064, P. R. China.
E-mail: kli@scu.edu.cn, xqyu@scu.edu.cn
b. Department of Pharmaceutical Sciences, College of Pharmacy, University of
Tennessee Health Science Centre Memphis, Tennessee 38163, the United States
† Electronic Supplementary Information (ESI) available: [details procedures, DFT
calculation and spectra]. See DOI: 10.1039/x0xx00000x
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Journal Name
NH2
N
a) Fluorescence "Turn-On" Detection of 5fC
View Article Online
DOI: 10.1039/D0CC04950F
NH2
O
N
N
PPh3
CH3OH, 60°C, 1 h
NC
O
N
O
N
HO
HO
280 nm, 1.5 h
O
O
OH
5fC Nucleoside
OH
b) Fluorescence "Turn-On" Detection of 5fC and Single-Base Sequencing of 5fC
O
O
Br
PPh3
O
NH2
HN
O
N
N
0.1 M NaHCO3, 37°C, 0.5 h
O
N
O
N
5fC-DNA
Scheme 1. (a) Previous reported strategy for fluorescence detection of 5fC nucleoside;
(b) Aqueous Wittig reaction under milder conditions for both single-base resolution
sequencing and fluorescence switch-on detection of 5fC-DNA.
t
Figure 1. (a) Chemical structures of phosphonium salts and Wittig reagents; (b) HPLC-UV
chromatograms of 5fC after incubation with these compounds for 1.5 h at 37C.
commercially available phosphonium salts, BuA+, appeared very
promising (Scheme 1). First, tBuA+ can be facilely dissolved in water
at a concentration of tens of millimoles per liter without any
cosolvent, which makes our method environment-friendly and
biocompatible. Second, BuA+ exclusively reacts with 5fC bearing a
characteristic 2-aminobenzaldehyde structure, while 5fU and abasic
sites (AP) do not interfere. Third, the Wittig-involved reaction can be
t
t
NaHCO3 to generate ylide BuA, then ylide BuA reacts with 5-CHO
of 5fC to form a carbon-carbon double bond, and finally, the ester
bond undergoes intramolecular nucleophilic substitution with the 4-
NH2 of 5fC to form a lactam ring. Density Functional Theory (DFT)
t
carried out at room temperature and the reaction time can be calculations at B3LYP/6-31++G(d,p) level provide additional
information on geometries and relative energies of (E)-5fC-tBuA, (Z)-
5fC-tBuA and cycle-C to support the proposed mechanism (Figure 2).
The energy level diagram reveals E-Z isomerization is an endergonic
process that can be achieved by daylighting or heating. Compared to
(E)-isomer, 4-NH2 is closer to the carbonyl group in (Z)-isomer, which
is advantageous for nucleophilic attack. The cyclization reaction is an
exergonic process by 29.0 kcal/mol indicating the formation of cycle-
C is energetically favourable. Promisingly, such a chemical cascade
reaction involving aqueous Wittig reaction could be developed as a
biorthogonal approach for chemoselective labelling of 5fC after
further condition optimization.
shorten to 0.5 h. Forth, the labeling reaction causes no observable
DNA degradation, which is desirable for analysis small amount of
sample DNA with high recovery. Finally, the generate 5-formyl-2’-
deoxycytidine-tBuA adduct contains a highly fluorescent scaffold
enabling quantification the overall levels of 5fC. Notably, this
‘‘covalent assembly’’ strategy that utilizes a substrate triggered
chemical cascade reaction to construct fluorophores provides “turn-
on” fluorescence with zero background and hence superior detection
t
sensitivity. Moreover, BuA+-mediated cyclization will protect 5fC
from deamination during bisulfite treatment and make it possible to
profile 5fC landscape at single base resolution.
From our previous research, ester-stabilized ylides have
relatively mild reactivity which may enable it to be stably present in
water and achieve 5fC labeling in DNA. Therefore, compound MA, EA,
tBuA and MEA were first investigated and compared, where different
substituents on both sides of the ester bond can result in differences
in reactivity and stability (Figure 1). Wittig derivatization of 5fC was
carried out with 100 eq. phosphoranes in pure water at 37C for 1.5
h. However, there were no new HPLC signals other than 5fC (T=3.63
min), indicating these ylides are almost incapable of labeling 5fC in
water. The resulting aqueous suspension suggested the possible
reason is their poor solubility. Given that phosphonium salts are
more water-soluble than their phosphorane counterparts and the
“one-pot” Wittig reactions between phosphonium salts and
aldehydes in the presence of bases proceed smoothly, we hence
reperformed our Wittig transformation in aq. NaHCO3 using
To achieve the best derivatization efficiency of 5fC, we
comprehensively screened the pH, molar ratio of tBuA+/5fC, reaction
temperature and reaction time. As shown in Figure S2a, 0.1 M
t
NaHCO3 is necessary to alkalize BuA+ to tBuA to ensure successful
Wittig olefination. Lowering the concentration of NaHCO3 or using
t
lower pH buffers doesn’t work. 60 eq. BuA+ is sufficient for fast
conversion of 5fC (Figure S2b). Notably, >99% conversion can be
achieved either the reaction time is shortened to 0.5 h (Figure S2c)
or the temperature is lowered to room temperature (Figure S2d).
This organic solvent-free, catalyst-free and by-product-free labelling
reaction is environmentally friendly, efficient and easy to operate,
showing great potential in 5fC analysis.
t
cycle-C, the reaction product of BuA+ with 5fC, emits strong
fluorescence at 393 nm upon excitation at 345 nm (Figure S3). Such
nucleoside-derived fluorophore possesses good water solubility,
extremely high quantum yield as well as nearly constant emission
intensity within the pH range of 6.5-8.5 (Figure S4). Based on the
above findings, we subsequently explored the feasibility of tBuA+ in
fluorescence-based turn-on detection of 5fC. The selectivity of tBuA+
t
compound MA+, EA+, BuA+ and MEA+, where ylides are prepared
t
in situ before reacting with 5fC. As expected, MA+, EA+, BuA+ can
be readily dissolved into water by vortexing, while MEA+ is still
partially suspended. More importantly, after 1.5 h incubation at 37C,
5fC was completely converted to its Wittig-product by tBuA+ without
consecutive steps: deprotonation of phosphonium salt tBuA+ with
2 | J. Name., 2012, 00, 1-3
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Figure 2. (a) Proposed formation mechanism of cycle-C; (b) The ground state geometries
and relative energies levels of (E)-5fC-tBuA, (Z)-5fC-tBuA and cycle-C optimized at
B3LYP/6-31++G(d,p) level of theory.
Figure 3. Quantitative and qualitative detection of 5fC. (a) Fluorescence spectra and (b)
emission intensity of tBuA+ after incubation with 5fC and other interfering species,
including A, G, C, T, U, 5mC, 5hmC, 5hmU, and 5fU; (c) Fluorescence spectra of ODNs that
contain 5fC, 5fU, AP or only canonical nucleobases after treatment with tBuA+; (d)
Fluorescence titration spectra and (e) linear relationship between emission intensity and
ODN-5fC concentration; (f) Quantification of 5fC in γ-irradiated ctDNA at different
irradiation doses.
for 5fC was first evaluated compared to five canonical
deoxynucleosides and their modifications under optimized
conditions. As shown in Figure 3a and 3b, only 5fC induced a
remarkable fluorescence enhancement at 393 nm; other potentially
interfering species, even 5fU, have little effect on the spectrum of
which is comparable to the values reported by Wagner et al. (0.45
5fC/106 bases/Gy). The digestion-free and purification-free
quantification of 5fC in γ-irradiated ctDNA at different irradiation
doses implied tBuA+ and our self-developed fluorescence “switch-on”
sensing method are highly reliable and practical.
t
tBuA+, indicating the superior specificity of BuA+ toward 5fC. This
result again verified that nucleosides without 2-aminobenzaldehyde
structure are incapable of reacting with tBuA+ to form highly
fluorescent products via intramolecular cyclization, and thus don’t
affect the qualitative and quantitative detection of 5fC at all. Then,
we treated a 15-mer oligonucleotide containing one 5fC site (ODN-
5fC) with tBuA+. Successful labeling of ODN-5fC was identified by ESI-
MS (Figure S5). Negative controls, including ODN-5fU, ODN-C, ODN-
AP (where the 5fC site is replaced by 5fU, C and abasic site,
Besides accurate determination the global levels of 5fC,
genome-wide profiling of 5fC is another indispensable method to
unravel its complex epigenetic mechanisms. Antibody-based
immunoprecipitation and chemical-assisted biotin pulldown with
aldehyde reactive probes (ARPs), such as o-(biotinylcarbazoylmethyl)
hydroxylamine, have been used to isolate and enrich 5fC-containing
DNA fragments in the genome followed by sequencing, but such
affinity-based approaches have limited resolution (100-400 bases). 14
In addition, the poor specificity of antibodies and ARPs for targeted
modifications always leads to false positive signals.15 Currently, the
gold standard established for detecting C modifications at single-
base resolution is bisulfite sequencing (BS-seq), where unmodified C,
5fC and 5caC, are efficiently converted to uracil (U) by sodium
bisulfite-mediated deamination and read as thymine (T) in
subsequent sequencing, while 5mC and 5hmC are resistant to this
chemical conversion and therefore still be read as C. That is, this
method does not distinguish 5fC from C and 5caC or 5mC from 5hmC.
Additional chemical derivatization steps before sodium bisulfite
treatment can be used to overcome these limitations. The most
recent breakthrough in this field came with several new methods
(Table S1), allowing selectively positional measurement of the four C
oxidations, respectively. Considering our cyclization-enabled
derivatization will protect 5fC against NaHSO3-induced deamination,
we set out to explore the potential of tBuA+ in mapping 5fC in DNA
t
respectively) were used to confirm the specificity of BuA+ through
direct fluorescence readouts (Figure 3c). Besides, a satisfactory linear
correlation (R2=0.9938) between the fluorescence intensity and
ODN-5fC concentration ranging from 0-500 nM was obtained (Figure
3d and 3e). The detection limit (3δ/slope) was calculated to be 2.3
nM, which is 15 times lower than that of our previously reported
PPh3=CN. Totally, all these experimental results suggest tBuA+ is very
promising for site-specific labelling and rapid quantification of 5fC
under mild conditions.
It was reported that 5mC in DNA fragments exposed to ionizing
radiation would be oxidize to 5fC.13 In order to apply our method to
more complex biological samples to determine the overall 5fC
abundance, we prepared γ-irradiated calf thymus DNA (ctDNA,
percentage of 5mC to C is 6.4%). Irradiations of ctDNA (1 mg/mL, pre-
dissolved in an oxygenated aqueous solution) were carried out with
a 60Co γ-source (16.7 Gy/min). After treatment with tBuA+, the yield
of 5fC, according to the linear fit equation of the calibration curve
(Figure 3e). Then, the formation rate of 5fC was calculated to be 0.29
5fC/106 bases/Gy from the slope of the graph shown in Figure 3f,
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M. Su, A. Kirchner, S. Stazzoni, M. Mueller, M. Wagner, A.
at single base resolution. A 76-mer dsDNA containing one 5fC site
was labelled with tBuA+ without noticeable DNA degradation (Figure
S6), and then sent for EpiTect Bisulfite Kit (QIAGEN) following the
supplier’s instruction. After PCR amplification with EpiMark Hotstart
Taq polymerase (NEB), the PCR product was purified and subjected
to Sanger sequencing. As expected, we obtained a high C signal in the
original 5fC site from the sequencing data (Figure 4), indicating that
tBuA+ treatment indeed effectively prevents 5fC from bisulfite-
mediated deamination. In other words, with the protocol describe
above (Wittig-Seq), 5fC, 5mC and 5hmC will all be identified as C. By
subtracting the C signals (the sum of 5mC and 5hmC) obtained from
traditional BS-Seq, base-resolution location of 5fC can be
determined. Compared to NaBH4 and EtONH2 used in fCAB-seq and
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Figure 4. Single-base resolution analysis of 5fC. (a) Illustration of a strategy for samples
containing 5fC moieties. (b) Readouts of 5fC-residue in traditional BS-Seq and WittigBS-
Seq. (c) Sanger sequencing data comparing 5fC-containing 76-mer DNA with (below) or
without (above) tBuA+ treatment.
In summary, we have developed an efficient derivatization
method for 5fC-containing DNA using phosphonium salt tBuA+ under
mild and biocompatible conditions. This organic solvent-free,
catalyst-free, room temperature-allowable, short time-needed and
high yield labelling reaction has superior selectivity for 5fC against
5fU, AP and other biological interferences. In addition, cycle-C is
capable of resisting NaHSO3-mediated deamination, which allows
analysing 5fC at single base resolution. This fluorescence “turn-on”
detection and single-base sequencing method could have wider
applications in epigenome research.
This work was financially supported by National Natural Science
Foundation of China (Nos. 21572147 and 21877082), and Special
Funds for Prevention and Control of COVID-19 of Sichuan University.
Conflicts of interest
There are no conflicts to declare.
Notes and references
1
J. S. Hardwick, A. N. Lane and T. Brown, Bioessays, 2018,
40,1700199.
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Aqueous Wittig reaction in nucleic acid under mild conditions for fluorogenic identification of
5fC and single-base resolution sequencing were studied.