Fluorogenic Labeling of 5-Formylpyrimidine Nucleotides in DNA and RNA

Article abstract of DOI:10.1002/anie.201508893

5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA. Shining a light on formylpyrimidines: Mild and orthogonal reaction conditions were established for fluorogenic aldol condensation reactions to generate hemicyanine-like chromophores in DNA and RNA for the residue-specific detection of 5-formyluracil and 5-formylcytosine, which are two naturally occurring oxidized analogues of 5-methylated pyrimidine nucleobases.

Full text of DOI:10.1002/anie.201508893

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International Edition: DOI: 10.1002/anie.201508893
German Edition: DOI: 10.1002/ange.201508893
Modified Nucleobases
Fluorogenic Labeling of 5-Formylpyrimidine Nucleotides in DNA and
RNA
Biswajit Samanta, Jan Seikowski, and Claudia Hçbartner*
Abstract: 5-Formylcytosine (5fC) and 5-formyluracil (5fU)
are natural nucleobase modifications that are generated by
oxidative modification of 5-methylcytosine and thymine (or 5-
methyluracil). Herein, we describe chemoselective labeling of
5-formylpyrimidine nucleotides in DNA and RNA by fluoro-
genic aldol-type condensation reactions with 2,3,3-trimethylin-
dole derivatives. Mild and specific reaction conditions were
developed for 5fU and 5fC to produce hemicyanine-like
chromophores with distinct photophysical properties. Residue-
specific detection was established by fluorescence readout as
well as primer-extension assays. The reactions were optimized
on DNA oligonucleotides and were equally suitable for the
modification of 5fU- and 5fC-modified RNA. This direct
labeling approach of 5-formylpyrimidines is expected to help
in elucidating the occurrence, enzymatic transformations, and
functional roles of these epigenetic/epitranscriptomic nucleo-
base modifications in DNA and RNA.
the methyl group of thymine is also susceptible to oxidation,
although less prominently by the action of enzymes, which
have been shown to produce 5-hydroxymethyluracil
(5hmU).[6] Instead, reactive oxygen species (ROS) are
known to yield mutagenic 5-formyluracil (5fU) in DNA.[7]
While 5-methyluracil (5mU) is a known modification of
tRNA and rRNA, the oxidized products 5hmU and 5fU have
not yet been described as natural modifications in RNA.
However, based on recent reports on the enzymatic oxidation
of 5mC in RNA to 5hmC,[8] it can be expected that 5hmU and
5fU in RNA are waiting to be discovered. Synthetic 5fU has
been used in several nucleic acid studies associated with
oxidative damage,[9] viral defense,[10] and affinity labeling.[11]
Aldehyde groups have been the abiding choice for
refashioning nucleic acids with spectroscopic labels or affinity
probes.[12] Amine, hydrazide, and aminoxyl derivatives have
been used to attach fluorescent probes[13] or small molecule
tags such as biotin to 5fC-modified DNA,[3a,4b, 14] and most
recently also to 5fU-modified DNA,[15] for detection, enrich-
ment and sequencing. Formation of benzimidazole, benzox-
azole, or benzothiazol heterocycles upon reaction of 5fU with
1,2-phenylendiamine or o-amino(thio)phenol derivatives
generates fluorescent or spin-labeled nucleosides.[16] The
synthesis of a benzothiazol chromophore was possible on
DNA, but similar reactions were unsuccessful with 5fC.[16a]
Alternative typical organic reactions of aldehydes should
therefore be considered for labeling these biologically
important nucleoside modifications.[17]
In this work, we explored an aldol-type condensation of
5fU and 5fC in synthetic DNA and RNA oligonucleotides
with 2,3,3-trimethylindole derivatives to generate hemicya-
nine (Hcy)-like chromophores (Figure 1). Based on the
inherently distinct electronic properties of cytosine and
uracil nucleobases, we proposed to create two fluorescent
nucleotides with different photophysical characteristics, with
potential for the residue-specific detection of 5-formylpyr-
imidines in DNA and RNA. In addition, new environmentally
sensitive fluorescent nucleotides may be generated that could
serve as probes for nucleic acid folding and ligand or protein
binding.
N
ucleobase modifications in DNA and RNA are associated
with fundamental biological processes and span a diverse
range of chemical functionalities.[1] Modification-specific
chemical reactions are required to elucidate the occurrence
and roles of naturally modified nucleotides.[2] Formyl modi-
fications at the 5-position of pyrimidine nucleobases have
recently become the focus of much interest in the field of
nucleic acid chemical biology. In DNA, 5-formylcytosine
(5fC) is an epigenetic mark that is produced by successive
oxidation of 5-methylcytosine (5mC) by TET-family dioxy-
genases.[3] 5fC is hypothesized to be an intermediate in active
demethylation of 5mC, as well as to possess regulatory roles in
cellular development.[4] In RNA, 5fC is a posttranscriptional
modification in mitochondrial tRNAs that is known to
modulate codon–anticodon interactions, but the exact bio-
synthetic pathway of 5fC installation in RNA remains to be
elucidated.[5] In addition to oxidative modification of 5mC,
[*] Dr. B. Samanta, Prof. Dr. C. Hçbartner
Institute for Organic and Biomolecular Chemistry
Georg-August-University Gçttingen
Tammannstr. 2, 37077 Gçttingen (Germany)
and
The feasibility of the proposed labeling reaction was
established by exploring the condensation of 5-formyl-2’-
deoxyuridine with the sulfonylindole 1a, which yielded the
new hemicyanine nucleoside Hcy1dU (Figure 2 and Figure S1
in the Supporting Information). Absorbance and emission
properties of Hcy1dU were investigated in aqueous buffered
solutions and both showed increasing intensity with increas-
ing pH (Figure 2b). Fluorescence emission maxima in meth-
anol and DMSO were slightly blue-shifted but of comparable
intensity to those measured in buffered aqueous solutions,
Center for Nanoscale Microscopy and Molecular Physiology of the
Brain (CNMPB)
Gçttingen (Germany)
E-mail: claudia.hoebartner@chemie.uni-goettingen.de
Dr. B. Samanta, J. Seikowski, Prof. Dr. C. Hçbartner
Research Group Nucleic Acid Chemistry
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201508893.
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Table 1: 5fU- and 5fC-modified oligonucleotides.
Name
5’-sequence-3’[a]
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
R1
CTCTTGAG(fU)GTTATG
CTCTTGAA(fU)ATTATG
CTCTTGAC(fU)CTTATG
CTCTTGAT(fU)TTTATG
GACTCAA(fU)AGCCGTG
CTCTTGAG(fC)GTTATG
CTCTTGAA(fC)ATTATG
CTCTTGAC(fC)CTTATG
CTCTTGAT(fC)TTTATG
CATAG(fC)GCTCAAGAGAAATCTCGATGGa
CG(fC)GGAGCTCGCTTGTCG
r(GGAAGAGA(fU)GGCGACGG)
r(GGAAGAGA(fC)GGUGAUGG)
R2
[a] Primer binding site is underlined.
Figure 1. a) 5-formyluridine (5fU) and b) 5-formylcytidine (5fC) (oligo)-
nucleotides and their conversion into hemicyanine(Hcy)-modified
oligonucleotides using c) N-ethyl-2,3,3-trimethylindoleninium 5-sulfo-
nate (1) or d) benzo(e)indoleninium reagents 2 and 3.
Figure 2. a) Synthesis of the Hcy1dU nucleoside. b) UV/Vis absorb-
ance spectra of 10 mm Hcy1dU in aqueous solutions. c) Fluorescence
emission spectra of Hcy1dU in various solvents (0.25 mm, excitation at
l=418 nm).
while the emission intensity was considerably reduced in
acetonitrile and in desalted aqueous solution (Figure 2c).
To label the 5fU modification in DNA, analogous
condensation reactions with 1a were examined under various
conditions with 5fU-containing DNA oligonucleotides D1–
D5, which were prepared by solid-phase synthesis[18] (Table 1
and Figure S2). Analyses of labeling reactions by RP-HPLC
(monitored at l = 260 and 445 nm) and ESI-MS revealed
efficient formation of the Hcy1U-labled DNA, showing
approximately 80% conversion after incubation at 458C for
6 h at pH 6.0 (Figure 3a). Analysis of the labeling reaction by
anion exchange HPLC under denaturing conditions (pH 8,
6m urea, 808C) showed only approximately 40% conversion
to new products, of which only a tiny fraction was detectable
Figure 3. a) RP-HPLC trace at l=260 nm (black) and 445 nm (blue) of
5fU-modified DNA (D1) and D1-Hcy1U generated by reaction with 1a
at pH 6.0, 458C, 6 h. b–d) Fluorescence emission spectra of HcyU-
labeled DNAs D1–D5. c=0.25 mm in 10 mm Tris, pH 9.0, 150 mm
NaCl, 258C, excitation at l=445 nm (Hcy1U) anf 465 nm (Hcy2U and
Hcy3U). e) Fluorescence intensity dependence on 5fU content in DNA.
f) Analysis of data shown in (e).
at l = 445 nm (Figure S3). This result can be explained by
reversible addition of urea to the Michael-acceptor moiety of
Hcy1U and retro-aldol reaction. The urea addition product
was identified by ESI-MS after PAGE purification. Upon
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treatment with acetic acid, the desired Hcy1U-labeled DNAs
were restored (Figure S4). For preparative labeling reactions,
excess reagent 1a was removed by precipitation, ultrafiltra-
tion, or size-exclusion chromatography, and the identity of
Hcy1U-labeled DNAs D1–D5 was confirmed by ESI-MS
(Table S2).
In analogy to the labeling of 5fU-DNA, we examined
similar fluorogenic reactions on 5fC-modified oligonucleo-
tides (Table S1 and Figure S2). The optimal labeling con-
ditions developed for 5fU-DNA yielded only approximately
24% conversion of 5fC-DNA (Figure S7). Increasing the pH
to 7.5 at 458C increased the yield of the new product to 65%.
Upon incubation at 708C and pH 7.5, a second product was
observed. Mass spectrometric analysis revealed that the first
product was formed upon aldol addition of 1a, but the
condensation was not completed, that is, water was not yet
eliminated. Based on ESI-MS data, the second product
contained an additional molecule of 1a. We hypothesize
that a partially conjugated cyanine-like structure is formed on
the nucleobase, although the structure of this extensive
modification could not yet be unequivocally established. We
thus focused on optimizing the reaction conditions to produce
the designed Hcy1C-labeled DNA. A comprehensive screen
of pH and buffer conditions with 32P-labeled 5fC-DNA
suggested that the desired product is preferentially formed
at pH 7.5 (Figure S8). To further increase the nucleophilicity
of the indole reagent, 1a was treated with NaOH followed by
neutralization with Dowex-H+ to generate the enamine
compound 1b with an exocyclic double bond (which was
confirmed by NMR spectroscopy). Using 1b at pH 7.5 and
458C, the single addition product was obtained in 80% yield.
Mass spectrometry of the isolated product revealed the non-
dehydrated intermediate, which was further converted into
the condensation product. The dehydration was successfully
mediated by 3% acetic acid at 458C (Figure S9). The desired
product was stable and could be purified by denaturing gel
electrophoresis to homogeneity (Figure 4).
The optimized reaction conditions (i.e., using 1b at pH 7.4
and 458C, followed by acid treatment) were used for
preparative labeling of DNA oligonucleotides D6–D9,
which contained 5fC between different adjacent nucleotides.
The excitation and emission maxima of Hcy1C-labeled DNAs
were significantly blue-shifted (l = 335 and 400–415 nm,
respectively) compared to the analogous Hcy1U-labeled
DNAs, and the emission intensity was reduced. The Hcy1C
label showed modest environmental sensitivity in single-
stranded and double-helical environments (Figure 4c and
Figure S10). In contrast to Hcy1U, no significant dependence
of emission properties on pH (between pH 7 and 9) or
incubation time was observed. The distinct excitation and
emission wavelengths of Hcy1U and Hcy1C allowed discrim-
ination of both modifications present in the same sample
(Figure S11).
The fluorescence emission of Hcy1U-labeled DNA oligo-
nucleotides was investigated and compared to that of the
Hcy1dU nucleoside described above. The excitation and
emission maxima were found to be l = 445 nm and 480 nm,
respectively. The emission intensity of Hcy1U-labeled DNA
was also dependent on the pH of the solution (Figure S5).
Almost 4-fold enhancement was observed upon increasing
the pH from 8.0 to 9.0. Furthermore, the fluorescence
emission intensity was influenced by the sequence context
of the labeled nucleotide (Figure 3b). The lowest emission
intensity was observed for D1, where Hcy1U is embedded
between guanine nucleotides, which are known to partially
quench fluorescence emission by photo-induced electron
transfer. The highest emission intensity was observed for
D4, which contained Hcy1U embedded between thymidine
nucleotides. Interestingly, the fluorescence signal reached its
maximum intensity only very slowly upon incubation at pH 9,
with observed rate constants in the range of 0.05–0.1 minÀ1
(Figure S5). No time-dependent fluorescence increase was
observed for the Hcy1dU mononucleoside (Figure 3c) in
buffered solution at pH 9. The molecular explanation for the
slow fluorescence increase is difficult to define and may
involve a combination of factors related to local electrostatic
or solvent-mediated effects. Covalent adduct formation with
buffer components and degradation of the labeled DNA were
ruled out by HPLC and MS analysis after incubation at pH 9
for several hours.
To tune the fluorescence properties of the labeled DNAs,
analogous condensation reactions were performed with the
extended benzo[e]indoleninium reagents
2 and 3. The
extended p-system resulted in the expected red-shift of the
emission maximum in both cases (Figure 3d). The sulfonyl
group on the aromatic core in Hcy2U lead to significantly
higher fluorescence compared to the sulfonate at the N-alkyl
substituent in Hcy3U. This result indicates that the substitu-
tion pattern substantially alters the brightness of the fluo-
rophore, likely also by influencing stacking interactions with
adjacent nucleotides.
Next, we demonstrated that the new fluorogenic reaction
can be used to obtain quantitative information on the extent
of 5fU present in a DNA sample. Several 5fU nucleotides
were incorporated by primer-extension reactions using
5fdUTP. Upon labeling with 1a, the emission intensity
correlated with the number of 5fU modifications present in
the DNA (Figure S6). To examine the sensitivity for 5fU
detection, we generated a model calibration curve by spiking
known amounts (0.3–5%) of 5fU-modified DNA into sam-
ples of the unmodified DNA analogue, followed by labeling
with 1a. A linear correlation was obtained between fluores-
cence intensity and % 5fU-DNA in the mixture (Figure 3e,f).
The lowest concentration of labeled DNA measured in this
experiment was 25 nm, which corresponds to 0.02% 5fU
nucleotides in the DNA sample.
To further explore the potential of the aldehyde-specific
labeling reaction beyond fluorescence readout, we examined
the site-specific analysis of 5fC DNA in primer-extension
assays. While 5-formyl modifications are usually bypassed by
DNA polymerases,[19] the hemicyanine-nucleosides may act as
a “roadblock” to abort primer extension and enable detection
and quantification of underlying 5fC modifications. While
unlabeled 5fC-DNA caused only minor pausing of Klenow
DNA polymerase, a strong stop was detected when the
labeled Hcy1C-DNA was used as template (Figure 4d and
Figures S12,S13). In a proof-of-principle setup, we prepared
samples with known 5fC content at a particular target
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In conclusion, we have demonstrated efficient labeling of
naturally occurring 5fC and 5fU in nucleic acids by a new
fluorogenic aldol-type condensation reaction under mild and
biocompatible reaction conditions. The inherent properties of
uracil and cytosine nucleobases were exploited for residue-
specific labeling and detection by fluorescence readout.
Interrogation of 5fC levels at a particular target site was
demonstrated by primer-extension stop assays. Besides pro-
viding new insight into the distinct reactivity of 5fU and 5fC,
the new labeling reaction will find direct applications in the
detection and mapping of formyl-modified nucleotides in
biological samples. In future studies, the new fluorescent
labels may be used to monitor 5fC to 5fU deamination, and
could potentially be tuned for metabolic labeling and optical
mapping.
Acknowledgements
This work was supported by the Cluster of Excellence and
DFG Research Center Nanoscale Microscopy and Molecular
Physiology of the Brain (CNMPB) and the DFG Priority
Program Chemical Biology of Native Nucleic Acid Modifi-
cations (SPP1784).
Keywords: DNA · epigenetics · fluorogenic probes ·
modified nucleobases · RNA
Figure 4. a) Labeling Scheme for 5fC-modified DNA. b) Anion-
exchange HPLC traces (monitored at l=260 nm). Inset: PAGE analy-
sis of reaction using 5’-32P-labeled D6. m.w. =molecular weight.
c) Fluorescence emission spectra of Hcy1C-labeled DNAs D6–D9; the
nucleotides flanking the Hcy1C nucleoside are given in parentheses.
5 mm DNA in K-phosphate buffer, pH 7, excitation at l=335 nm.
d) Primer-extension assay with Klenow exo- DNA polymerase. Tem-
plates: i) unmodified DNA; ii) D10-5fC; iii) D10-Hcy1C. Right-hand gel:
increasing 5fC content in the template from 0–100%. Experimental
details are given in the Supporting Information.
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