New AdoMet Analogues as Tools for Enzymatic Transfer of Photo-Cross-Linkers and Capturing RNA–Protein Interactions

Article abstract of DOI:10.1002/chem.201605663

Elucidation of biomolecular interactions is of utmost importance in biochemistry. Photo-cross-linking offers the possibility to precisely determine RNA–protein interactions. However, despite the inherent specificity of enzymes, approaches for site-specifi

Full text of DOI:10.1002/chem.201605663

A Journal of
Accepted Article
Title: Novel AdoMet analogues as tools for enzymatic transfer of photo-
crosslinkers and capturing RNA-protein interactions
Authors: Fabian Muttach, Florain Mäsing, Armido Studer, and Andrea
Rentmeister
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To be cited as: Chem. Eur. J. 10.1002/chem.201605663
Link to VoR: http://dx.doi.org/10.1002/chem.201605663
Supported by

Chemistry - A European Journal
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COMMUNICATION
Novel AdoMet analogues as tools for enzymatic transfer of photo-
crosslinkers and capturing RNA-protein interactions
[a]
[b]
[b]
[a,c]
Fabian Muttach, Florian Mäsing, Armido Studer and Andrea Rentmeister*
[
8]
Abstract: Elucidation of biomolecular interactions is of utmost
importance in biochemistry. Photo-crosslinking offers the possibility
to precisely determine RNA-protein interactions. However, despite
the inherent specificity of enzymes, approaches for site-specific
introduction of photo-crosslinking moieties into nucleic acids are
scarce. Methyltransferases in combination with synthetic analogues
of their natural cosubstrate S-adenosyl-L-methionine (AdoMet) allow
for the post-synthetic site-specific modification of biomolecules. We
report on three novel AdoMet analogues bearing the most
widespread photo-crosslinking moieties (aryl azide, diazirine and
benzophenone). We show that these photo-crosslinkers can be
enzymatically transferred to the methyltransferase target, i.e. the
mRNA cap, with high efficiency. Photo-crosslinking of the resulting
modified mRNAs with the cap interacting protein eIF4E was
successful with aryl azide and diazirine but not benzophenone,
reflecting the affinity of the modified 5' caps.
synthesis and was used for interstrand photo-crosslinking as
[
9]
well as for crosslinking to DNA-binding proteins. DNA was also
modified with four different photo-reactive moieties including
benzophenone and crosslinked to yeast RNA polymerase III.
RNA with photo-crosslinking moieties at defined positions could
be synthesized as well. Diazirines were incorporated into a
chemically synthesized microRNA and used for photo-
[10]
[
11]
crosslinking with RNA-interacting proteins.
Aryl azides were
employed for crosslinking in an RNA duplex and for probing of
[
12]
[
13]
a Diels-Alderase ribozyme.
However, only few enzymatic approaches for site-specific
introduction of photo-crosslinkers have been reported. A 5' cap
analogue
containing
6-thioguanosine
was
chemically
synthesized and enzymatically incorporated at the 5' end of RNA
during in vitro transcription. Upon irradiation at 365 nm,
[
14]
intrastrand photo-crosslinking of RNA was observed.
Site-
specific enzymatic incorporation of a photo-crosslinking moiety
at an internal position was achieved in a different study using an
unnatural base-pairing system.
We thought that enzymatic conversions should be ideal for site-
specific introduction of photo-crosslinking moieties if an
Photo-crosslinking has become an enabling technique for
mapping interactions of biomolecules – even if they are weak or
transient – and for clarifying structures of bigger complexes. In
the field of proteins, site-specific incorporation of amino acids
with photo-crosslinking moieties advanced the field significantly
because pinpointing the sites on a protein in the vicinity of the
interacting protein became possible. In the field of nucleic
acids, irradiation with short-wave UV-light has long been used
for crosslinking and immunoprecipitation (CLIP) methods, even
though it damages nucleic acids at the required wavelengths
[15]
appropriate
biocatalyst
is
available.
In
particular,
methyltransferases (MTases) have been successfully used for
specific functionalization of biomolecules. Instead of transferring
a methyl group from their natural cosubstrate S-adenosyl-L-
methionine (AdoMet) to their target molecule, several MTases
can be used or engineered to transfer other functional moieties
[1]
[16]
from synthetic analogues. During the past few years, AdoMet
[
2]
(
~254 nm). Advanced CLIP based techniques therefore rely on
analogues with extended side-chains bearing different alkene,
alkyne, amino, azido or 4-vinyl benzyl functionalities have been
[
3]
incorporation of 4-thiouridine or 6-thioguanosine compatible
with longer wavelengths (~365 nm). Importantly, CLIP methods
allow for transcriptome-wide identification of RNA-binding sites
and are compatible with living cells and could be coupled with
high-resolution mass-spectrometry.
developed and used in combination with highly promiscuous
[17]
MTases.
The transferred moieties were used to label the
[
3]
[18]
[19]
MTase targets , to alter their biological functions or for pull-
down studies.
[
4]
[20]
In a completely different approach, S-
Akin to the protein-centred approaches, site-specific instead of
randomly distributed installation of photo-crosslinkers into
nucleic acids helps to precisely assign interactions. The most
widespread photo-crosslinking moieties available to date are aryl
adenosylhomocysteine (SAH) analogues with crosslinking
moieties have been developed. These molecules are known
MTase inhibitors and were used for binding and profiling
[21]
MTases.
However, AdoMet analogues containing photo-
[
5]
azides, benzophenones and diazirines. Additionally, furan-
moieties allowed for the mild red-light dependent nucleic acid
crosslinkers have not been described to the best of our
knowledge, nor has the post-synthetic enzymatic transfer of
photo-crosslinkers to biomolecules been reported. We aimed to
address this shortcoming by synthesizing AdoMet analogues
suitable for transfer of the most widespread photo-crosslinking
groups, namely aryl azides, diazirines and benzophenones. We
then wanted to test whether such bulky moieties are still suitable
for enzymatic transfer and if they would thus allow for site-
specific post-synthetic modification of biomolecules. Lastly, we
wanted to see whether the installed moieties would be functional
for photo-crosslinking based on a known interaction partner.
[
6]
[7]
interstrand and nucleic acid-protein crosslinking. DNA with
diazirines at specific positions is accessible by solid-phase
[
a]
Fabian Muttach, Prof. Dr. A. Rentmeister, University of Münster,
Department of Chemistry, Institute of Biochemistry,
Wilhelm-Klemm-Str. 2, 48149 Münster (Germany)
E-mail: a.rentmeister@uni-muenster.de
[
[
b]
c]
Florian Mäsing, Prof. Dr. A. Studer, University of Münster,
Department of Chemistry, Institute of Organic Chemistry,
Corrensstr. 40, 48149 Münster (Germany)
Prof. Dr. A. Rentmeister
Cells-in-Motion Cluster of Excellence (EXC 1003—CiM)
University of Münster (Germany)
Results and Discussion
For MTase-based enzymatic transfer of photo-crosslinking
groups, suitable AdoMet analogues and a promiscuous MTase
are required. For efficient transfer of the AdoMet analogue side-
Supporting information for this article is given via a link at the end of
the document.
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Chemistry - A European Journal
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COMMUNICATION
chain an unsaturated bond is required in ß-position of the
sulfonium center, most likely due to a stabilizing conjugation of
the p orbital with the antibonding π orbital in the transition
[
17a]
state.
It is known that alkyl chains other than methyl (e.g.
[
17a]
ethyl or propyl) are barely transferred by methyltransferases.
Based on these considerations we designed three novel AdoMet
analogues bearing the most widespread photo-crosslinking
moieties in the side-chain (Scheme 1).
Scheme 1. Chemical structure of novel AdoMet analogues with photo-
crosslinking side-chains.
Scheme 2. Synthesis of the novel AdoMet analogues AdoArAz (1a) (A),
AdoDiaz (1b) (B) and AdoBP (1c) (C), bearing photo-crosslinking moieties in
their side-chain.
Since these AdoMet analogues are very bulky, they will not be
accepted by every MTase but require a highly promiscuous
enzyme for transfer. We recently reported on the very high
substrate promiscuity of cap N7 methyltransferase Ecm1, which
accepted alkynes, azides, and even aromatic moieties with
To elucidate whether our new AdoMet analogues will be suitable
for enzymatic conversions, we tested their stability under typical
reaction conditions and analyzed degradation pathways. To this
end, 1a-c were incubated in a buffer consisting of 200 mM
NaCl/1mM EDTA/10 % glycerol at 37 °C and pH = 8 and
analyzed at different time points by HPLC. Quantification of
remaining 1a-c over time revealed half-lives of ~2.3 h for 1a,
[
19]
uncompromised activity.
MTase for further studies.
Ecm1 was therefore chosen as the
Scheme 2 shows the synthesis of the three novel AdoMet
analogues. To obtain the aryl-azide bearing AdoMet analogue
1a, we started from 3-aminobenzyl alcohol (2) and converted it
~2 h for 1b, and ~1.5 h for 1c, respectively (ESI Figure S6).
to the respective azide 3 (Scheme 2A, experimental details in
ESI). Compound 3 was then converted to the mesylate 4 and
reacted with S-adenosyl-L-homocysteine 5 to 1a. Preparation of
diazirine-containing AdoMet analogue 1b required synthesis of
These half-lives are in the range of previously reported AdoMet
analogues and should thus be sufficient for methyltransferase-
[22]
based conversions.
Using LC/MS, we were also able to
identify the decay products of 1a-c. In line with previous reports,
we observed formation of 5, but also adenine resulting from
cleavage of the glycosidic bond, releasing adenine (ESI Figure
3
-[(4-hydroxymethyl)phenyl]-3-trifluoromethyl-3H-diazirine (6) in
[
11]
seven steps according to a published procedure.
The benzyl
[
22]
alcohol 6 was then converted to the respective mesylate 7 and
reacted with (Scheme 2B). For the synthesis of
S7 and S8).
5
benzophenone-containing AdoMet analogue 1c we simply
Enzymatic transfer of photo-crosslinking moieties
reacted commercially available 4-(bromomethyl)benzophenone
Next, we tested whether 1a-c are suitable cosubstrates for
enzymatic transfer of photo-crosslinking moieties. To this end,
we used the methyltransferase Ecm1, which shows remarkable
(
4
8) with 5 (Scheme 2C). In this case, AgClO was added to
precipitate AgBr to increase the conversion. While in the case of
mesylates, reaction times of 4 h were sufficient, reactions were
performed overnight if a bromide was used. AdoMet analogues
[
19]
cosubstrate promiscuity.
Ecm1 naturally transfers methyl
groups to the N7 position of the 5' cap of mRNA but also acts on
[
23]
1
a-c were purified via RP-HPLC and characterized by HPLC,
a minimal dinucleotide cap GpppA 9.
GpppA were reacted with 1-2 mM 1a-c and 20 mol% Ecm1. The
enzymes 5'-methylthioadenosine/S-adenosylhomocysteine
Typically, 300 µM
1
MS, and H-NMR (ESI Figure S3, S4, S16-19). These data were
backed up by collision induced decay (CID)-MS/MS and the
fragment spectra could be assigned to the respective AdoMet
analogues (ESI Figure S5). The concentration of AdoMet
analogues was determined by weighing of the compounds. It is
important to point out that all AdoMet analogues were isolated
as equal mixtures of the S- and R-epimers, meaning that the
biologically effective concentration is actually reduced to half.
We always indicate the total concentrations used in the
respective conversions.
nucleosidase (MTAN) and LuxS were added to all reactions in
order to enzymatically degrade the by-product S-
adenosylhomocysteine which is
methyltransferases.
a
strong inhibitor of
For all three AdoMet analogues tested, efficient transfer of the
respective photo-crosslinking group to 9 was observed (Figure
1A). The product peaks detected in HPLC analysis were also
analyzed by MS to validate formation of 10a-c, respectively (see
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Chemistry - A European Journal
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COMMUNICATION
Figure 1B and C). Specifically, quantitative conversions could be
achieved under the conditions described above and using 2 mM
of the AdoMet analogue. To further characterize the enzymatic
transfer reaction, we performed time course experiments under
the conditions mentioned above and using 1 mM of the
respective AdoMet analogue. These measurements revealed
that Ecm1-catalyzed transfer reactions are surprisingly fast and
efficient considering the bulkiness of 1a-c. For 1a-b, transfer
was complete after 60-90 minutes based on the consumption of
GpppA and appearance of a new peak which could be assigned
to the modified cap analogue (see Figure 1D). For 1c, however,
only 60 % modification product was observed under these
conditions suggesting that the benzophenone moiety partially
impedes transfer, most likely due to its extreme sterical demand.
Furthermore, we found the enzymatic transfer using Ecm1 to be
7
highly regiospecific. When the cap analogue m GpppA (N7
position not accessible) was used in combination with Ecm1 no
enzymatic transfer of the photo-crosslinking moiety was
observed (ESI Figure S20).
Taken together, these data suggest that the new AdoMet
analogues 1a-c are sufficiently stable and suitable for enzymatic
conversions resulting in postsynthetic modification of the 5' cap
with photo-crosslinking moieties. Since multiple other MTases
with similar architecture (i.e. an open binding site) and high
cosubstrate promiscuity are known, we anticipate that 1a-c will
also be suitable for enzymatic modification of different classes of
[
22, 24]
target molecules.
Figure 1. (A) Enzymatic transfer of photo-crosslinking moieties to the cap
analogue GpppA (300 µM) using the respective AdoMet analogue (2 mM) and
the methyltransferase Ecm1 (60 µM). (B) HPLC analysis of enzymatic transfer
reactions using the photo-crosslinking AdoMet analogues 1a-c. (C) Mass
spectrometric analysis (LC-ESI/MS) of the enzymatic transfer reactions.
2
+
+
+
Expected mass for C27
H
34
N
C
13
O
17
P
3
= 452.5699 [M +H ], found: 452.5709
2+
+
+
(1a); expected mass for
29
H
34
F
3
N
12 17
O P
3
= 486.0859 [M +H ], found:
2+ + +
4
86.0667 (1b); expected mass for C34
H
39
N
10 18
O P
3
= 484.0823 [M +H ],
found: 484.0889 (1c); (D) Time course experiments for enzymatic transfer of
the novel AdoMet analogues 1a-c (1mM) using Ecm1 and the cap analogue
GpppA. All reactions were performed in duplicates and average values are
depicted.
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COMMUNICATION
Labelling with functional probes using aromatic azide 1a
Aromatic azides are not only suitable for photo-crosslinking
purposes but also for bioorthogonal reaction in
a strain-
promoted azide-alkyne cycloaddition (SPAAC reaction) using a
strained alkyne. To evaluate the reactivity of the aromatic azide
we transferred the aromatic azide of 1a to GpppA 9 followed by
direct reaction of the azido-modified cap analogue 10a with the
cyclooctyne MegaStokes dye 608 (Sigma). Separation of the
reaction mixture by PAGE and in-gel fluorescence analysis
yielded a fluorescent band only if all components were present
(
ESI Figure S21A). Control reactions omitting either Ecm1 or the
AdoMet analogue in the enzymatic reaction did not give a
fluorescent band. Additionally, we reacted the azido-modified
cap 10a with DBCO-biotin for 1 h at 37 °C and confirmed
product formation by LC-MS analysis (expected mass for
2
3
+
+
+
C
69
H
90
N
19
O
25
P
3
S
= 886.7360 [M +H ], found: 886.7321; see
ESI Figure S22). We also extended our labelling strategy to the
modification of a 24 nt model RNA. To this end we enzymatically
capped the RNA, subjected it to enzymatic modification and
SPAAC reaction and after PAGE separation and SYBR-Gold
staining analyzed the RNA. Again, a fluorescent band was only
observed if all components were present (ESI Figure S21B). It
should however be noted that aryl azides are susceptible to
reduction as observed in an LC-MS analysis (ESI Figure S9).
Reduction of the azido moiety could be circumvented by
switching to a DTT-free reaction buffer.
Crosslinking
To assess whether the photo-crosslinking groups are functional
and the reactive species is produced, we tested the photo-
reactivity of the novel AdoMet analogues. To this end, 1a-c were
continuously irradiated at their respective wavelengths (312 nm
for 1a, 365 nm for 1b and both wavelengths for 1c). At different
time points, samples were taken and their absorption spectra
measured (Figure 2 and ESI Figure S10). In each case, changes
in absorption spectra were observed indicating photo-reactivity
of the AdoMet analogues.
Figure 2. Representative absorption spectra of AdoMet analogues 1a-c before
and after photo-irradiation for 20 min at either 312 nm (1a) or 365 nm (1b and
.
1c). After indicated time-points samples were taken and absorption spectra
were recorded. Changes in the absorption spectra indicate photo-reactivity of
the respective AdoMet analogues (A) 1a with irradiation at 312 nm (poly-styrol
filtered light) (B) 1b with irradiation at 365 nm (C) 1c with irradiation at 365 nm.
To test whether the photo-crosslinking moieties are still
functional after transfer to the MTase target (i.e. the modified 5'
cap), we prepared 24 nt model RNAs with different 5' caps. The
RNAs were produced enzymatically (T7 transcription) and
capped using the Vaccinia capping system. To produce RNAs
with modified caps, the AdoMet was omitted at this step.
Uncapped RNAs were enzymatically degraded using
a
combination of 5'-polyphosphatase (Epicentre) and terminator
exoribonuclease XRN-1 (NEB) (ESI Figure S24 and S25). The
remaining RNA was then enzymatically modified using Ecm1
and 1a-c, respectively using conditions that should yield
complete conversion.
These capped 24 nt RNAs (termed 10a-c-RNAs) were then
tested in binding and crosslinking studies with a known cap
interacting protein eIF4E (Figure 3A). The eIF4E from S.
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Chemistry - A European Journal
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COMMUNICATION
cerevisiae was recombinantly expressed in E. coli and purified.
Identity and purity of eIF4E (≥75 %) was confirmed by SDS-
PAGE and LC-ESI-MS (ESI Figure S2). S. cerevisiae eIF4E was
amounts of sample and fluorescent labelling of one binding
partner. To this end we prepared 10a-c as described above.
[
25]
Purified eIF4E was labelled with a Cy5-NHS dye and titrated to
7
7
7
incubated with the 10a-c-RNAs or m GpppG-RNA as control
different concentrations of m GTP, m GpppA, 9, or 10a-c,
and irradiated. Specifically, the 10a-RNA mixture was irradiated
for 20 min at 312 nm using an UV-table. The 10b-c-RNAs were
irradiated for 30 min at 365 nm using an LED (1000 mW).
Separation on a 10 % denat. PAA gel revealed an additional
band with lower electrophoretic mobility for 10a- and 10b-RNAs
in the presence of eIF4E and after irradiation, whereas for 10c-
RNA an additional band could not be unambiguously detected
respectively. From these data, we could determine K
using the integrated software.
d
-values
However, we observed that in different sets of experiments the
-values varied depending on details of the protein labelling
procedure and the measuring mode. This limits the accuracy of
the absolute K -values obtained by microscale thermophoresis,
as recently documented in detail.
comparison of binding affinities of different cap analogues is
d
possible, although the absolute numbers obtained for the K -
K
d
d
[26]
Nevertheless, a reliable
(
Figure 3B). The additional band does not occur if the mixture of
0a-b-RNA and eIF4E (at identical concentrations) was not
1
irradiated, indicating that photo-crosslinking had occurred.
Interestingly, for 10b-RNA a shifted band was also observed
when irradiation was performed in the absence of the cap
binding protein eIF4E, suggesting that 10b-RNA might react with
values should not be over interpreted. Representative
thermophoresis measurements are given in the supplementary
information (ESI Table S1 and Figure S23).
In a comparative evaluation of all modified cap analogues used
[
11]
7
7
itself in the absence of a suitable binding partner.
This
in this study, binding tests of eIF4E with m GTP and m GpppA
clearly showed the lowest K -values, as would be expected for
potential self-crosslinking band however showed a different
length from the RNA-protein crosslink band and did not occur in
the presence of eIF4E.
d
7
the natural binding partners. The dissociation constant of m GTP
7
(~ 1 µM) was lower than for m GpppA (~ 5 µM), confirming
[
27]
previous reports based on different methods.
For 10a-b, the
K
d
-values for binding to eIF4E were markedly increased, namely
in the range of ~50 µM (Figure 4). For 9 and 10c, however, no
binding to eIF4E could be measured (ESI Figure S23). For 9,
this is in accordance with the literature (reported values for
7
[28]
m GpppG range from 0.2 to 2.5 µM) and for 10c this explains
why no crosslinking of 10c-RNA with eIF4E could be seen.
Measurements were also performed using eIF4E from D.
melanogaster, giving comparable
d
K -values and thereby
confirming our thermophoresis experiments obtained with S.
cerevisiae eIF4E (data not shown). Based on these data, we
conclude that the modifications in 10a-RNA and 10b-RNA
impair binding to eIF4E, while in 10c-RNA binding is abolished.
At high concentrations of eIF4E there seems to be enough 10a-
b-RNA bound eIF4E to obtain crosslinking, indicating that the
crosslinking groups per se are functional.
Figure 3. Reactions of differently photo-crosslinker modified 5' RNA caps. (A)
Concept of methyltransferase-based transfer of photo-crosslinking moieties
and subsequent photo-crosslinking to an interacting protein. (B) Photo-
crosslinking of differently cap-modified 24 nt RNAs to eIF4E. Aryl-azide-,
diazirine- or benzophenone-modified 24 nt RNA (10a-c-RNA) was subjected to
photo-crosslinking with the yeast cap binding protein eIF4E (~100 ng RNA and
appr. 4 equiv. eIF4E). The RNA was resolved on a 15 % denat. PAGE (run for
appr. 2 h, 15 W, 1×TBE), stained with SYBR-gold and scanned on a Typhoon
FLA9500 laser scanner (λex = 473 nm; λem = 510 nm LP).
The lack of a photo-crosslinking band for 10c-RNA prompted us
to test whether the modified caps were still binding to eIF4E. In
general, photo-crosslinking based on benzophenone would be
expected to give higher crosslinking yields, because
benzophenone in the excited state may return to the ground
state in case hydrogen abstraction on the electron-deficient ketyl
oxygen does not take place. However, the benzophenone-
group is also the sterically most demanding moiety in our set.
Although this had only little effect on the enzymatic transfer from
Figure 4. Trend of dissociation constants for the interaction of eIF4E with
different N7-modified cap analogues determined by microscale
thermophoresis. As controls, binding of eIF4E to m GpppA, m GTP (positive
controls), and GpppA (negative control) was determined.
7
7
[
5]
Conclusions
1c to the 5' cap, the binding of 10c or 10c-RNA could be
We herein described the chemical synthesis of three novel
AdoMet analogues with photo-crosslinking properties. The
photo-crosslinking moieties were enzymatically transferred to
the N7 position of the mRNA cap with high efficiencies.
Diazirine- and aryl azide-modified cap analogues 10a-b retained
abolished and thus prevent crosslinking.
To benchmark the effect of the photo-crosslinking groups at the
N7 position of the 5' cap on binding to eIF4E, we performed
microscale thermophoresis measurements, which require low
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Chemistry - A European Journal
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COMMUNICATION
[
3] M. Hafner, M. Landthaler, L. Burger, M. Khorshid, J. Hausser, P. Berninger,
the ability to bind to the cap binding protein eIF4E, whereas 10c
was not bound. We also produced 24 nt long model mRNAs with
the differently modified caps and performed photo-crosslinking
studies to eIF4E. Herein, crosslinking of 10a-b-RNAs with eIF4E
was observed, whereas 10c-RNA showed no crosslinking, in
line with the binding constants. Since the wavelength required
for photo-crosslinking is longer and thus less damaging for the
diazirine compared to the aryl azide, the AdoMet analogue 1b is
currently the best choice for enzymatic transfer and photo-
crosslinking to a directly interacting protein.
Of course, the direct modification of a protein binding site (i.e.
the N7 position of the cap for eIF4E used in this study) is not an
ideal system for photo-crosslinking studies but should rather be
regarded as a proof of concept. We anticipate that our photo-
crosslinking approach will be particularly useful if sites adjacent
to the protein binding-site of interest can be enzymatically
modified. This can be achieved by smart choices of MTase and
target site or by developing new AdoMet analogues causing less
steric hindrance.
A. Rothballer, M. Ascano, Jr., A. C. Jungkamp, M. Munschauer, A. Ulrich, G. S.
Wardle, S. Dewell, M. Zavolan and T. Tuschl, Cell 2010, 141, 129-141.
[4] K. Kramer, T. Sachsenberg, B. M. Beckmann, S. Qamar, K. L. Boon, M. W.
Hentze, O. Kohlbacher and H. Urlaub, Nat. Methods 2014, 11, 1064-1070.
[
5] G. W. Preston and A. J. Wilson, Chem. Soc. Rev. 2013, 42, 3289-3301.
[6] a) M. Op de Beeck and A. Madder, J. Am. Chem. Soc. 2012, 134, 10737-
0740; b) L. L. Carrette, E. Gyssels, J. Loncke and A. Madder, Org. Biomol.
Chem. 2014, 12, 931-935.
7] L. L. Carrette, E. Gyssels, N. De Laet and A. Madder, Chem. Commun.
2016, 52, 1539-1554.
8] Z. Qiu, L. Lu, X. Jian and C. He, J. Am. Chem. Soc. 2008, 130, 14398-
4399.
[9] U. K. Shigdel, J. Zhang and C. He, Angew. Chem. Int. Ed. 2008, 47, 90-93.
10] J. J. Tate, J. Persinger and B. Bartholomew, Nucleic Acids Res. 1998, 26,
421-1426.
11] K. Nakamoto and Y. Ueno, J. Org. Chem. 2014, 79, 2463-2472.
1
[
[
1
[
1
[
[12] K. L. Buchmueller, B. T. Hill, M. S. Platz and K. M. Weeks, J. Am. Chem.
Soc. 2003, 125, 10850-10861.
[
[14] M. Nowakowska, J. Kowalska, F. Martin, A. d'Orchymont, J. Zuberek, M.
Lukaszewicz, E. Darzynkiewicz and J. Jemielity, Org. Biomol. Chem. 2014, 12,
13] R. Wombacher and A. Jäschke, J. Am. Chem. Soc. 2008, 130, 8594-8595.
4
[
841-4847.
15] M. Kimoto, M. Endo, T. Mitsui, T. Okuni, I. Hirao and S. Yokoyama, Chem.
Biol. 2004, 11, 47-55.
[
[
16] X. Cheng and R. J. Roberts, Nucleic Acids Res. 2001, 29, 3784-3795.
17] a) C. Dalhoff, G. Lukinavicius, S. Klimasauskas and E. Weinhold, Nat.
Chem. Biol. 2006, 2, 31-32; b) G. Lukinavicius, V. Lapiene, Z. Stasevskij, C.
Dalhoff, E. Weinhold and S. Klimasauskas, J. Am. Chem. Soc. 2007, 129,
2
758-2759; c) S. Willnow, M. Martin, B. Lüscher and E. Weinhold,
Experimental Section
Chembiochem 2012, 13, 1167-1173; d) K. Islam, I. Bothwell, Y. Chen, C.
Sengelaub, R. Wang, H. Deng and M. Luo, J. Am. Chem. Soc. 2012, 134,
5
909-5915; e) J. M. Holstein, D. Stummer and A. Rentmeister, Chem. Sci.
Experimental details can be found in the supplementary information.
2015, 6, 1362-1369.
[
2
18] a) D. Schulz, J. M. Holstein and A. Rentmeister, Angew. Chem. Int. Ed.
013, 52, 7874-7878; b) W. Peters, S. Willnow, M. Duisken, H. Kleine, T.
Macherey, K. E. Duncan, D. W. Litchfield, B. Lüscher and E. Weinhold, Angew.
Chem. Int. Ed. 2010, 49, 5170-5173; c) B. J. C. Law, A.-W. Struck, M. R.
Bennett, B. Wilkinson and J. Micklefield, Chem. Sci. 2015, 6, 2885-2892.
Acknowledgements
[
19] J. M. Holstein, L. Anhäuser and A. Rentmeister, Angew. Chem. Int. Ed.
2016, 55, 10899-10903.
20] R. Wang, K. Islam, Y. Liu, W. Zheng, H. Tang, N. Lailler, G. Blum, H.
A.R. thanks the DFG for support by an Emmy Noether fellowship
[
(
(
RE2796/2-1) and the Fonds der Chemischen Industrie
Dozentenpreis). F.M. is supported by the DFG Priority
Deng and M. Luo, J. Am. Chem. Soc. 2013, 135, 1048-1056.
[21] a) C. Dalhoff, M. Huben, T. Lenz, P. Poot, E. Nordhoff, H. Koster and E.
Weinhold, Chembiochem 2010, 11, 256-265; b) B. D. Horning, R. M. Suciu, D.
A. Ghadiri, O. A. Ulanovskaya, M. L. Matthews, K. M. Lum, K. M. Backus, S. J.
Brown, H. Rosen and B. F. Cravatt, J. Am. Chem. Soc. 2016, 138, 13335-
13343.
Programme SPP1784 (RE2796/3-1). This work was supported
by the SFB858. We thank Ann-Marie Lawrence-Dörner for
excellent technical support, Dr. Wolfgang Dörner for help with
LC-MS analysis and Henning Klaasen for assistance with
synthesis. The NMR (Dr. Klaus Bergander) and mass
spectrometry facilities (Dr. Matthias Letzel) at the Organisch-
Chemisches Institut, Münster are acknowledged for analytical
services.
[
22] G. Lukinavicius, M. Tomkuviene, V. Masevicius and S. Klimasauskas,
ACS Chem. Biol. 2013, 8, 1134-1139.
[23] S. Hausmann, S. Zheng, C. Fabrega, S. W. Schneller, C. D. Lima and S.
Shuman, J. Biol. Chem. 2005, 280, 20404-20412.
[
24] a) A. Plotnikova, A. Osipenko, V. Masevicius, G. Vilkaitis and S.
Klimasauskas, J. Am. Chem. Soc. 2014, 136, 13550-13553; b) 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; c) K. Islam, Y. Chen, H. Wu, I. R. Bothwell, G. J.
Blum, H. Zeng, A. Dong, W. Zheng, J. Min, H. Deng and M. Luo, Proc. Natl.
Acad. Sci. U S A 2013, 110, 16778-16783; d) K. Islam, W. Zheng, H. Yu, H.
Deng and M. Luo, ACS Chem. Biol. 2011, 6, 679-684.
Keywords: AdoMet analogue • RNA labelling • photo-
crosslinking • RNA-protein interactions
[
25] a) M. Jerabek-Willemsen, C. J. Wienken, D. Braun, P. Baaske and S.
Duhr, Assay Drug Dev. Technol. 2011, 9, 342-353; b) C. J. Wienken, P.
Baaske, U. Rothbauer, D. Braun and S. Duhr, Nat. Commun. 2010, 1, 100.
[
1] a) H. Baruah, S. Puthenveetil, Y. A. Choi, S. Shah and A. Y. Ting, Angew.
Chem. Int. Ed. 2008, 47, 7018-7021; b) N. Hino, Y. Okazaki, T. Kobayashi, A.
Hayashi, K. Sakamoto and S. Yokoyama, Nat. Methods 2005, 2, 201-206; c) J.
W. Chin, A. B. Martin, D. S. King, L. Wang and P. G. Schultz, Proc. Natl. Acad.
Sci. U S A 2002, 99, 11020-11024; d) H. W. Ai, W. Shen, A. Sagi, P. R. Chen
and P. G. Schultz, Chembiochem 2011, 12, 1854-1857; e) M. J. Schmidt and
D. Summerer, Angew. Chem. Int. Ed. 2013, 52, 4690-4693.
[
26] T. H. Scheuermann, S. B. Padrick, K. H. Gardner and C. A. Brautigam,
Anal. Biochem. 2016, 496, 79-93.
27] A. Niedzwiecka, J. Marcotrigiano, J. Stepinski, M. Jankowska-Anyszka, A.
[
Wyslouch-Cieszynska, M. Dadlez, A. C. Gingras, P. Mak, E. Darzynkiewicz, N.
Sonenberg, S. K. Burley and R. Stolarski, J. Mol. Biol. 2002, 319, 615-635.
[
28] Y. Jia, V. Polunovsky, P. B. Bitterman and C. R. Wagner, Med. Res. Rev.
[
2] a) M. Ascano, M. Hafner, P. Cekan, S. Gerstberger and T. Tuschl, Wiley
2012, 32, 786-814.
Interdiscip. Rev. RNA 2012, 3, 159-177; b) R. P. Sinha and D. P. Hader,
Photochem. Photobiol. Sci. 2002, 1, 225-236.
.
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