Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA

Article abstract of DOI:10.1002/anie.201800188

m⁶A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m⁶A sites is of utmost importance. However, m⁶A does not interfere with Watson–Crick base pairing, making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl groups can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m⁶A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-l-selenohomocysteine and validated different types of known rRNA methylation sites.

Full text of DOI:10.1002/anie.201800188

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Accepted Article
Title: Enzymatic or in vivo installation of propargyl groups in
combination with click chemistry enables enrichment and
detection of methyltransferase target sites in RNA
Authors: Katja Hartstock, Benedikt Nilges, Anna Ovcharenko, Nicolas
Cornelissen, Nikolai Puellen, Sebastian Leidel, and Andrea
Rentmeister
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201800188
Angew. Chem. 10.1002/ange.201800188
Link to VoR: http://dx.doi.org/10.1002/anie.201800188
http://dx.doi.org/10.1002/ange.201800188

10.1002/anie.201800188
Angewandte Chemie International Edition
COMMUNICATION
Enzymatic or in vivo installation of propargyl groups in
combination with click chemistry enables enrichment and
detection of methyltransferase target sites in RNA
Katja Hartstock[a], Benedikt Nilges[b], Anna Ovcharenko[a], Nicolas Cornelissen[a], Nikolai
Püllen[a], Ann-Marie Lawrence-Dörner[a], Sebastian A. Leidel,[b] and Andrea Rentmeister*[a]
Dedication ((optional))
Abstract: m6A is the most abundant internal modification in
eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes
mRNA metabolism, impacting cell differentiation and development.
Precise transcriptome-wide assignment of m6A sites is of utmost
importance. However, m6A does not interfere with Watson-Crick
base pairing making polymerase-based detection challenging. We
developed a chemical biology approach for the precise mapping of
methyltransferase (MTase) target sites based on the introduction of
a bioorthogonal propargyl group in vitro and in cells. We show that
propargyl can be introduced enzymatically by wild-type METTL3-
METTL14. Reverse transcription terminated up to 65 % at m6A sites
after bioconjugation and purification, hence enabling detection of
METTL3-METTL14 target sites by next generation sequencing.
Importantly, we implemented metabolic propargyl labeling of RNA
MTase target sites in vivo based on propargyl-L-selenohomocysteine
and validated different types of known rRNA methylation sites.
assignment are based on immunoprecipitation (IP) using m6A-
specific antibodies and subsequent RNA sequencing (RNA-
Seq).[7] The resolution of these methods was improved by
crosslinking inducing transitions and truncations detectable by
RNA-Seq.[8] However, due to the size of the antibody, the target
nucleotide can often not be assigned precisely. Furthermore,
anti-m6A antibodies likely have a preference for neighboring
nucleotides, potentially introducing biases. Indeed, different anti-
m6A-antibodies showed varying cross-linking-induced single-
nucleotide substitution patterns.[8a] While SCARLET can
independently validate and quantify specific m6A sites this
method is impractical to be used at a large number of candidate
sites.[9]
To identify m6A sites directly, the profile of reverse
transcriptases has been investigated in detail. Indeed, reverse
transcription (RT) can yield different rates of misincorporation at
modified RNA sites leading to nucleotide transitions.[10]
Furthermore, an RT-active KlenTaq DNA polymerase was
engineered and shown to exhibit up to 15 % misincorporation
opposite m6A.[11]
We sought to develop a chemical biology approach for the
direct detection of m6A-sites based on synthetic analogs of the
natural cosubstrate S-adenosyl-L-methionine (AdoMet).[12] In
many cases, AdoMet analogs with small side-chains and an
unsaturated C-atom in β-position – such as allyl or propargyl –
are accepted by MTases.[13] The propargyl group appeared
particularly promising because it is bioorthogonal and can be
reacted with azides in the copper-catalyzed azide-alkyne
cyloaddition (CuAAC) allowing for a facile generation of sterically
more demanding groups that (i) may impair reverse transcription
and (ii) allow for affinity purification (Scheme 1A). The transfer
efficiency can be increased by using a more stable selenium-
based cosubstrate (SeAdoYn, 1).[14]
N6-Methyladenosine (m6A) is the most abundant internal
modification of eukaryotic mRNA and has sparked particular
interest because it is reversible, suggesting a regulatory role.
Several recent studies implicated m6A involvement in circadian
rhythm maintenance, cell-cycle regulation, cell differentiation
and reprogramming, maintenance of a leukemic state, stress
responses, and spermatogenesis.[1] At the molecular level, m6A
was shown to alter RNA folding and structure by subtly changing
base-pairing stability.[2] Moreover, reader proteins of m6A were
3]
identified that determine mRNA-fates.[1c,
Interestingly, m6A
promotes both cap-dependent and cap-independent translation
4]
via recruitment of eIF3.[3c,
Binding of m6A can also target
mRNAs to the decay pathway.[3b] In Drosophila, m6A affects
female-specific alternative splicing of the sex determination
factor Sex lethal.[5] Additional functions of m6A have been
inferred from knockdown studies but are not understood at the
molecular level.[6]
To test whether non-natural modifications at the N6 position
of adenosine increase termination during RT, we performed
primer extension assays with different RT enzymes on a test
RNA produced by in vitro transcription using N6-modified ATP
(Figure 1A-D, Figure S1 and S3-4). For N6-propargyl and N6-
benzyl adenosine, gel electrophoretic analysis revealed
increased termination at or upstream of the modification (19 +/-
2 % and 30 +/- 5 %, respectively), whereas termination was
barely detectable for unmodified adenosine or m6A (Figure 1C-
D). N6-Azidohexyl adenosine also increased termination albeit at
a less defined position (Figure 1D). We found this to be the case
for different RT enzymes (Figures 1C and S4). These data show
that increasing the size of the modification at N6 of adenosine
bears potential to determine the site of modification via RT.
Methods for the precise assignment of m6A sites are highly
sought after. Since m6A is not distinguished from A by standard
polymerases, most transcriptome-wide methods for m6A site
[a]
[b]
Department of Chemistry, University of Münster
Institute of Biochemistry
Wilhelm-Klemm-Straße 2, 48149 Münster (Germany)
E-mail: a.rentmeister@uni-muenster.de
Max Planck Research Group for RNA Biology
Max Planck Institute for Molecular Biomedicine
Röntgenstraße 20, 48149 Münster (Germany).
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201800188
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Scheme 1. Concept for enrichment and assignment of MTase-target sites in
vitro and in vivo. A) METTL3-METTL14 catalyzes N6-propargylation of
adenosine with SeAdoYn (1) as cosubstrate. Bioconjugation of sterically
demanding groups (R1) by reaction with an azide causes reverse
transcriptases to terminate opposite the modification. B) Propargyl-L-
selenohomocysteine (2) can be fed to mammalian cells as the metabolic
precursor of 1, which is then used by cellular MTases. Total RNA can be
isolated and enriched for various RNA-MTase target sites. Abbreviations:
MAT: methionine adenosyltransferase, NGS: next generation sequencing.
Figure 1. Effect of different N6-modifications of adenosine on reverse
transcription (RT) and their enzymatic introduction by METTL3-METTL14. A)
Scheme of the primer extension assay to test the effect of modifications on
RT. The N6-modification of adenosine is shown as a red square. B) N6-
modifications investigated in this study. C) Primer extension assay for Maxima
H Minus. A representative denaturing PAA gel is shown. Positions in the RNA
sequence corresponding to elevated termination are color-coded. D)
Quantification of primer extension assay as shown in C) after 20 min reaction
from three independent experiments. Position of termination is color-coded as
in C). E) Enzymatic N6-modification of adenosine in target RNA by
recombinant METTL3-METTL14. Cosubstrates AdoMet, AdoPropen, and
SeAdoYn (1) result in formation of m6A, N6-allyl-adenosine (a6A), and N6-
propargyl-adenosine (p6A), respectively. LC-MS analyses of ribonucleosides
after enzymatic degradation are shown. Extracted ion chromatograms for m6A
(C11H15N5O4 [M+H]+ 282.120 ± 0.005), a6A (C13H17N5O4 [M+H]+ 308.135 ±
0.005), p6A (C13H15N5O4 [M+H]+ 306.120 ± 0.005) are shown in grey, purple,
and orange, respectively. Guanosine is shown as control (C10H13N5O5 [M+H]+
284.099 ± 0.005).
To determine whether these non-natural modifications can be
introduced enzymatically, we synthesized AdoMet analogs and
tested whether they are converted by METTL3-METTL14, which
was recombinantly produced in Sf21 insect cells[15] (Figure S5).
Indeed, LC-MS analysis confirmed that SeAdoYn was well
accepted as cosubstrate, yielding N6-propargyl-adenosine in the
27 nt substrate RNA (Figure 1E, Figure S7-8, Table S1).
Similarly, AdoPropen was accepted – albeit less efficiently
(Figure 1E, Figure S8) – yielding N6-allyl-adenosine, which was
recently reported to be suitable for RNA labeling and
identification by mutation assay.[16] The higher activity with 1 is
likely the consequence of selenium being less electronegative
than sulfur, thus, extending its lifetime in physiological
buffers.[14a, 17]
Encouraged by the efficient termination of p6A-containing RNA
after bioconjugation in primer extension assays, we anticipated
our approach to be compatible with NGS. We therefore
combined our in vitro assay with an adapted low bias approach
of library preparation[18] to evaluate N6-modified adenosine in
RNA. Fragments containing N6-biotin-modified adenosine were
coupled to streptavidin magnetic beads (Figure 2A). After linker
ligation to the bead-bound RNA and RT, the resulting cDNA was
circularized, amplified by PCR and sequenced. The reads were
mapped to the target RNA sequence and relative fragment
coverage, as well as relative fragment end frequency were
plotted (Figure 2D). The NGS data showed that in the case of
bioconjugated p6A-RNA, most fragments terminated 1 or 2 nt
downstream of the N6-modified adenosine in line with our results
of the primer extension assay (Figure 2B). In contrast, the m6A-
containing sample showed an even distribution of sequence
termination (Figure 2D). Here, only ~10 % of sequence reads
terminated at or downstream of the modification. Similarly, we
observed only a minor effect for unconjugated p6A-RNA (Figure
S11).
Since the propargyl group can be efficiently introduced
by wild-type METTL3-METTL14 but only shows mild termination
in RT, we next sought to increase the size of the N6-adenosine
modification by reacting propargylated RNA with biotin-azide in a
CuAAC reaction (Figure S2). In RT, the resulting N6-biotinylated
RNA caused ~40 % termination, predominantly at the position
directly downstream of the modification (Figure 2B-C).
Immobilization of the biotinylated RNA on streptavidin-bound
magnetic beads prior to RT further increased the extent of
termination to >65 % (Figure 2B-C). Termination in primer
extension assays was also observed when the propargyl group
was introduced post-synthetically (Figure S13). Taken together,
our data indicate that the enzymatic addition of a propargyl
group by METTL3-METTL14 is feasible and that significant
termination can be achieved through bioconjugation to biotin and
coupling to streptavidin beads.
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10.1002/anie.201800188
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Figure 3. In vivo identification of RNA-MTase target sites based on metabolic
labeling with propargyl-L-selenohomocysteine. A) LC-MS analysis of total RNA
from cells fed with 2 (red) and control cells fed with L-methionine (blue). EIC
for propargyl-adenosine is shown (C13H15N5O4 [M+H]+ 306.12 ± 0.01). B) NGS
of rRNA with and without metabolic labelling. Coverage of a section of 28S
rRNA from labeled and purified (top) and methylated rRNA (bottom) is shown.
Representative examples of enriched and depleted regions are marked. All
methylated rRNA positions are indicated above. C) Frequency of known
methylation sites in 28S and 18S rRNA found in 12 enriched regions and 7
depleted regions alongside frequencies of methylation in randomly tested
regions.
Figure 2. Precise and direct assignment of m6A sites based on bioconjugation
and next generation sequencing. A) Scheme of bioconjugation of N6-
propargylated RNA via CuAAC with biotin-azide (biotin: orange triangle)
enabling enrichment via streptavidin (SA)-coated magnetic beads (orange
circle) prior to reverse transcription. B) Primer extension assay for Maxima H
Minus. A representative denaturing PAA gel is shown. Positions in the RNA
sequence corresponding to elevated termination are color-coded. C)
Quantification of primer extension assay as shown in B) after 20 min reaction.
Position of termination is color-coded as in B). D) Next generation sequencing.
Upper panel: m6A-containing RNA. Lower panel: p6A-containing RNA after
bioconjugation as illustrated in A). Fragment coverage for the RNA is shown
(grey bars). Red line indicates the fraction of fragments terminating at a
respective position.
Finally, we tested whether our strategy can be used to identify
RNA MTase target sites in vivo. To this end, we established
metabolic labeling of MTase target sites via biosynthesis of
AdoMet analogs from methionine analogs and ATP by
methionine adenosyltransferase (MAT) (Scheme 1B).[19]
Specifically, we fed mammalian HeLa cells with propargyl-L-
selenohomocysteine (2), then isolated total RNA and validated
formation of 2′-O-propargyl-adenosine by LC-MS/MS (Figures
3A and S12). Next, we clicked the total RNA to a biotin-azide
and generated NGS libraries. We mapped the reads to
ribosomal RNA, which is known to contain different types of
nucleotide methylations. We assigned peaks enriched in RNA
from metabolic propargyl labeling as well as RNA from control
cells and compared their position to known methylation sites in
human rRNA (Figure 3B, Table S2, Figure S14).[20] Strikingly, we
found that 83 % of the peaks contained one or more methylation
events and only 17 % of enriched regions are devoid of
predicted methylations (Figure 3C). In contrast, peaks that were
depleted, i.e. found in the control but not in the metabolically
labeled samples, did not overlap with any methylation site
(Figure 3C). As an independent control we randomly sampled
We further analyzed whether non-natural modifications at m6A
sites might lead to elevated levels of misincorporation, as
recently reported for a different non-natural N6-modification of
adenosine and engineered polymerases.[11, 16] Interestingly, we
found only 0–3 % of misincorporation in both m6A and the non-
natural N6-modification indicating that misincorporation rates are
not increased in our setup (Figure S9). Taken together, these
data show that post-synthetic modification at METTL3-METTL14
target sites in combination with click chemistry is compatible with
NGS and can be used to assign the m6A sites with a resolution
of 2 nucleotides.
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Zambrano, S. C. Robson, D. Aspris, V. Migliori, A. J. Bannister, N.
Han, E. De Braekeleer, H. Ponstingl, A. Hendrick, C. R. Vakoc, G. S.
Vassiliou and T. Kouzarides, Nature 2017, 552, 126-131.
[2] N. Liu, Q. Dai, G. Zheng, C. He, M. Parisien and T. Pan, Nature
2015, 518, 560-564.
regions of rRNA for RNA methylation. Only approximately 30 %
of these randomly assigned regions mapped to known
methylation sites, establishing that our strategy strongly enriches
for MTase target sites independent of the type of methylation.
In summary, we showed that the propargyl-group can be
efficiently introduced by WT METTL3-METTL14 from the
synthetic AdoMet analog SeAdoYn (1). In combination with
bioconjugation and NGS, this presents a new avenue for direct
and precise assignment of m6A sites by blocking reverse
transcription independent of potential antibody biases.
Compared to a recently described method, based on iodination
of N6-allyladenosine,[16] our approach has several advantages:
(i) the CuAAC reaction is highly selective and enables RNA
enrichment, (ii) the propargyl group is more efficiently introduced
than the allyl group, because we use a selenium-based AdoMet
analog and (iii) we could introduce non-natural modifications in
vivo. For the first time, we show that a propargyl-group can be
used for metabolic labeling of RNA MTase target sites by
feeding cells with the respective amino acid precursor 2. In
combination with our bioconjugation and NGS protocol, we
confirmed known MTase target sites in rRNA. Adaptations for
mRNA sequencing will likely enable detection of m6A sites in
vivo. Finally, our strategy paves the way to identify target sites of
different RNA MTases in a transcriptome-wide manner.
[3] a) C. Zhang, Y. Chen, B. Sun, L. Wang, Y. Yang, D. Ma, J. Lv, J.
Heng, Y. Ding, Y. Xue, X. Lu, W. Xiao, Y. G. Yang and F. Liu, Nature
2017, 549, 273-276; b) X. Wang, Z. Lu, A. Gomez, G. C. Hon, Y.
Yue, D. Han, Y. Fu, M. Parisien, Q. Dai, G. Jia, B. Ren, T. Pan and
C. He, Nature 2014, 505, 117-120; c) X. Wang, B. S. Zhao, I. A.
Roundtree, Z. Lu, D. Han, H. Ma, X. Weng, K. Chen, H. Shi and C.
He, Cell 2015, 161, 1388-1399; d) W. Xiao, S. Adhikari, U. Dahal, Y.
S. Chen, Y. J. Hao, B. F. Sun, H. Y. Sun, A. Li, X. L. Ping, W. Y. Lai,
X. Wang, H. L. Ma, C. M. Huang, Y. Yang, N. Huang, G. B. Jiang, H.
L. Wang, Q. Zhou, X. J. Wang, Y. L. Zhao and Y. G. Yang, Mol. Cell
2016, 61, 507-519; e) Y. Yang, X. Fan, M. Mao, X. Song, P. Wu, Y.
Zhang, Y. Jin, Y. Yang, L. L. Chen, Y. Wang, C. C. Wong, X. Xiao
and Z. Wang, Cell Res. 2017, 27, 626-641.
[4] a) K. D. Meyer, D. P. Patil, J. Zhou, A. Zinoviev, M. A. Skabkin,
O. Elemento, T. V. Pestova, S. B. Qian and S. R. Jaffrey, Cell 2015,
163, 999-1010; b) H. Du, Y. Zhao, J. He, Y. Zhang, H. Xi, M. Liu, J.
Ma and L. Wu, Nat. Commun. 2016, 7, 12626.
[5] a) T. Lence, J. Akhtar, M. Bayer, K. Schmid, L. Spindler, C. H.
Ho, N. Kreim, M. A. Andrade-Navarro, B. Poeck, M. Helm and J. Y.
Roignant, Nature 2016, 540, 242-247; b) I. U. Haussmann, Z. Bodi,
E. Sanchez-Moran, N. P. Mongan, N. Archer, R. G. Fray and M.
Soller, Nature 2016, 540, 301-304.
[6] a) G. Zheng, J. A. Dahl, Y. Niu, P. Fedorcsak, C. M. Huang, C. J.
Li, C. B. Vagbo, Y. Shi, W. L. Wang, S. H. Song, Z. Lu, R. P.
Bosmans, Q. Dai, Y. J. Hao, X. Yang, W. M. Zhao, W. M. Tong, X. J.
Wang, F. Bogdan, K. Furu, Y. Fu, G. Jia, X. Zhao, J. Liu, H. E.
Krokan, A. Klungland, Y. G. Yang and C. He, Mol. Cell 2013, 49, 18-
29; b) J. M. Fustin, M. Doi, Y. Yamaguchi, H. Hida, S. Nishimura, M.
Yoshida, T. Isagawa, M. S. Morioka, H. Kakeya, I. Manabe and H.
Okamura, Cell 2013, 155, 793-806.
[7] a) D. Dominissini, S. Moshitch-Moshkovitz, S. Schwartz, M.
Salmon-Divon, L. Ungar, S. Osenberg, K. Cesarkas, J. Jacob-
Hirsch, N. Amariglio, M. Kupiec, R. Sorek and G. Rechavi, Nature
2012, 485, 201-206; b) K. D. Meyer, Y. Saletore, P. Zumbo, O.
Elemento, C. E. Mason and S. R. Jaffrey, Cell 2012, 149, 1635-
1646.
Experimental Section
Experimental Details can be found in the Supplementary Information.
All sequencing data are available at the Gene Expression Omnibus
(Accession number: GSE109298).
[8] a) B. Linder, A. V. Grozhik, A. O. Olarerin-George, C. Meydan, C.
E. Mason and S. R. Jaffrey, Nat. Methods 2015, 12, 767-772; b) K.
Chen, Z. Lu, X. Wang, Y. Fu, G. Z. Luo, N. Liu, D. Han, D.
Dominissini, Q. Dai, T. Pan and C. He, Angew. Chem. Int. Ed. 2015,
54, 1587-1590.
Acknowledgements
This work was funded by the SPP1784 of the DFG (RE2796/3-1
and LE 3260/2-1) and EXC 1003 Cells in Motion – Cluster of
Excellence. A. O. and B. S. N. gratefully acknowledge
fellowships of the Graduate School of the Cells-in-Motion Cluster
of Excellence (EXC 1003 - CiM), University of Münster,
Germany. A.R. thanks the Fonds der Chemischen Industrie for
financial support (Dozentenpreis). We thank Dr. W. Dörner, F.
Muttach, Kristina Rau, and S. Wulff for excellent experimental
assistance, and Prof. Gunter Meister (Regensburg) for the
pFastBacDual_METTL3_METTL14 plasmid.
[9] N. Liu, M. Parisien, Q. Dai, G. Zheng, C. He and T. Pan, RNA
2013, 19, 1848-1856.
[10] E. M. Harcourt, T. Ehrenschwender, P. J. Batista, H. Y. Chang
and E. T. Kool, J. Am. Chem. Soc. 2013, 135, 19079-19082.
[11] J. Aschenbrenner, S. Werner, V. Marchand, M. Adam, Y.
Motorin, M. Helm and A. Marx, Angew. Chem. Int. Ed. 2018, 57,
417-421.
[12] a) C. Dalhoff, G. Lukinavicius, S. Klimasauskas and E.
Weinhold, Nat. Protoc. 2006, 1, 1879-1886; b) C. Dalhoff, G.
Lukinavicius, S. Klimasauskas and E. Weinhold, Nat. Chem. Biol.
2006, 2, 31-32; c) Y. Motorin, J. Burhenne, R. Teimer, K. Koynov, S.
Willnow, E. Weinhold and M. Helm, Nucleic Acids Res. 2010, 39,
1943-1952; d) D. Schulz, J. M. Holstein and A. Rentmeister, Angew.
Chem. Int. Ed. 2013, 52, 7874-7878.
[13] a) K. Islam, W. Zheng, H. Yu, H. Deng and M. Luo, ACS Chem.
Biol. 2011, 6, 679-684; b) R. Wang, W. Zheng, H. Yu, H. Deng and
M. Luo, J. Am. Chem. Soc. 2011, 133, 7648-7651; c) K. Islam, I.
Bothwell, Y. Chen, C. Sengelaub, R. Wang, H. Deng and M. Luo, J.
Am. Chem. Soc. 2012, 134, 5909-5915; d) L. Anhauser, F. Muttach
and A. Rentmeister, Chem. Commun. 2018, 54, 449-451; e) J. M.
Holstein, L. Anhauser and A. Rentmeister, Angew. Chem. Int. Ed.
2016, 55, 10899-10903; f) J. M. Holstein, D. Stummer and A.
Rentmeister, Protein Eng., Des. Sel. 2015, 28, 179-186; g) J. M.
Holstein, D. Schulz and A. Rentmeister, Chem. Commun. 2014, 50,
4478-4481; h) F. Muttach, N. Muthmann, D. Reichert, L. Anhauser
and A. Rentmeister, Chem. Sci. 2017, 8, 7947-7953; i) J. M.
Keywords: N6-Methyladenosine • ribose methylation • RNA
modification• metabolic labeling • next generation sequencing
[1] a) B. S. Zhao, I. A. Roundtree and C. He, Nat. Rev. Mol. Cell Biol.
2017, 18, 31-42; b) K. Xu, Y. Yang, G. H. Feng, B. F. Sun, J. Q.
Chen, Y. F. Li, Y. S. Chen, X. X. Zhang, C. X. Wang, L. Y. Jiang, C.
Liu, Z. Y. Zhang, X. J. Wang, Q. Zhou, Y. G. Yang and W. Li, Cell
Res. 2017, 27, 1100-1114; c) P. J. Hsu, Y. Zhu, H. Ma, Y. Guo, X.
Shi, Y. Liu, M. Qi, Z. Lu, H. Shi, J. Wang, Y. Cheng, G. Luo, Q. Dai,
M. Liu, X. Guo, J. Sha, B. Shen and C. He, Cell Res. 2017, 27,
1115-1127; d) I. Barbieri, K. Tzelepis, L. Pandolfini, J. Shi, G. Millan-
This article is protected by copyright. All rights reserved.

10.1002/anie.201800188
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Holstein, D. Stummer and A. Rentmeister, Chem. Sci. 2015, 6,
1362-1369.
[14] a) S. Willnow, M. Martin, B. Luscher and E. Weinhold,
Chembiochem 2012, 13, 1167-1173; b) J. M. Holstein, F. Muttach,
S. H. H. Schiefelbein and A. Rentmeister, Chem. Eur. J. 2017, 23,
6165-6173.
[15] a) J. Liu, Y. Yue, D. Han, X. Wang, Y. Fu, L. Zhang, G. Jia, M.
Yu, Z. Lu, X. Deng, Q. Dai, W. Chen and C. He, Nat. Chem. Biol.
2014, 10, 93-95; b) E. Scholler, F. Weichmann, T. Treiber, S. Ringle,
N. Treiber, A. Flatley, R. Feederle, A. Bruckmann and G. Meister,
RNA 2018.
[18] A. Lecanda, B. S. Nilges, P. Sharma, D. D. Nedialkova, J.
Schwarz, J. M. Vaquerizas and S. A. Leidel, Methods 2016, 107, 89-
97.
[19] a) F. Muttach and A. Rentmeister, Angew. Chem. Int. Ed. 2016,
55, 1917-1920; b) F. Muttach and A. Rentmeister, Methods 2016,
107, 3-9; c) R. Wang, K. Islam, Y. Liu, W. Zheng, H. Tang, N. Lailler,
G. Blum, H. Deng and M. Luo, J. Am. Chem. Soc. 2013, 135, 1048-
1056; d) S. Singh, J. Zhang, T. D. Huber, M. Sunkara, K. Hurley, R.
D. Goff, G. Wang, W. Zhang, C. Liu, J. Rohr, S. G. Van Lanen, A. J.
Morris and J. S. Thorson, Angew. Chem. Int. Ed. 2014, 53, 3965-
3969.
[16] X. Shu, Q. Dai, T. Wu, I. R. Bothwell, Y. Yue, Z. Zhang, J. Cao,
Q. Fei, M. Luo, C. He and J. Liu, J. Am. Chem. Soc. 2017, 139,
17213-17216.
[20] S. K. Natchiar, A. G. Myasnikov, H. Kratzat, I. Hazemann and B.
P. Klaholz, Nature 2017, 551, 472-477.
[17] M. Tomkuviene, B. Clouet-d'Orval, I. Cerniauskas, E. Weinhold
and S. Klimasauskas, Nucleic Acids Res. 2012, 40, 6765-6773.
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