Trapping Transient RNA Complexes by Chemically Reversible Acylation

Article abstract of DOI:10.1002/anie.202010861

RNA-RNA interactions are essential for biology, but they can be difficult to study due to their transient nature. While crosslinking strategies can in principle be used to trap such interactions, virtually all existing strategies for crosslinking are poorly reversible, chemically modifying the RNA and hindering molecular analysis. We describe a soluble crosslinker design (BINARI) that reacts with RNA through acylation. We show that it efficiently crosslinks noncovalent RNA complexes with mimimal sequence bias and establish that the crosslink can be reversed by phosphine reduction of azide trigger groups, thereby liberating the individual RNA components for further analysis. The utility of the new approach is demonstrated by reversible protection against nuclease degradation and trapping transient RNA complexes of E. coli DsrA-rpoS derived bulge-loop interactions, which underlines the potential of BINARI crosslinkers to probe RNA regulatory networks.

Full text of DOI:10.1002/anie.202010861

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
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Accepted Article
Title: Trapping Transient RNA Complexes by Chemically Reversible
Acylation
Authors: Willem A Velema, Hyun Shin Park, Anastasia Kadina, Lucian
Orbai, and Eric T. Kool
This manuscript has been accepted after peer review and appears as an
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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
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202010861
Link to VoR: https://doi.org/10.1002/anie.202010861

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Trapping Transient RNA Complexes by Chemically Reversible
Acylation
Willem A. Velema, Hyun Shin Park, Anastasia Kadina, Lucian Orbai and Eric T. Kool*
nucleobases such as 4-thiouridine,[30] phenyl selenide uridine[31]
Abstract: RNA-RNA interactions are essential for biology, yet they
and carbazole derivatives,[32] but the need to incorporate these
can be difficult to study due to their transient nature. While
modifications in the RNA of interest prior to use hinders their
crosslinking strategies can in principle be used to trap such
application in native settings. Furthermore, most of these
interactions, virtually all existing strategies for crosslinking are poorly
methods form irreversible crosslinks, which can interfere with
reversible, chemically modifying the RNA and hindering molecular
downstream analysis of crosslinked RNA complexes. This
analysis. Here, we describe a soluble crosslinker design (BINARI) that
reacts with RNA via acylation. We find that it efficiently crosslinks
noncovalent RNA complexes with miminal sequence bias and
establish that the crosslink can be reversed by phosphine reduction
of azide trigger groups, liberating the individual RNA components for
further analysis. Use of the new approach is demonstrated by
reversible protection against nuclease degradation and by trapping
transient RNA complexes of E. coli DsrA-rpoS derived bulge-loop
interactions, which underlines the potential of BINARI crosslinkers to
probe RNA regulatory networks.
problem can be overcome by applying small molecule
crosslinkers such as psoralen,[33,34] which was discovered to
crosslink opposing pyrimidines in duplex nucleic acids upon
irradiation with 365 nm light.[33] Exposure to 254 nm light reverses
the crosslink, and this forms the basis for several recent
transcriptome-wide methods for interrogation of RNA-RNA
interactions like psoralen analysis of RNA interactions and
structures (PARIS)[35] and ligation of interacting RNA followed by
high-throughput sequencing (LIGR-seq).[36] However, psoralen
crosslinking can suffer from low crosslink efficiency[37] and modest
recovery yields,[38] and is biased towards pyrimidine bases.[39]
To overcome these obstacles, we describe here a new
molecular approach to crosslinking transient RNA-RNA
interactions. Our design involves bis-nicotinic azide reversible
interaction (BINARI) probes bearing two electrophilic moieties
that acylate 2'-OH groups of nearby ribonucleotides,[5] resulting in
a covalent link between interacting RNA strands (Fig. 1).
Phosphine-mediated reduction of azide trigger groups
conveniently reverses the crosslink, enabling downstream
analysis of individual components of RNA complexes. We find
that crosslinks are formed in high yields of up to 84% with minimal
sequence bias and are readily reversed at up to 70% yield.
BINARI is also functional in complex biological media such as cell
lysates. We show that RNA can be temporarily protected from
nuclease degradation by BINARI crosslinking, which might prove
useful for RNA handling and storage applications. Finally, the
utility of BINARI is demonstrated by trapping transient RNA-RNA
interactions of hairpins derived from the E. coli DsrA-rpoS RNA
complex. The captured RNA-RNA complexes are readily
reversed to identify the individual components. We expect that
this method will find use for studying both inter- and intra-
molecular RNA-RNA interactions.
Recent studies have revealed the existence of numerous and
complex biological roles of RNA,[1–3] eliciting the need for chemical
tools that can aid in analyzing biomolecular structure and
interactions.[4,5] RNA has been shown to play a central role in
many important regulatory networks.[6,7] For example, interactions
between non-coding RNAs (ncRNA) and proteins are crucial for
transcriptional regulation,[8] chromatin modifications[9] and post-
translational modifications,[10] among others.[10] Riboswitches
interact with small-molecule substrates to regulate gene
expression,[11] such as the thiamine pyrophosphate (TPP)
riboswitch in bacteria and several eukaryotes.[12,13] Further, RNA
can interact with other RNA molecules to regulate RNA
splicing,[14] translation[15] and modification.[16] For example, small
nucleolar RNAs (snoRNAs) have been shown to guide
modifications on ribosomal RNA (rRNA) through base pairing.[16]
Other prominent examples include the binding of miRNAs to
messenger RNAs to control translation,[17] and the dimerization of
viral RNA genomes.[18] However, it has been challenging to study
such RNA-RNA interactions due to the transient nature of most of
these complexes,[19–21] and many such interactions likely remain
uncharacterized.
The majority of methods for studying nucleic acid
interactions rely on chemical crosslinking.[22–25] The most
widespread example of this is formaldehyde crosslinking,[26] which
involves adducts to exocyclic amine groups; while it is routinely
used for preserving RNA in clinical settings and for studying
biomolecular interactions,[27] it suffers from low recovery
yields.[27,28] Other amine-reactive aldehydic crosslinkers include
1,4-phenyl-diglyoxal.[29] More selective crosslinking methods
have been developed more recently, including the use of modified
Our design of a chemically reversible crosslinker relies on
high-yield chemistry for 2’-OH acylation as well as a strategy for
efficient reversal. We recently reported the azide-substituted RNA
H.S. Park, Dr. A. Kadina, Prof. Dr. E.T. Kool
Department of Chemistry
Stanford University
Stanford, CA 94305, USA
E-mail: kool@stanford.edu
Figure 1. RNA crosslinking through reversible bis-acylation. A) Schematic
diagram of RNA strands crosslinked with BINARI probes and later crosslink
reversal upon phosphine treatment. B) Molecular structure of BINARI probes
and crosslinking through bis-acylation of 2'-OH groups of spatially proximal
nucleotides. Phosphine-mediated reduction of the azide groups results in
intramolecular lactam formation and liberation of the crosslinked RNA strands.
Dr. W.A. Velema
Institute for Molecules and Materials
Radboud University
Nijmegen, 6525 AJ, The Netherlands
Supporting information for this article is given via a link at the end of the
document
Dr. Lucian Orbai
Cell Data Sciences
46127 Landing Pkwy
Fremont, CA 94538, USA
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10.1002/anie.202010861
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COMMUNICATION
acylating reagent NAI-N3,[40] which was able to modify a large
fraction of 2'-OH positions of model RNA strands,[41] and this
reaction was shown to be reversible upon treatment with
phosphines.[41] For the new design, we envisioned chemically
related reversible acylating moieties that are linked via
polyethylene glycol (PEG) spacers. Three crosslinkers were
prepared with increasing spacer length: PEG1, PEG3 and PEG5
(Fig. 1B). The compounds were synthesized from ethyl 5-nitro-2-
methyl-nicotinate (1) (Scheme 1), which was converted to
phenolic compound 3 via a consecutive reduction and Sandmeyer
reaction. TBDMS protection and bromination afforded compound
5, which was reacted with sodium azide and deprotected with
TBAF to obtain intermediate 7. Reaction with electrophile-
modified PEG linkers resulted in compounds 8a, 8b and 8c, which
were converted to the final crosslinkers by hydrolyzing the esters
and activating the acid with carbonyldiimidazole.
Figure 2. RNA crosslinking. A) Sequences of self-complementary RNA strands
in this experiment. B) PAGE analysis of BINARI 3 crosslinked RNA 1. C)
Crosslinking yields of the different BINARI probes with self-complementary RNA
1. D) Crosslinking yields of BINARI 5 at different concentrations. E) Crosslinking
yields of BINARI 5 at different incubation times. Error bars represent s.d. of
triplicate experiments.
Crosslinking conditions were optimized using BINARI 5
with the dimeric RNA 1. Testing increasing concentrations of
crosslinker showed that the greatest amount of crosslinked RNA
was formed, not surprisingly, at the highest concentration tested
(100 mM) (Fig. 2D and Fig. S2), and increased incubation time
resulted in more efficient crosslinking, which plateaued after
about 4 hours (Fig. 2E and Fig. S3). Next, we tested if the
crosslinked RNA complex could be reversed into the original
single stranded RNA (ssRNA) upon phosphine treatment. 4-
(Diphenylphosphino)benzoic acid (DPBA), triphenylphosphino-3-
sulfonic acid sodium salt (TPPMS) and tris(hydroxypropyl)
phosphine (THPP) (Fig. 3A) as initial candidates for reductants[41]
were incubated with the crosslinked RNA 1 complex for 2 hours
at room temperature and analyzed by PAGE (Fig. 3B).
Scheme 1. Synthesis of BINARI probes. Reagents and conditions: (i) Pd/C 10%,
H2, MeOH, 40 °C, 48 h; (ii) NaNO2, H2O, H2SO4, 0 °C-100 °C, 30 min; (iii)
TBDMSCl, imidazole, CH2Cl2, rt, 2h; (iv) NBS, AIBN, CCl4, 70 °C, 3 h; (v) NaN3,
DMF, rt, 16 h; (vi) TBAF, THF, rt, 48 h; (vii) Tf(C2H4O2)n, DIPEA, CH2Cl2, 0 °C-
rt, 2 h; (viii) NaOH, H2O, MeOH, rt, 16 h; (ix) CDI, DMSO, rt, 1 h.
An in vitro test with model RNA strands was executed to
assess the performance of the crosslinkers. The partially self-
complementary short RNA 1 (10 μM), which is complementary via
only 6 base pairs, (Fig. 2A) was incubated with BINARI 1, 3, or 5
(100 mM) in 100 mM MOPS buffer (pH= 7.5, 6 mM MgCl2, 100
mM NaCl) for 4 hours at room temperature. RNA was purified by
precipitation and the amount of crosslinking was analyzed by 20%
polyacrylamide gel-electrophoresis (PAGE) (Fig. 2B), quantified
by the intensity of the bands. Under these conditions BINARI 3
and BINARI 5 achieved a high degree of crosslinking into the
dimeric form (84% and 75% respectively), while BINARI 1
provided a lesser 45% crosslinking yield, which may reflect less-
than-optimal linker length to bridge the two reactive RNA groups
(Fig. 2C and Fig. S1). These results suggest that significant
distance between the two acylating groups is required for most
efficient crosslinking. This is in accordance with earlier findings
that acylation of RNA mainly occurs in non-base-paired regions,[5]
and as such the reactive 2'-OH groups may be further apart than
they are in RNA duplexes. This hypothesis is further supported by
experiments showing decreased crosslinking efficiency when the
optimized conditions were applied to RNA 2 (Fig. S4), which has
the same base pairing sequence as RNA 1, but lacks the
noncomplementary tails. The experiments indicate that
crosslinking mostly occurs in non-base paired regions, but does
rely on duplex formation to bring opposing strands in close
proximity. When the analogous DNA variant of RNA 1 was used,
no crosslinking was observed (Fig. S4), which demonstrates that
BINARI is selective for RNA-RNA interactions. To conclusively
demonstrate that the lower mobility band observed by PAGE is
crosslinked RNA, Cy5-labeled RNA S2 was treated with BINARI
3 alone or in the presence of complementary RNA S1 (Fig. S5).
Imaging using a Cy5-fluorescence filter set showed that the lower
mobility band is only observed when both complementary RNA
strands are present, implying that this band is indeed crosslinked
RNA and not monofunctionalized RNA (Fig. S5).
Figure 3. Reversal of RNA crosslinks. A) Structures of tested phosphines. B)
PAGE analysis of BINARI 3 crosslinked RNA 1 and THPP reversal of
crosslinked RNA 1. C) Amount of uncrosslinked- monofunctionalized and
crosllinked RNA 1 obtained after incubation with each of three phosphines. D)
Yields of the crosslink reversal reactions at varied concentrations of THPP. E)
Amount of uncrosslinked RNA obtained as a function of incubation times with
THPP. Error bars represent s.d. of triplicate experiments.
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10.1002/anie.202010861
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Treatment with DPBA and TPPMS resulted in low yields
of 2% and 15% of unmodified RNA 1 respectively, while THPP
efficiently reversed the crosslink, resulting in 70% recovery of
unmodified RNA 1 (Fig. 3C and Fig. S6). The difference in
reversal efficiency between the tested phosphines can potentially
be explained by the more flexible nature and lower steric bulk of
THPP compared to DPBA and TPPMS, which could allow for it to
access sterically hindered crosslinks.
RNA from nuclease degradation; future experiments will explore
the applications of BINARI for RNA preservation purposes, which
is highly important for clinical sample storage[28]
.
One outstanding challenge in RNA structural and
functional analysis is to capture and analyze transient RNA
complexes.[45,46] One such class of interactions are loop-loop
interactions in bacterial and viral systems, which can occur when
two hairpins have sequence complementarity in their loops.[47] For
example, the HIV-1 virus genomic RNA is dimerized via “kissing
loop” interactions.[48] A second case is DsrA RNA in Escherichia
coli (E.coli), which has been shown to regulate translation of rpoS
mRNA, a sigma factor, through a bulge-loop interaction, which
initiates a strand-displacement reaction.[49,50] To test the utility of
BINARI probes for analysis of such a transient and relatively weak
RNA interaction, we applied the crosslinking approach to capture
this RNA-RNA complex. It was hypothesized that BINARI can
acylate 2´-OH groups near the complementary region of the
complex. Three hairpin RNAs were designed based on the DsrA-
rpos interaction[50] (Fig. 5A). HP1 has full complementarity to HP3
in its loop region, while HP2 has two nucleotide deletions in its
loop. Importantly, none of the potential complexes have
complementarity in the stem region, excluding the formation of an
extended duplex such as occurs with HIV dimerization. One
challenging aspect of studying transient complexes is to identify
the individual components after capturing the interaction. This is
made difficult in part with poorly reversible crosslinking
chemistries, where the chemical modification can prevent
molecular analysis, and is also particularly challenging when
interactions between multiple components are possible, which
would be the case in a cellular context.
Increasing the concentration of THPP showed that
maximum reversal was reached at a concentration of 20 mM (Fig.
3D and Fig. S7). Since THPP can oxidize during the reversal
reaction, we checked whether a second treatment with 20 mM
THPP can result in higher crosslinking reversal, which was not the
case. Increasing the THPP incubation time showed that maximum
reversal was reached after 2 hours (Fig. 3E and Fig. S8). The
optimized conditions resulted in up to 84% crosslinking and 70%
reversal, which compares favorably to known psoralen crosslink
reactions that yield mostly below 50% and more typically less than
10% crosslinking[37,42] and exhibit low reversal efficiency.[43]
Figure 4. Gel electrophoretic analysis of temporary protection against
nucleases. S1 nuclease and RNAse T1 fully digest RNA1, whereas BINARI 3
crosslinked RNA 1 remains largely intact. Reversal of the crosslink with THPP
(20 mM) efficiently liberates native RNA 1.
The ribose structure of RNA renders it susceptible to
nuclease catalyzed hydrolysis.[44] This makes handling and
storing RNA for biomedical applications particularly challenging.
We envisioned that by applying BINARI, RNA could temporarily
be stabilized and protected from nuclease degradation.
Subsequent reversal of the crosslink reaction would liberate the
native ssRNA for further downstream applications. Since BINARI
likely locks RNA in a partly double stranded complex, it was
anticipated that it could provide protection against single-strand-
selective nucleases. Using the optimized crosslinking conditions,
we tested RNAse T1 and S1 nuclease. Five μM RNA 1 or BINARI
3-crosslinked RNA 1 in the appropriate buffer (see SI for details)
was incubated with S1 Nuclease or RNAse T1 for 10 minutes at
room temperature. The reaction was stopped by the addition of 7
M urea and cooled on ice. PAGE analysis of the enzymatic
reactions (Fig. 4) showed that both nucleases fully digested
unmodified RNA 1. Significantly, crosslinked RNA 1 remained
largely intact upon treatment with nucleases, providing evidence
that the BINARI crosslink does provide protection against
nuclease degradation. RNAse T1 appears to hydrolyze mono-
functionalized RNA 1 as well (Fig. 4), whereas S1 nuclease
mostly hydrolyzes unmodified RNA 1.
Figure 5. Structures of DraS-rpoS derived hairpins and analysis of their
transiently formed complexes with BINARI. A) Sequence and structure of the
DraS-rpoS derived hairpins HP1, HP2 and HP3. HP1 and HP3 have full
complementary in their loop regions, while HP2 has a two-nucleotide deletion.
B) PAGE analysis of the BINARI 3-captured RNA complex. THPP reversal
reveals that the formed complex solely consists of HP1 and HP3.
To test this challenge, HP1, HP2 and HP3 were mixed
at 10 μM in 100 mM MOPS buffer (pH= 7.5, 6 mM MgCl2, 100 mM
NaCl) for 4 hours at room temperature with BINARI 3 (100 mM).
Analysis of the reaction by 20% PAGE showed a clear band with
decreased mobility (Fig. 5B), indicative of a captured RNA dimeric
complex. The band was excised from the gel and the extracted
RNA complex was treated with THPP (100 mM) in water for 2
hours at room temperature to reverse the crosslink. Interestingly,
when the isolated and reversed complex was analyzed by PAGE,
two clear bands were observed that correspond in mobility to HP1
and HP3, which is in accordance with superior sequence
complementarity of HP1-HP3 compared to a potential HP2-HP3
Subsequent treatment of crosslinked RNA 1 with THPP
(20 mM) in water liberated the native ssRNA 1 (Fig. 4).These
results indicate that BINARI could potentially be used to protect
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COMMUNICATION
[8]
Y. Long, X. Wang, D. T. Youmans, T. R. Cech, Sci. Adv. 2017, 3,
eaao2110.
P. Han, C.-P. Chang, RNA Biol. 2015, 12, 1094–1098.
S. Geisler, J. Coller, Nat. Rev. Mol. Cell Biol. 2013, 14, 699.
A. Serganov, E. Nudler, Cell 2013, 152, 17–24.
G. Mayer, M.-S. L. Raddatz, J. D. Grunwald, M. Famulok, Angew. Chemie
Int. Ed. 2007, 46, 557–560.
S. Li, R. R. Breaker, Nucleic Acids Res. 2013, 41, 3022–3031.
A. J. Matlin, F. Clark, C. W. J. Smith, Nat. Rev. Mol. Cell Biol. 2005, 6,
386–398.
M. Selmer, C. M. Dunham, F. V Murphy, A. Weixlbaumer, S. Petry, A. C.
Kelley, J. R. Weir, V. Ramakrishnan, Science (80-. ). 2006, 313, 1935–
1942.
N. J. Watkins, M. T. Bohnsack, Wiley Interdiscip. Rev. RNA 2012, 3, 397–
414.
complex as well as three other possible self-dimeric complexes.
Potential crosslinking of self-dimeric complexes was ruled out by
treating individual HPs with BINARI 3 (Fig. S9). This experiment
illustrates the need for efficient crosslinking to capture a transient
complex, and underlines the potential for employing BINARI
crosslinkers to probe RNA-RNA transient and weak interactions
more widely. The reversibility of the method allows for further
downstream analysis.
[9]
[10]
[11]
[12]
[13]
[14]
BINARI crosslinking and reversal efficiency was
[15]
[16]
compared to psoralen with two additional partially self-
complementary RNA sequences. The effects of different
overhanging bases neighboring a duplex (RNA 1-A) and different
internal sequences (RNA S3) were examined. BINARI displayed
high crosslinking yield for all three sequences, and the crosslinks
were reversed successfully with THPP with relatively small
sequence bias (Fig. S10). On the other hand, psoralen displayed
lower crosslinking yields across all sequences and even failed to
crosslink RNA 1-A, confirming psoralen‘s bias for uridines (Fig.
S11). Whereas BINARI uncrosslinking showed little RNA
degradation, reversal of psoralen crosslinks with 254 nm UV
resulted in poor recovery of the original RNA bands, likely due to
UV induced degradation.[51–53] One key advantage of psoralen is
its compatibility with biological environments (e.g.such as cell
lysate or intact cells.[35,36] To test whether BINARI is also
functional in such complex mixtures, RNA crosslinking was
conducted in HeLa whole cell lysate (Fig. S12). BINARI 3
[17]
[18]
S. Djuranovic, A. Nahvi, R. Green, Science (80-. ). 2012, 336, 237–240.
N. Dubois, R. Marquet, J.-C. Paillart, S. Bernacchi, Front. Microbiol.
2018, 9, 527.
[19]
S. Y. Yang, P. Lejault, S. Chevrier, R. Boidot, A. G. Robertson, J. M. Y.
Wong, D. Monchaud, Nat. Commun. 2018, 9, 4730.
J. Gong, Y. Ju, D. Shao, Q. C. Zhang, Quant. Biol. 2018, 6, 239–252.
Z. Lu, H. Y. Chang, Curr. Opin. Struct. Biol. 2016, 36, 142–148.
I. S. Hong, M. M. Greenberg, J. Am. Chem. Soc. 2005, 127, 3692–3693.
U. K. Shigdel, J. Zhang, C. He, Angew. Chemie Int. Ed. 2008, 47, 90–93.
J. Liu, L. Cai, W. Sun, R. Cheng, N. Wang, L. Jin, S. Rozovsky, I. B.
Seiple, L. Wang, Angew. Chemie - Int. Ed. 2019, n/a, DOI
10.1002/anie.201910135.
[20]
[21]
[22]
[23]
[24]
[25]
M. Shelbourne, T. Brown, A. H. El-Sagheer, T. Brown, Chem. Commun.
2012, 48, 11184–11186.
[26]
[27]
[28]
H. Huang, P. B. Hopkins, J. Am. Chem. Soc. 1993, 115, 9402–9408.
H. Do, A. Dobrovic, Clin. Chem. 2015, 61, 64–71.
S. Karmakar, E. M. Harcourt, D. S. Hewings, F. Scherer, A. F. Lovejoy, D.
M. Kurtz, T. Ehrenschwender, L. J. Barandun, C. Roost, A. A. Alizadeh, E.
T. Kool, Nat. Chem. 2015, 7, 752.
R. Wagner, R. A. Garrett, Nucleic Acids Res. 1978, 5, 4065–4076.
K. Chen, Z. Lu, X. Wang, Y. Fu, G.-Z. Luo, N. Liu, D. Han, D.
Dominissini, Q. Dai, T. Pan, C. He, Angew. Chemie Int. Ed. 2015, 54,
1587–1590.
efficiently cross-linked both
a fluorescently labeled self-
complementary RNA (Cy5-RNA 1) and separate complementary
RNAs (RNA S1 and S2) without loss of efficiency when compared
to buffer. Taken together, BINARI displays superior perfomance
with minimal sequence bias for crosslinking and high reversal
recovery as compared with psoralen.
[29]
[30]
[31]
[32]
J. L. Sloane, M. M. Greenberg, J. Org. Chem. 2014, 79, 9792–9798.
K. Fujimoto, A. Yamada, Y. Yoshimura, T. Tsukaguchi, T. Sakamoto, J.
Am. Chem. Soc. 2013, 135, 16161–16167.
In summary, the BINARI probe design constitutes a new
reversible crosslinking strategy for RNA. The presented approach
offers superior crosslinking yields compared to existing methods
with minimal sequence bias, and is selective for RNA-RNA
interactions with no detectable crosslinking of duplex DNA. It is
also compatible with complex biological media such as cell
lysates. We have shown that BINARI-crosslinked RNA is
temporarily protected from nuclease degradation and that
crosslink reversal liberates the native RNA, suggesting future
applications in storage and preservation of RNA. Furthermore,
BINARI was applied successfully to capture transient RNA
complexes of E.coli DsrA-rpos derived hairpins, and the efficient
crosslink reversal reaction enabled the identification of individual
RNA components of the complex. We anticipate that the future
combination of this reversible crosslinking approach with high-
throughput sequencing technologies could aid in mapping the
RNA interactome.
[33]
[34]
J. P. Calvet, T. Pederson, Proc. Natl. Acad. Sci. 1979, 76, 755–759.
T. Stafforst, D. Hilvert, Angew. Chem. Int. Ed. Engl. 2010, 49, 9998–
10001.
Z. Lu, Q. C. Zhang, B. Lee, R. A. Flynn, M. A. Smith, J. T. Robinson, C.
Davidovich, A. R. Gooding, K. J. Goodrich, J. S. Mattick, J. P. Mesirov, T.
R. Cech, H. Y. Chang, Cell 2016, 165, 1267–1279.
E. Sharma, T. Sterne-Weiler, D. O’Hanlon, B. J. Blencowe, Mol. Cell
2016, 62, 618–626.
[35]
[36]
[37]
W. P. Zhen, O. Buchardt, H. Nielsen, P. E. Nielsen, Biochemistry 1986, 25,
6598–6603.
[38]
[39]
J. M. Stadler, T. Stafforst, Org. Biomol. Chem. 2014, 12, 5260–5266.
L. Good, S. K. Awasthi, R. Dryselius, O. Larsson, P. E. Nielsen, Nat.
Biotechnol. 2001, 19, 360–364.
R. C. Spitale, R. A. Flynn, Q. C. Zhang, P. Crisalli, B. Lee, J.-W. Jung, H.
Y. Kuchelmeister, P. J. Batista, E. A. Torre, E. T. Kool, H. Y. Chang,
Nature 2015, 519, 486–90.
A. Kadina, A. M. Kietrys, E. T. Kool, Angew. Chemie - Int. Ed. 2018, 57,
3059–3063.
R. Kirsch, V. J. Olzog, S. Bonin, C. E. Weinberg, H. Betat, P. F. Stadler,
M. Mörl, Biotechniques 2019, 67, 178–183.
[40]
[41]
[42]
[43]
[44]
F. Gasparro, Psoralen Dna Photobiology, Boca Raton: CRC Press, 1988.
V. Genna, M. Marcia, M. De Vivo, J. Am. Chem. Soc. 2019, 141, 10770–
10776.
J. Šponer, G. Bussi, M. Krepl, P. Banáš, S. Bottaro, R. A. Cunha, A. Gil-
Ley, G. Pinamonti, S. Poblete, P. Jurečka, N. G. Walter, M. Otyepka,
Chem. Rev. 2018, 118, 4177–4338.
Acknowledgements
[45]
[46]
We are grateful for support from the U.S. National Institutes of
Health (R01GM127295 and R43ES028559).
R. T. Batey, R. P. Rambo, J. A. Doudna, Angew. Chemie Int. Ed. 1999, 38,
2326–2343.
[47]
[48]
Y. Eguchi, J. Tomizawa, Cell 1990, 60, 199–209.
J. C. Paillart, E. Skripkin, B. Ehresmann, C. Ehresmann, R. Marquet, Proc.
Natl. Acad. Sci. 1996, 93, 5572–5577.
N. Majdalani, C. Cunning, D. Sledjeski, T. Elliott, S. Gottesman, Proc.
Natl. Acad. Sci. 1998, 95, 12462–12467.
N. Salim, R. Lamichhane, R. Zhao, T. Banerjee, J. Philip, D. Rueda, A. L.
Feig, Biophys. J. 2012, 102, 1097–1107.
A. Ariza-Mateos, S. Prieto-Vega, R. Díaz-Toledano, A. Birk, H. Szeto, I.
Mena, A. Berzal-Herranz, J. Gómez, Nucleic Acids Res. 2012, 40, 1748–
1766.
Keywords: Acylation • Crosslinking • RNA-RNA interactions •
RNA complexes • Nucleic acids
[49]
[50]
[51]
[1]
[2]
[3]
[4]
T. R. Cech, J. A. Steitz, Cell 2014, 157, 77–94.
C. He, Nat Chem Biol 2010, 6, 863–865.
R.-W. Yao, Y. Wang, L.-L. Chen, Nat. Cell Biol. 2019, 21, 542–551.
F. Li, J. Dong, X. Hu, W. Gong, J. Li, J. Shen, H. Tian, J. Wang, Angew.
Chemie - Int. Ed. 2015, 54, 4597–4602.
[52]
[53]
Z. Jericević, I. Kućan, R. W. Chambers, Biochemistry 1982, 21, 6563–
6567.
E. J. Wurtmann, S. L. Wolin, Crit. Rev. Biochem. Mol. Biol. 2009, 44, 34–
[5]
[6]
[7]
W. A. Velema, E. T. Kool, Nat. Rev. Chem. 2020, 4, 22–37.
L. S. Waters, G. Storz, Cell 2009, 136, 615–628.
E. Anastasiadou, L. S. Jacob, F. J. Slack, Nat. Rev. Cancer 2017, 18, 5.
This article is protected by copyright. All rights reserved.

10.1002/anie.202010861
Angewandte Chemie International Edition
COMMUNICATION
49.
This article is protected by copyright. All rights reserved.

10.1002/anie.202010861
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Xlink’s Awakening: Crosslinking of
Willem A. Velema, Hyun Shin Park,
Anastasia Kadina, Lucian Orbai and Eric
T. Kool*
transient RNA complexes is achieved
using
dimeric
2’-OH
acylating
reagents. The crosslinks of captured
complexes are efficiently reversed by
Page No. – Page No.
phosphine-mediated
azide-trigger groups,
convenient downstream analysis.
reduction
of
Trapping Transient RNA Complexes
by Chemically Reversible Acylation
enabling
This article is protected by copyright. All rights reserved.