A chemical labeling of N6-formyl adenosine (f6A) RNA

Article abstract of DOI:10.1016/j.cclet.2021.09.028

N⁶-methyl adenosine (m⁶A) is an eminent epigenetic mark in mRNAs that affects a broad range of biological functions in diverse species. However, the chemically inert methyl group prevents a direct labeling of this modification for subsequent detection and sequencing. Therefore, most current approaches for the labeling of m⁶A still have limitations of relying on the utilization of corresponding methyltransferases, which resulted in the lacking of efficiency. Here we present an approach which selectively alkylated the N⁶-formyl adenosine (f⁶A), the key intermediate during chemical oxidation of m⁶A, with an alkyne functionality that can be further labeled with click reactions. This covalent labeling approach will be able to facilitate in the affinity purification, detection and genome-wide profiling studies.

Full text of DOI:10.1016/j.cclet.2021.09.028

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A chemical labelling of N6-formyl adenosine (f6A) RNA
Li-jun Xie , Cui-Lian Lin , Li Liu , Liang Cheng
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Please cite this article as: Li-jun Xie , Cui-Lian Lin , Li Liu , Liang Cheng ,
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Communication
A chemical labelling of N6-formyl adenosine (f6A) RNA
Li-jun Xiea, Cui-Lian Lina,b, Li Liua, b, Liang Chenga,b
aBeijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Laboratory of Molecular Recognition and Function, CAS Research/Education Center
for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
b University of Chinese Academy of Sciences, Beijing 100049, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
N6-methyl adenosine (m6A) is an eminent epigenetic mark in mRNAs that affects a broad range
of biological functions in diverse species. However, the chemically inert methyl group prevents
a direct labelling of this modification for subsequent detection and sequencing. Therefore, most
current approaches for the labelling of m6A still have limitations of relying on the utilization of
corresponding methyltransferases, which resulted in the lacking of efficiency. Here we present
an approach which selectively alkylated the N6-formyl adenosine (f6A), the key intermediate
during chemical oxidation of m6A, with an alkyne functionality that can be further labelled with
click reactions. This covalent labelling approach will be able to facilitate in the affinity
purification, detection and genome-wide profiling studies.
Available online
Keywords:
RNA epigenetics
Chemical modulation
Labelling
Nucleic acids
Alkylation
N6-Methyl adenosine (m6A) is a widely studied epigenetic
mark that was discovered in the early 1970s in messenger RNAs
(mRNAs) from eukaryotes [1-4]. The methylation process is
catalyzed by a multicomponent methyltransferase complex,
including METTL3, METTL14, WTAP and other “writers” [5, 6].
FTO and ALKBH5 are so-far two identified m6A demethylases
(“erasers”) that can remove m6A methylated groups from RNA,
which makes the epigenetic modification a dynamic reversible
process [7-9]. On the other hand, regulatory proteins (“readers”)
like YTHDF and YTHDC subtypes can bind to the m6A
modification site in RNA and initiate different downstream
effects [10-12]. However, the comprehensive biological functions
of m6A modification are not fully understood at present [13, 14],
largely because of the difficulties of identifying m6A sites. The
N6-methyl substituent does not affect its reverse transcription
during the PCR process. Therefore, traditional m6A RNA
alkyne/azide containing thiols can be exploited to collect those
fragments and then subjected to library construction and
deep-sequencing (Scheme 1A). Similarly, Liu et al. adapted the
enzymatic methylation process by replacing the natural methyl
donor SAM to Se-allyl-L-selenohomocysteine (Ally-SeAM) [21].
Under the promotion of the methyltransferase METTL3, the
original m6A sites will be replaced with N6-allyl adenosines (a6A).
With the chemically functional alkenyl substituent in hand, they
initiated the iodine-catalyzed intramolecular cyclization to
generate the N1,N6-cyclized adenosine (cyc-A) followed by
sequencing (Scheme 1B). In spite of above two methods, further
technology development of a robust, efficient, unbiased approach
for whole-genome methylation profiling of m6A is still highly
desirable. The development of such an approach without using
antibodies or modifying enzymes will aid the general community
in consistent profiling of RNA epigenetic modifications, and in
developing disease-specific diagnoses as well as establishing
biomarkers.
Here we propose a new approach inspired by the discovery
that m6A can be chemically oxidized by the flavin
mononucleotide (FMN) promoted oxidation [22]. The inert C-H
bonds at the N6-methyl sites can be selective activated to generate
hm6A, just like FTO enzyme. On the other hand, hm6A can be
further converted to N6-formyl adenosine (f6A). We and others
have invented chemical-labelling approaches to selectively label
C5-formyl cytidines (f5C/5fC) with functional groups, such as
amines, for robust affinity enrichment, determination and
sequencing [23-27]. We envisioned that such a chemical labelling
strategy could be combined with FMN-mediated conversion of
m6A to f6A for a selective labelling of m6A for genome-wide
detection and profiling (Scheme 1C). In our new approach, we
took advantage of the electron-withdrawing propriety of the
formyl group by employing the nucleophilic substitution.
Utilizing Huisgen cycloaddition (click) chemistry [28], a possible
fragments are usually captured and
enriched by
immunoprecipitation and then identified by second-generation
sequencing [15-19]. However, these approaches are limited to the
sources of antibodies or recognizing enzymes (reading proteins
or restriction enzymes) and the specific sites in the transcriptome.
Thus, there is a great need for a simple, sensitive, antibody-free
method for m6A detection.
Labeling of nucleic acids is required for many studies aiming
to elucidate their functions and dynamics in vitro and in live cells.
To date, two different strategies have been developed to label,
profile and analysis genome-wide m6A methylation patterns in
live cells (Scheme 1). Jia et al. utilized m6A demethylease FTO
that are responsible to m6A demethylation at the RRm6ACH
sequence and converted the inert methyl substituent to
chemically reactive hydroxymethyl group (hm6A), which was
sensitive to nucleophilic substitution with thiol compounds like
dithiothreitol (DTT) to afford the N6-dithiolsitolmethyl adenosine
(dm6A) [20]. Thus, bioorthgonally functional groups like
 Corresponding author.
E-mail address: chengl@iccas.ac.cn

tag (or any chemical tag) may be installed, thus facilitating an
efficient and unbiased labelling of the original m6A-containing
RNA fragments for detection and genome-wide profiling. This
new approach may provide a wider coverage of m6A-containing
genomic regions compared with other affinity-enrichment
methods.
Scheme 1. Previously reported labelling of m6A and our strategy.
To investigate the reactivity of f6A, we firstly prepared the
adenosine derivative in a gram scale (Scheme 2) [8]. The
group with Wittig olefination, nucleophilic addition and other
related reactions. However, due to the amide resonance with its
enolate isomer, the carbonyl was stabilized in the N-formyl
functionality and thus can not be labelled under extreme mild
conditions (see Supporting information for details). We thus
tuned to the N-formyl itself by adapting the N-alkylation of
peptides/proteins. The N-H in f6A was influenced by the
heterocyclic adenine and the attached carbonyl group and should
be acidic enough to be easily deprotonated. Thus by using mild
potassium carbonate as the base with the crown ether
18-crown-6, we were able to screen a bunch of electrophiles, in
most cases, alkyl halides 4-15. With the simplest iodomethane 4,
the N6-formyl-N6-methyl adenosine 16 was obtained in a 30%
yield with 3 h (Scheme 3B, Table S1 in Supporting information).
However, no de-formylation product 16' was observed,
indicating that the alkylation indeed could selectively label f6A
while inhibiting its hydrolysis. A much more reactive benzyl
bromide 5 would lead to a higher yield of the corresponding
di-substituted adenosine in 40 min. Considering the bioorthgonal
property of alkyne functionality, we next tested several alkynyl
halides 6 and 7 and sulfonates 8-13. While the propargyl chloride
6 afforded the N6-formyl-N6-propargyl adenosine 18 in a 33%
yield, its bromide analogue 7 resulted in a much faster alkylation
(within 15 min) along with a few de-formylation product 18'
(15%). We thus assume that the leaving group might play a
crucial role in the efficiency of the propargylation and may
influence the stability of the product 18 during the alkylation.
Indeed, simple propargyl mesylate 8 and benzenesulfonate 9
generated uneven distribution of 18 and 18'. Interestingly, the
p-toluenesulfonate 10, p-methoxyl benzenesulfonate 11 and
p-fluorobenzenesulfonate 12, all afforded the N6-propargyl
hydroxyls
at
adenosine
were
protected
with
tert-butyl(dimethyl)silyl (TBS) group, and then the free amine in
1 was reacted with N,N-dimethyformamide diethy acetal to
generate the dimethylformimidamide 2. The latter was then
subjected to hydrolysis with hydroxybenzotriazole to release the
N-formyl group. We modified this step by using dichloromethane
instead of methanol, which significantly improved the yield to
45%. Upon treatment with triethylamine trihydrofluoride, the
unprotected f6A was obtained in 23% yield (4 steps).
Scheme 2 Synthesis of f6A.
With the sufficient amount of f6A in hand, we then
investigated the chemical labelling of this central intermediate
(Scheme 3A). We firstly intended to functionalize the carbonyl

adenosine 18' in inferior yields (24%-28%), while the o-nitro
benzenesulfonate 13 delivered moderate amount of 18 (31%). We
also verified if allylic motif can be introduced to f6A since the
terminal alkene is also a bioorthgonal handler in biocongugation
chemistry. Thus f6A was subjected to react with allylic bromide
14, affording the corresponding labelled product 19 in good yield
(72%) without any noticeable de-formylation. Another
interesting example was using Morita-Baylis-Hillman adduct 15
as the electrophile. In that case, the acrylate motif was selectively
introduced to the N6-position, which might afford another type of
chemical enrichment by conjugate addition. Thus different
labelling groups could be hosted by varying the electrophiles.
aforementioned labelling to this oligo (see Supporting
information for details). Unfortunately, the f6A oligo remained
untouched or decomposed completely under these conditions (pH
8.0 or pH 9.0), no matter what electrophile we have used. Thus, a
more efficient method would be needed for RNA samples
isolated from live cells. This optimization is currently underway
in our laboratory and will be reported in due courses.
(A)
(B)
(C)
m6A RNA
f6A RNA
8
4
f6A RNA
A RNA
A RNA
100
50
0
0
20
10
0
0
10
20
30
0
2
4
6
8
Retention time (min)
Time (h)
Scheme 5. (A) The preparation of f6A RNA; (B) HPLC analysis of
an ssRNA 5'-AUUCUCAm6AC-3' after treatment with FMN and
irradiation with blue LED light of 470 nm under oxygen for 30 min
(red line), compared to A RNA (5'-AUUCUCAAC-3') and m6A RNA
(blue line); (C) Stability test of f6A RNA (red line) at room
temperature and the generation curve of A RNA (black line).
(B)
80
N6,N6-formyl alkyl adenosines 16-20
N6-alkyl adenosines 16'-20'
In summary, we have presented a practical approach for the
selective labelling of f6A, an essential intermediate during the
oxidative demethylation of m6A, with simple and easily available
small organic molecules. The alkylation proceeded rapidly and
selectively under mild conditions to covalently link a bunch of
bioorthogonal components. With the successful establishment of
click reaction pertaining to f6A derivatives, it is expected to
provide a useful strategy for chemically uniform and highly
selective labelling of m6A RNAs in an enzyme-free and covalent
selective fashion.
60
40
20
0
4
5
6
7
8
9
0
1
2
3
4
5
1
1
1
1
1
1
Scheme 3 Chemical labelling of f6A.
As a demonstration of the utilization of this powerful reaction
toward m6A enrichment, we applied the 1,3-dipolar cycloaddition
of the product 18 with ethyl azidoacetate 21 (Scheme 4). The
click reaction smoothly afforded the triazole product 22 in good
yield (45%). Interestingly, the formyl group was removed during
the post-modification process. In a word, we have developed a
wide range of labelling intermediates with f6A via simple steps
and rapidly creating new products for further applications.
Declaration of Interest Statement
We wish to confirm that there are no known
conflicts of interest associated with this publication
and there has been no significant financial support
for this work that could have influenced its
outcome.
Acknowledgments
This work was supported by the National Key R&D Program
of China (Nos. 2017YFA0208100 and 2020YFA0707901),
National Natural Science Foundation of China (Nos. 22022704,
91853124, 21977097, and 21778057), Postdoctoral Innovative
Talents Support Program (No. BX20200337, Dr. L.J. Xie) and
Chinese Academy of Sciences.
Scheme 4 The click reaction of 18 with ethyl azidoacetate.
In order to explore the possibility of this approach at
transcription RNAs, we prepared an m6A containing oligo
5'-AUUCUCAm6AC-3' and subjected it to the standard chemical
demethylation conditions (Scheme 5A). We have carefully
optimized the amount of FMN and the oxidation time. Finally we
were able to generate the f6A oligo 5'-AUUCUCAf6AC-3' in 36%
yield according to HPLC analysis (Scheme 5B). It was reported
in literature that f6A was unstable in aqueous solutions and
rapidly hydrolyzed to adenosine. However, under our reaction
conditions, f6A oligo was stable in 8 h, with less than 30% of
decomposition (Scheme 5C). With that in hand, we applied the

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Graphical Abstract
We present here an approach which selectively alkylated the N6-formyl adenosine f6A, the key intermediate during chemical
oxidation of N6-methyl adenosine m6A, with an alkyne functionality that can be further labelled with click reactions.