Tag-Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases

Article abstract of DOI:10.1002/anie.202013936

The mRNA modification N⁶-methyladenosine (m⁶A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3-METTL14 and METTL16 act as “writers” for different target transcripts and sequence motifs. The modification is perceived by dedicated “reader” and “eraser” proteins, but not by polymerases. We report that METTL3-14 shows remarkable cosubstrate promiscuity, enabling sequence-specific internal labeling of RNA without additional guide RNAs. The transfer of ortho-nitrobenzyl and 6-nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase-catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo-enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo-enzymatic modification of different sites in a single RNA.

Full text of DOI:10.1002/anie.202013936

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Tag-free internal RNA labeling and photocaging based on mRNA
methyltransferases
Authors: Anna Ovcharenko, Florian P. Weissenboeck, and Andrea
Rentmeister
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013936
Link to VoR: https://doi.org/10.1002/anie.202013936

10.1002/anie.202013936
Angewandte Chemie International Edition
RESEARCH ARTICLE
Tag-free internal RNA labeling and photocaging based on mRNA
methyltransferases
Anna Ovcharenko,[a,b] Florian P. Weissenboeck[a,b] and Andrea Rentmeister*[a,b]
[a]
A. Ovcharenko, F. Weissenboeck and Prof. Dr. A. Rentmeister
Department of Chemistry, Institute of Biochemistry
University of Münster
Corrensstrasse 36, 48149 Münster Germany
E-mail: a.rentmeister@uni-muenster.de
A. Ovcharenko, F. Weissenboeck and Prof. Dr. A. Rentmeister
Cells in Motion Interfaculty Center
[b]
University of Münster
Waldeyerstraße 15, 48149 Münster Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: The mRNA modification m6A is associated with multiple
roles in cell function and disease. The methyltransferases METTL3-
METTL14 and METTL16 act as “writers” for different target transcripts
and sequence motifs. The modification is perceived by dedicated
“reader” and “eraser” proteins, but not by polymerases. We report that
METTL3-14 shows remarkable cosubstrate promiscuity, enabling
sequence-specific internal labeling of RNA without additional guide
RNAs. The transfer of ortho-nitrobenzyl and 6-nitropiperonyl groups
allowed enzymatic photocaging of RNA in the consensus motif, which
impaired polymerase-catalyzed primer extension in a reversible
manner. METTL16 was less promiscuous but suitable for chemo-
enzymatic labeling using different types of click chemistry. Since both
enzymes act on distinct sequence motifs, their combination allowed
orthogonal chemo-enzymatic modification of different sites in a single
RNA.
have proven useful for labeling larger RNAs and even been
applied in the cellular context. Notable examples are TGT, Tias
and various approaches with methyltransferases (MTases).[8]
However, all of these required a minimal structure motif that had
to be appended to the RNA or acted on all RNAs with a specific
feature, such as the 5′ cap or a free 2′-OH group at the 3′ end.[8-9]
Sequence-specific modification required a guide-RNA in addition
to the MTase.[8k] Nucleic acid enzymes have been selected for
site-specific post-transcriptional labeling of RNAs at the 2′ position
or 3′ end by conjugating an additional appropriately labeled
substrate, typically derived from an NTP analogue.[10] The
recently described RAIL approach allows to site-selectively
functionalize RNA via DNA-induced structure, but faces
limitations for functionalizing very short RNAs.[11]
For RNA photocaging, the same concepts and limitations
apply. Here, the introduction of photocaging groups has been
largely limited to chemical synthesis whereas post-transcriptional
12]
enzymatic modification was dependent on tags.[3a,
We and
Introduction
others recently introduced MTases as tools to photocage DNA
sequence-specifically,[13] but enzymatic sequence-specific
photocaging of RNA sequences – be it by protein- or nucleic acid-
based enzymes – has not been reported to the best of our
knowledge.
Covalent modifications of RNA represent the basis of numerous
approaches aiming to make RNA accessible to biophysical and
cellular studies by fluorescent, FRET, spin or biotin labeling.[1]
Applications range from investigating the functional interplay of
domains to studying transcriptional dynamics and tracking the
localization in cells or in vivo.[2] The installation of photocleavable
groups aims to control functions of RNA.[3] Ideally, labels should
not interfere with the natural function and photocleavable groups
should release the native biomolecule after irradiation, giving
preference to modifications installed directly at the RNA without
the need for additional tags.
Solid phase synthesis provides the most comprehensive
toolbox for installation of functional groups in RNA and does not
face limitations with respect to sequence or structural elements.[4]
However, the size is limited to approximately 100 nt and long
RNAs require chemical or enzymatic ligation of the synthetic
fragment with a polymerase-derived RNA.[5] Chemo-enzymatic
methods do not face length limitations and can be based on co-
or post-transcriptional introduction of modifications. While co-
transcriptional labeling was originally nucleoside- but not position-
specific, the development of unnatural base pairs has greatly
expanded the possibilities but is still tedious to realize.[2f, 6] The
use of specific polymerases like poly(A)-polymerase can also
provide position-specificity.[7] Post-transcriptional modifications
The recent rise of epitranscriptomics has led to reports on
mRNA modifications and the responsible methyltransferases,
including their recombinant production and crystal structures ([14]
,
Figure S1). m6A is the most prevalent internal modification in
eukaryotic mRNAs and has been linked to fundamental biological
processes, like neuronal development,[15] cell differentiation,[16]
the circadian cycle[17] and diseases, including cancer.[18] In
contrast to snoRNPs and tRNA-methyltransferases, these mRNA
methyltransferases seem to require no additional guide RNA and
not rely on pre-existing modifications. These enzymes might
therefore be ideal for labeling mRNAs without the need to attach
sequence or structure motifs and without the requirement of
additional guide RNAs, but this potential has not been explored to
date.
Results and Discussion
We chose to assess the potential of the two currently known
mRNA methyltransferases responsible for m6A formation,
METTL3-METTL14 (abbrev. METTL3-14) and METTL16, for
1
This article is protected by copyright. All rights reserved.

10.1002/anie.202013936
Angewandte Chemie International Edition
RESEARCH ARTICLE
Table 1. Conversions observed for different MTase-substrate combinations.
Conditions: 3 µM RNA, 3 µM METTL16 or METTL3-METTL14, 1 mM AdoMet
or AdoMet analog, incubation for 1 h at 37 °C.
sequence-specific labeling and photocaging of RNAs (Scheme 1).
METTL3-14 is a heterodimer with preference for the DRACH motif
(Figure S1A).[14, 19] The available crystal structures of SAM-bound
METTL3-14 in pre-catalytic state suggest that the AdoMet
analogs could fit into the free space in the binding pocket (Figure
S1A). In cells, METTL3-14 functions in complex with other
proteins,[20] but the heterodimer alone is active in vitro and able to
transfer allyl and propargyl groups from analogs of the natural
cosubstrate S-adenosyl-L-methionine (AdoMet).[21] METTL16
requires a different consensus motif (UACAGAGAA) that needs
to be presented as a loop in a hairpin structure.[22] METTL16 was
shown to methylate only two target RNAs, but additional target
transcripts are likely.[22] Neither of these enzymes requires an
additional guide RNA to target the consensus motif.
(
)
Theor.
Rel.
mod. spec.
[%] mod. A [%]
Site-
mod.A
[%]
Entry Substr. yield RNA MTase Prod.
[%]
1
2
3
4
5
6
7
8
9
1a
100
I
I
3-14
3-14
3-14
3-14
3-14
3-14
3-14
3-14
16
2a
2b
2c
2d
2e
2f
55
45
tr.
100
55
45
-
Se-1b 100
82
tr
1c
1d
1e
1f
100
100
100
100
100
100
5.9
I
I
tr.
tr
-
I
32
30
tr
58
55
tr
32
30
-
I
Scheme 1. A) Methyltransferases METTL3-14 and METTL16 used for
sequence-specific modification of RNA without dedicated tags. B) Photocaging
(PC) groups block reverse transcription in a reversible manner. Bioorthogonal
groups can be used for RNA labeling using different types of click chemistry.
1g
1h
1a
I
2g
2h
2a
2b
2c
2f
I
29
3.6
2.5
tr
53
100
69
tr
29
61
42
-
II
II
II
II
10 Se-1b 5.9
16
11
12
13
1c
1f
5.9
5.9
16
To explore the cosubstrate scope of METTL3-14 and METTL16,
we designed three different substrate RNAs containing the
respective consensus motifs (I-III, Table S1). METTL3-14 was
tested with RNA I containing four repeats of the GGACU
consensus motive. Using RNA I and AdoMet (1a) resulted in 55%
of m6A, according to HPLC analysis after digestion to single
nucleosides (Table 1). SeAdoYn (Se-1b) as cosubstrate yielded
45% of 2b, corresponding to 82% relative to AdoMet. AdoMet
analogs with longer alkyl chains like azidobutenyl- (1c) or
hexenynyl- (1d) were not efficiently converted. However, the
benzylic AdoMet analogs with vinylbenzyl- (1e) and ortho-
nitrobenzyl- (1f) groups (ONB) gave high relative conversions
(58% and 55% compared to AdoMet). Moreover, we synthesized
a new AdoMet analog carrying a 6-nitropiperonyl (NP) group (1h),
which gave a 53% conversion relative to AdoMet. All N6-modified
adenosine products were confirmed by LC-MS analysis (Figure
S2). Controls using heat-inactivated enzyme did not yield product
peaks (Figure S3). The kinetics of the transfer reactions indicated
that, as expected, natural AdoMet is a better cosubstrate than all
AdoMet analogs tested (Figure S4, Table S4). Nevertheless, the
data summarized in Table 1 show that METTL3-14 is able to
transfer short alkyl and benzylic groups to the N6-position of
adenosine making RNA with the consensus motif GGACU
accessible to sequence-specific labeling and photocaging.
16
n.d.
7.0
2.8
tr
n.d.
100
40
tr
n.d.
49
19
-
1a
14.3 III
16
2a
2b
2c
2d
2f
14 Se-1b 14.3 III
16
15
16
17
18
1c
1d
1f
14.3 III
14.3 III
14.3 III
14.3 III
16
16
tr
tr
-
16
n.d.
n.d.
n.d.
n.d.
-
1g
16
2g
-
Next, we tested the cosubstrate scope of METTL16,
recombinantly expressed in insect cells (Figure S5), using RNAs
of different lengths containing the target sequence (RNA II: 61 nt
and RNA III: 26 nt).[22b,
23]
These RNAs contain 17 and 7
adenosines, respectively, but only a single target adenosine each.
Therefore, the total amount of site-specifically modified m6A
would correspond to 14.3% or 5.9%, respectively (Table 1).
Using the natural cosubstrate (1a) RNA II or III yielded 61 or
49 % of site-specifically methylated m6A (Table 1), with the longer
RNA II being a better substrate than RNA III. Using SeAdoYn (Se-
1b) yielded 42% and 19% of site-specifically modified 2b for RNA
II and III, respectively (Figure S6), corresponding to 40-69%
relative yield compared to AdoMet. In addition, METTL16 was
able to transfer the 4-azido-but-2-enyl group from 1c yielding the
corresponding product 2c, albeit in low yields. All N6-modified
products were confirmed by LC-MS (Figure S6). Control reactions
with inactivated enzyme did not yield products (Figure S7). The
kinetics of the transfer reactions were measured (Figure S4, Table
S4). In contrast to METTL3-14, METTL16 did not transfer benzylic
groups (Table 1). These data show that METTL16 can transfer
2
This article is protected by copyright. All rights reserved.

10.1002/anie.202013936
Angewandte Chemie International Edition
RESEARCH ARTICLE
longer alkyl chains including bioorthogonal groups to the
sequence motif UACAGAGAA, which is distinct from METTL3-14.
However, its promiscuity does not comprise benzylic groups. In
contrast to METTL3-14, there are no crystal structures available
for SAM-bound METTL16 in pre-catalytic state, so its substrate
promiscuity cannot be readily assessed from the available
structural data (Figure S1B).
Uncaging at higher wavelengths is desirable to reduce photo-
damage and phototoxicity, especially in a cellular context. Gel
analysis showed that the RNA was not degraded by irradiation
under used conditions (Figure 2C, Figure S12D,E). Importantly,
after light-triggered removal of the photocaging groups, the RNA
could again be enzymatically remodified (Figure S13), further
supporting intactness of RNA after irradiation.
To assess whether the post-synthetically modified RNAs
I-III could be further functionalized, we used the copper-catalyzed
Next, we were interested to see whether the ONB or NP
groups at the N6-position of adenosine would affect a biological
function. Recent work showed that N6-methylation of adenosine
does not have significant effects on wild-type polymerases,
(CuAAC)
and
strain-promoted
(SPAAC)
azide-alkyne
cycloaddition for functionalization with Cy5 as a fluorescent dye
or biotin as an affinity handle (Figure 1). Successful clicking of the
N6-propargylated RNA I was confirmed by LC-MS for biotin-azide
(Figure 1B, Figure S8A,B) and in-gel fluorescence for Cy5-azide,
which also proved that the RNA remained intact during the click
procedure (Figure 1C). Similarly, RNA with N6-(azido-but-2-enyl)-
adenosine was labeled via SPAAC using DBCO-Cy5, whereas
controls with heat inactivated enzymes were not (Figure S9).
Since every long RNA is likely to contain DRACH motives, we
used METTL3-14 to label mRNA produced by in vitro transcription
(IVT) coding for the reporter protein firefly luciferase (FLuc mRNA,
1900 nt long) with Cy5 azide (Figure S10).
whereas extended residues such as triazoles or engineered
25]
polymerases impeded the reverse transcription.[21b,
We
therefore designed a longer RNA containing two METTL3-14
consensus motifs and primer binding site for reverse
a
transcription (Table S1). This RNA IV was made by IVT, modified
post-transcriptionally using Se-1b or 1f, respectively, and then
used for reverse transcription (RT) (Figure 2D). In this experiment,
RNA V served as a control, with GGACU motif replaced by
GGCCU. A control RT with unmodified RNA revealed that out of
the two GGACU consensus motifs, the one harbouring the target
adenosine at 35 nt was suitable for analysis by RT because it did
not show termination for unmodified control RNA (Figures S14).
When RNA IV was treated with 1f and METTL3-14, the RT
showed a termination band at 36 nt, corresponding to one
nucleotide downstream of the modified adenosine. This band was
almost absent when the sample was irradiated for 2 min at 365
nm or 405 nm prior to RT (Figure 2D, Figure S14, S16). According
to the LC-MS analysis, these irradiation conditions completely
removed the ONB-modifications and according to PAA gel
analysis they did not lead to degradation of the RNA (Figure 2B-
C, Figures S14-16). Similar results were observed when RNA IV
was treated with 1h and METTL3-14 while the control RNA V
showed no termination at 36 nt (Figure S16A). In this case,
irradiation for 1 min at 420 nm was sufficient to remove the NP-
modification, according to LC-MS (Figure S16B). Additionally, all
(
modified RNAs contained
a termination band at 34 nt,
))
corresponding to one nucleotide upstream of the modification
(Figure 2D, Figure S14, S16). These data show that the ONB and
NP groups can be removed by brief irradiation with UV light
without degrading the RNA. Placing the ONB/NP group at the N6-
position of adenosine significantly increases termination opposite
this modification. For the first time, a post-synthetically installed
ONB/NP group in RNA can be removed by light.
Figure 1. A) CuAAC of RNA
I
containing N6-propargyl-adenosine from
enzymatic modification via METTL3-14 using Cy5-azide or biotin-azide. B) LC-
QTOF-MS analysis of RNA after enzymatic digestion to ribonucleosides before
and after CuAAC using biotin-azide. Left: EIC of N6-propargyladenosine (2b,
C
C
13H15N5O4 [M+H]+ 306.120±0.005), right: EIC for N6-biotinylated adenosine (2i,
26H37N11O6S [M+H]+ 632.272 ± 0.005, also see Figure S8A). C) PAA gel
analysis of RNA after CuAAC with Cy5-azide on SybrGold and Cy5 channels.
Control contained heat-inactivated enzyme.
When biotin-azide was clicked to the enzymatically proparglyated
RNA, we observed strong termination in a reverse transcription
assay (Figure S11). Based on our finding that METTL3-14
accepts 1f and 1h and transfers the ONB and NP groups to the
N6-position of adenosine, we sought to explore the photocaging/-
uncaging ability for the respective RNA (Figure 2A). While MTase-
based transfer and removal of photocaging groups has been
reported previously for DNA,[13a] enzymatic transfer and light-
triggered removal of the ONB or NP derivatives was not reported
for RNA to date. Here, irradiation of RNAs containing the ONB
group installed by METTL3-14 completely removed the ONB
group after 2 min at either 365 nm or 405 nm (Figure 2B, Figure
S12A,B). The NP group was completely removed after 1 min
irradiation at 420 nm, according to LC-MS (Figure S12C), in line
with previous reports about the NP group for DNA photocaging.[24]
Figure 2. A) Scheme of photocleavage of the ONB group from enzymatically
modified target RNA. B) Analysis of photocleavage shown in (A) using LC-MS
after digestion to ribonucleosides. EICs for 2f are shown (C17H18N6O6 [M+H]+
403.136 ± 0.005). C) Gel analysis of samples from (A) before and after
3
This article is protected by copyright. All rights reserved.

10.1002/anie.202013936
Angewandte Chemie International Edition
RESEARCH ARTICLE
were found in mammals,[27] suggesting that most mRNAs would
be available for labeling/manipulation with this enzyme complex
without the need to append a tag nor add a guide RNA. Finally,
we provide proof of concept that the two MTases can be used for
orthogonal labeling of RNA at different sites. This can be useful
irradiation (15% denat. PAGE). D) Reverse transcription (RT) of RNA IV
modified using METTL3-14 and Se-1b or 1f. For 1f a new termination band is
observed at 36 nt, 1 nt downstream of the modified A. This termination band is
reduced if the RNA was irradiated before RT. Ctrl shows RT for unmodified RNA
IV. Full gel is shown in Figure S14B.
for
double
labeling
(including
FRET
labels
or
fluorophore/quencher pairs for biophysical studies) or labeling an
mRNA that should also become activated by light (e.g. to activate
and track RNA).
Since METTL3-14 and METTL16 target distinct sequence motifs,
it should be possible to sequence-specifically label RNAs
containing both target motifs. To assess this option, we designed
a chimeric RNA containing both the METTL3-14 and the
METTL16 consensus sequence as well as control chimeras with
point mutations in either the METTL3-14 or the METTL16 target
sites (Table S1).[14b, 26] As expected, these control chimeras were
only modified by the MTase whose target motif remained intact,
confirming specificity of the enzymes (Figures 3A, 3B).
Knowing that MTase-based modification is sequence-
specific, we tested dual modification of the chimeric RNA by first
applying Se-1b and METTL16 (step 1 in Figure 3C/D) followed by
treatment with 1f and METTL3-14 (step 2 in Figure 3C/D). After
each step, LC-MS analysis confirmed that the modification was
successful (Figure 3D, S17). Indeed, the double modification of
the chimeric RNA was possible by sequential use of METTL16
and METTL3-14 using different AdoMet analogs. Finally, we also
showed that the transferred ONB group could be selectively
removed by light from the double modified chimeric RNA (step 3
in Figure 3C/D). During all steps, the RNA remained intact (Figure
3E).
Conclusions
Taken together, our data show that mRNA methyltransfersases
provide a means to label RNAs containing their motifs at internal
sites. In the case of METTL3-14, a ~1900 nt FLuc-mRNA could
be labeled without the need to append a structural hallmark (like
the 5′ cap) or secondary structure element (like for TGT or TIAS).
This is possible because the cosubstrate scope of human m6A
MTases is broader than previously expected, especially for
METTL3-14, which even accepts photocaging groups. Looking at
the crystal structures of METTL3-14 in complex with AdoMet the
accommodation of large substituents can be anticipated (Figure
S1). The sequence-specific chemo-enzymatic labeling via
propargylation and subsequent click chemistry is highly efficient.
The transfer of photocaging groups is ~50% compared to AdoMet,
which was sufficient to impair reverse transcription. Protein
engineering should allow to further increase the yield and provide
a way to control biological functions at exactly the sites, where
modifications occur in nature. As a proof of concept, we showed
that photocaging groups block extension during reverse
transcription, but we anticipate effects on stability and binding of
proteins and miRNAs, as previously shown for chemically
photocaged short nucleic acids[3a, 12a]. In particular, for studying
the function of epitranscriptomic modifications, we envision that
blocking MTase target sites and the interaction with their “reader”
proteins could become a very attractive tool. Sequence-specific
labeling has been achieved previously with MTases but required
the addition of a guide-RNA complicating the system.[8k] The
Figure 3. Orthogonal labeling approach of an RNA bearing METTL3-14 and/or
METTL16 consensus motifs (shown in red and gray, respectively). A) Enzymatic
modification of RNA bearing a mutation in the METTL3-14 consensus motif
(control chimera 1). Extracted ion chromatograms for N6-propargyladenosine
(2b, m/z = 306.1). B) Enzymatic modification of RNA bearing a mutation in the
METTL16 consensus motive (control chimera 2). LC-MS analysis as in (A). C)
Scheme of the reversible double modification. D) LC-QQQ-MS analysis after
each
modification
step.
Extracted
ion
chromatograms
for
N6-propargyladenosine (2b, m/z = 306.1) and N6-(o-nitrobenzyl)adenosine (2f,
m/z = 403.1) shown in red and black, respectively. E) Analysis of the modified
RNA on 15% PAA gel.
target requirements of METTL3-14 and METTL16 are small, Acknowledgements
especially METTL3-14 only requires a 5 nt consensus motif
(DRACH) that leaves room for variations. Based on m6A
sequencing, 12,000 METTL3-14 target sites in over 7,000 genes
A.R. gratefully acknowledges funding by the DFG (RE2796/6-1,
RE2796/7-1) and the ERC (772280). A.O. is supported by the
4
This article is protected by copyright. All rights reserved.

10.1002/anie.202013936
Angewandte Chemie International Edition
RESEARCH ARTICLE
Cerniauskas, E. Weinhold and S. Klimasauskas, Nucleic Acids Research
2012, 40, 6765-6773.
Graduate School of the Cells-in-Motion Cluster of Excellence
(EXC 1003–CiM), University of Münster, Germany. We thank S.
Wulff, Dr. W. Dörner, Dr. P. Špaček, N. Muthmann, D. Reichert,
K. Hartstock, A.-M. Lawrence-Dörner, F. Vinke and Dr. L.
Anhäuser for excellent experimental assistance, Prof. Dr. Markus
Bohnsack (University of Göttingen) for providing the METTL16-
encoding plasmid and Prof. Dr. Gunter Meister (University of
Regensburg) for the pFastBacDual_METTL3_METTL14 plasmid
in the framework of SPP1784.
[9] Y. Motorin, J. Burhenne, R. Teimer, K. Koynov, S. Willnow, E. Weinhold and
M. Helm, Nucleic Acids Research 2011, 39, 1943-1952.
[10] a) T. J. Carrocci, L. Lohe, M. J. Ashton, C. Höbartner and A. A. Hoskins,
Chemical Communications 2017, 53, 11992-11995; b) L. Büttner, F.
Javadi-Zarnaghi and C. Höbartner, Journal of the American Chemical
Society 2014, 136, 8131-8137; c) B. Samanta, D. P. Horning and G. F.
Joyce, Nucleic Acids Res 2018, 46, e103; d) M. Ghaem Maghami, C. P.
M. Scheitl and C. Höbartner, Journal of the American Chemical Society
2019, 141, 19546-19549; e) M. Ghaem Maghami, S. Dey, A.-K. Lenz and
C. Höbartner, Angewandte Chemie International Edition n/a.
Keywords: RNA labeling • METTL3-METTL14 • photocaging •
[11] L. Xiao, M. Habibian and E. T. Kool, Journal of the American Chemical
Society 2020, 142, 16357-16363.
AdoMet analogs
[12] a) V. Mikat and A. Heckel, RNA 2007, 13, 2341-2347; b) A. Heckel and G.
Mayer, Journal of the American Chemical Society 2005, 127, 822-823;
c) A. Heckel, M. C. R. Buff, M. S. L. Raddatz, J. Muller, B. Potzsch and
G. Mayer, Angewandte Chemie-International Edition 2006, 45, 6748-
6750; d) J. M. Govan, D. D. Young, H. Lusic, Q. Y. Liu, M. O. Lively and
A. Deiters, Nucleic Acids Research 2013, 41, 10518-10528; e) D. Zhang,
C. Y. Zhou, K. N. Busby, S. C. Alexander and N. K. Devaraj, Angewandte
Chemie International Edition 2018, 57, 2822-2826; f) G. H. Zheng, L.
Cochella, J. Liu, O. Hobert and W. H. Li, Acs Chemical Biology 2011, 6,
1332-1338.
[1] N. Muthmann, K. Hartstock and A. Rentmeister, WIREs RNA 2020, 11,
e1561.
[2] a) G. Sicoli, F. Wachowius, M. Bennati and C. Höbartner, Angewandte
Chemie International Edition 2010, 49, 6443-6447; b) F. Wachowius and
C. Hobartner, Chembiochem 2010, 11, 469-480; c) K. J. Westerich, K. S.
Chandrasekaran, T. Gross-Thebing, N. Kück, E. Raz and A. Rentmeister,
Chemical Science 2020; d) E. L. Zajaczkowski, Q. Y. Zhao, Z. H. Zhang,
X. Li, W. Wei, P. R. Marshall, L. J. Leighton, S. Nainar, C. Feng, R. C.
Spitale and T. W. Bredy, ACS Chemical Neuroscience 2018, 9, 1858-
1865; e) A. A. Sawant, P. P. Mukherjee, R. K. Jangid, S. Galande and S.
G. Srivatsan, Organic and Biomolecular Chemistry 2016, 14, 5832-5842;
f) C. Domnick, F. Eggert, C. Wuebben, L. Bornewasser, G. Hagelueken,
O. Schiemann and S. Kath-Schorr, Angewandte Chemie International
Edition n/a; g) C. Manz, A. Y. Kobitski, A. Samanta, B. G. Keller, A.
Jäschke and G. U. Nienhaus, Nature Chemical Biology 2017, 13, 1172-
1178; h) M. Zhao, F. D. Steffen, R. Börner, M. F. Schaffer, Roland K O.
Sigel and E. Freisinger, Nucleic Acids Research 2017, 46, e13-e13.
[3] a) Q. Y. Liu and A. Deiters, Accounts of Chemical Research 2014, 47, 45-
55; b) C. Brieke, F. Rohrbach, A. Gottschalk, G. Mayer and A. Heckel,
Angewandte Chemie-International Edition 2012, 51, 8446-8476.
[4] L. Anhauser and A. Rentmeister, Current Opinion in Biotechnology 2017, 48,
69-76.
[13] a) L. Anhauser, F. Muttach and A. Rentmeister, Chemical Communications
2018, 54, 449-451; b) M. Heimes, L. Kolmar and C. Brieke, Chemical
Communications 2018, 54, 12718-12721.
[14] a) X. Wang, J. Feng, Y. Xue, Z. Guan, D. Zhang, Z. Liu, Z. Gong, Q. Wang,
J. Huang, C. Tang, T. Zou and P. Yin, Nature 2016, 534, 575-578; b) P.
Wang, K. A. Doxtader and Y. Nam, Mol Cell 2016, 63, 306-317.
[15] J. Widagdo and V. Anggono, J Neurochem 2018, 147, 137-152.
[16] a) P. J. Batista, B. Molinie, J. Wang, K. Qu, J. Zhang, L. Li, D. M. Bouley,
E. Lujan, B. Haddad, K. Daneshvar, A. C. Carter, R. A. Flynn, C. Zhou,
K. S. Lim, P. Dedon, M. Wernig, A. C. Mullen, Y. Xing, C. C. Giallourakis
and H. Y. Chang, Cell Stem Cell 2014, 15, 707-719; b) S. Geula, S.
Moshitch-Moshkovitz, D. Dominissini, A. A. Mansour, N. Kol, M. Salmon-
Divon, V. Hershkovitz, E. Peer, N. Mor, Y. S. Manor, M. S. Ben-Haim, E.
Eyal, S. Yunger, Y. Pinto, D. A. Jaitin, S. Viukov, Y. Rais, V. Krupalnik,
E. Chomsky, M. Zerbib, I. Maza, Y. Rechavi, R. Massarwa, S. Hanna, I.
Amit, E. Y. Levanon, N. Amariglio, N. Stern-Ginossar, N. Novershtern, G.
Rechavi and J. H. Hanna, Science 2015, 347, 1002-1006.
[5] a) E. Paredes, M. Evans and S. R. Das, Methods 2011, 54, 251-259; b) A.
H. El-Sagheer and T. Brown, Accounts of Chemical Research 2012, 45,
1258-1267; c) S. Keyhani, T. Goldau, A. Blümler, A. Heckel and H.
Schwalbe, Angewandte Chemie International Edition 2018, 57, 12017-
12021.
[17] a) C. Y. Wang, J. K. Yeh, S. S. Shie, I. C. Hsieh and M. S. Wen, Biochem
Biophys Res Commun 2015, 465, 88-94; 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.
[6] a) F. Eggert, K. Kurscheidt, E. Hoffmann and S. Kath-Schorr, ChemBioChem
2019, 20, 1642-1645; b) R. Kawai, M. Kimoto, S. Ikeda, T. Mitsui, M.
Endo, S. Yokoyama and I. Hirao, Journal of the American Chemical
Society 2005, 127, 17286-17295; c) Y. J. Seo, D. A. Malyshev, T.
Lavergne, P. Ordoukhanian and F. E. Romesberg, Journal of the
American Chemical Society 2011, 133, 19878-19888.
[18] a) S. Wang, C. Sun, J. Li, E. Zhang, Z. Ma, W. Xu, H. Li, M. Qiu, Y. Xu, W.
Xia, L. Xu and R. Yin, Cancer Lett 2017, 408, 112-120; b) J. Luo, H. Liu,
S. Luan, C. He and Z. Li, Int J Mol Sci 2018, 19; c) D. Dai, H. Wang, L.
Zhu, H. Jin and X. Wang, Cell Death Dis 2018, 9, 124; d) L. He, J. Li, X.
Wang, Y. Ying, H. Xie, H. Yan, X. Zheng and L. Xie, J Cell Mol Med 2018,
22, 4630-4639.
[7] a) M.-L. Winz, A. Samanta, D. Benzinger and A. Jäschke, Nucleic Acids
Research 2012, 40, e78; b) L. Anhäuser, S. Hüwel, T. Zobel and A.
Rentmeister, Nucleic Acids Research 2019, 47, e42-e42.
[8] a) J. M. Holstein, D. Stummer and A. Rentmeister, Chemical science 2015,
6, 1362-1369; b) J. M. Holstein, D. Schulz and A. Rentmeister, Chemical
Communications 2014, 50, 4478-4481; c) D. Schulz, J. M. Holstein and
A. Rentmeister, Angewandte Chemie International Edition 2013, 52,
7874-7878; d) F. Muttach, N. Muthmann, D. Reichert, L. Anhauser and
A. Rentmeister, Chemical Science 2017, 8, 7947-7953; e) J. M. Holstein,
F. Muttach, S. H. H. Schiefelbein and A. Rentmeister, Chemistry – A
European Journal 2017, 23, 6165-6173; f) J. M. Holstein, L. Anhäuser
and A. Rentmeister, Angewandte Chemie International Edition 2016, 55,
10899-10903; g) F. H. Li, J. S. Dong, X. S. Hu, W. M. Gong, J. S. Li, J.
Shen, H. F. Tian and J. Y. Wang, Angewandte Chemie-International
Edition 2015, 54, 4597-4602; h) S. C. Alexander, K. N. Busby, C. M. Cole,
C. Y. Zhou and N. K. Devaraj, J Am Chem Soc 2015, 137, 12756-12759;
i) C. Y. Zhou, S. C. Alexander and N. K. Devaraj, Chem Sci 2017, 8,
7169-7173; j) A. Osipenko, A. Plotnikova, M. Nainyte, V. Masevicius, S.
Klimasauskas and G. Vilkaitis, Angewandte Chemie-International Edition
2017, 56, 6507-6510; k) M. Tomkuviene, B. Clouet-d'Orval, I.
[19] P. Śledź and M. Jinek, eLife 2016, 5, e18434.
[20] S. Schwartz, M. R. Mumbach, M. Jovanovic, T. Wang, K. Maciag, G. G.
Bushkin, P. Mertins, D. Ter-Ovanesyan, N. Habib, D. Cacchiarelli, N. E.
Sanjana, E. Freinkman, M. E. Pacold, R. Satija, T. S. Mikkelsen, N.
Hacohen, F. Zhang, S. A. Carr, E. S. Lander and A. Regev, Cell Rep
2014, 8, 284-296.
[21] a) X. Shu, Q. Dai, T. Wu, I. R. Bothwell, Y. Yue, Z. Zhang, J. Cao, Q. Fei,
M. Luo, C. He and J. Liu, Journal of the American Chemical Society 2017;
b) K. Hartstock, B. S. Nilges, A. Ovcharenko, N. V. Cornelissen, N. Pullen,
A. M. Lawrence-Dorner, S. A. Leidel and A. Rentmeister, Angew Chem
Int Ed Engl 2018, 57, 6342-6346.
[22] a) K. E. Pendleton, B. Chen, K. Liu, O. V. Hunter, Y. Xie, B. P. Tu and N. K.
Conrad, Cell 2017, 169, 824-835.e814; b) H. Shima, M. Matsumoto, Y.
Ishigami, M. Ebina, A. Muto, Y. Sato, S. Kumagai, K. Ochiai, T. Suzuki
and K. Igarashi, Cell Rep 2017, 21, 3354-3363; c) A. Ruszkowska, M.
Ruszkowski, Z. Dauter and J. A. Brown, Sci Rep 2018, 8, 5311; d) K. A.
Doxtader, P. Wang, A. M. Scarborough, D. Seo, N. K. Conrad and Y.
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202013936
Angewandte Chemie International Edition
RESEARCH ARTICLE
Nam, Mol Cell 2018, 71, 1001-1011 e1004; e) A. S. Warda, J.
Kretschmer, P. Hackert, C. Lenz, H. Urlaub, C. Höbartner, K. E. Sloan
and M. T. Bohnsack, EMBO reports 2017, 18, 2004-2014.
[23] M. Mendel, K. M. Chen, D. Homolka, P. Gos, R. R. Pandey, A. A. McCarthy
and R. S. Pillai, Mol Cell 2018, 71, 986-1000 e1011.
[24] Z. Vanikova, M. Janouskova, M. Kambova, L. Krasny and M. Hocek, Chem
Sci 2019, 10, 3937-3942.
[25] J. Aschenbrenner, S. Werner, V. Marchand, M. Adam, Y. Motorin, M. Helm
and A. Marx, Angew Chem Int Ed Engl 2018, 57, 417-421.
[26] S. Singh, J. J. Zhang, T. D. Huber, M. Sunkara, K. Hurley, R. D. Goff, G. J.
Wang, W. Zhang, C. M. Liu, J. Rohr, S. G. Van Lanen, A. J. Morris and
J. S. Thorson, Angewandte Chemie-International Edition 2014, 53, 3965-
3969.
[27] 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.
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202013936
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
The m6A methyltransferase METTL3-METTL14 showed remarkable cosubstrate promiscuity, enabling sequence-specific internal
labeling of RNA. The transfer of ortho-nitrobenzyl and 6-nitropiperonyl groups allowed enzymatic photocaging of RNA. METTL16 was
less promiscuous but suitable for chemo-enzymatic labeling via click chemistry. Since both enzymes act on distinct sequence motifs,
their combination allowed to modify different sites in a single RNA.
Institute and/or researcher Twitter usernames: @RentmeisterLab
7
This article is protected by copyright. All rights reserved.