A Covalent Approach for Site-Specific RNA Labeling in Mammalian Cells

Article abstract of DOI:10.1002/anie.201410433

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comp

Full text of DOI:10.1002/anie.201410433

Angewandte
Chemie
DOI: 10.1002/anie.201410433
RNA Labeling Very Important Paper
A Covalent Approach for Site-Specific RNA Labeling in Mammalian
Cells**
Fahui Li, Jianshu Dong, Xiaosong Hu, Weimin Gong, Jiasong Li, Jing Shen, Huifang Tian, and
Jiangyun Wang*
Abstract: Advances in RNA research and RNA nanotechnol-
ogy depend on the ability to manipulate and probe RNA with
high precision through chemical approaches, both in vitro and
in mammalian cells. However, covalent RNA labeling methods
with scope and versatility comparable to those of current
protein labeling strategies are underdeveloped. A method is
reported for the site- and sequence-specific covalent labeling of
RNAs in mammalian cells by using tRNAIle2-agmatidine
synthetase (Tias) and click chemistry. The crystal structure of
Tias in complex with an azide-bearing agmatine analogue was
solved to unravel the structural basis for Tias/substrate
recognition. The unique RNA sequence specificity and plastic
Tias/substrate recognition enable the site-specific transfer of
azide/alkyne groups to an RNA molecule of interest in vitro
and in mammalian cells. Subsequent click chemistry reactions
facilitate the versatile labeling, functionalization, and visual-
ization of target RNA.
approaches,[2] bioorthogonal chemistry[3] relies upon the
specific and covalent attachment of probe molecules to the
biomacromolecule of interest (BOI). As a result, it offers
several advantages: 1) the affinity can be considered to be
infinity, therefore stringent wash conditions can be applied to
rigorously purify the BOI; 2) the high affinity allows for the
visualization of low-abundance BOIs, which is particularly
important for non-coding RNAs;[1a] 3) this method is
extremely versatile; probe molecules harboring fluorescence,
NMR, IR, or EPR functional groups can be conveniently
conjugated to the BOI.[3] Because of these unique features,
bioorthogonal chemistry has been successfully employed in
protein and glycan functional studies to visualize their
expression, localization, and interaction partners in mamma-
lian cells.[3]
While engineering of the translational[4] or post-transla-
tional machinery[5] in combination with bioorthogonal
chemistry has enabled protein labeling with unprecedented
precision and versatility,[6] comparable methods for the site-
and sequence-specific tagging of RNAs in mammalian cells is
currently underdeveloped.[7] To achieve this, we need to
engineer unique components of the post-transcriptional
machinery and employ bioorthogonal chemistry. First, an
RNA-modifying enzyme must recognize a specific site in
a unique RNA sequence in the transcriptome. Second, this
enzyme must be able to transfer a small molecule bearing
a unique chemical handle (such as an azide or alkyne group)
to the specific site. Subsequent covalent labeling through
bioorthogonal chemistry then leads to the site-specific con-
jugation of reporter groups, thus enabling fluorescence,
NMR, EPR, or IR spectroscopy measurements (Figure 1).
To search for a post-transcriptional modification module
that can be used for precise RNA labeling in mammalian cells,
we turned to the tRNA-modifying enzymes, which are known
to catalyze around 100 kinds of tRNA modification.[8] Faithful
translation of the genetic code relies on the chemical
modification of tRNA,[9] especially at the wobble position 34
of the anticodon.[10] Many tRNA-modifying enzymes acting
on this position are found only in specific domains, thus
implying that they may have evolved independently.[11]
Notably, the C34 position of the AUA-decoding tRNAIle2 is
modified with lysine by the enzyme lysidine synthetase (Tils)
in bacteria,[12] but with agmatine (AGM, 1; Scheme 1) by the
enzyme tRNAIle2-agmatidine synthetase (Tias) in arch-
aea.[13,14] In eukaryotes, tRNAIle bearing pseudouridine (Y)
or inosine (I) at the wobble position decodes the AUA
codon.[9,15] As shown previously, Archaeoglobus fulgidus (Af)
Tias recognition of tRNAIle2 requires seven nucleotides: G1,
G2, C34, U36, A37, C71, and C72 (Figure S13A in the
I
t has recently emerged that RNA has functions as diverse
and important as those of proteins.[1] To unravel the dynamic
trafficking, localization, and interactions of RNA in living
cells, numerous methods have been developed for the non-
covalent labeling of RNAs in mammalian cells, and dramatic
progress has been made.[2] Compared to the noncovalent
[*] F. H. Li,[+] J. S. Dong,[+] W. M. Gong, J. S. Li, J. Y. Wang
Laboratory of RNA Biology and Laboratory of Quantum Biophysics
Institute of Biophysics, Chinese Academy of Sciences
15 Datun Road, Chaoyang District, Beijing, 100101 (China)
E-mail: jwang@ibp.ac.cn
J. Y. Wang
Beijing National Laboratory for Molecular Sciences (BNLMS)
Institute of Chemistry, Chinese Academy of Sciences
Beijing, 100190 (China)
X. S. Hu[+]
Department of Chemistry, School of Chemistry
Chemical Engineering and Life Sciences
Wuhan University of Technology, Wuhan 430070 (China)
J. Shen, H. F. Tian
The Laboratory of Carcinogenesis and Translational Research
(Ministry of Education)
Core Laboratory, Peking University School of Oncology
Beijing Cancer Hospital & Institute, Beijing, 100142 (China)
[+] These authors contributed equally to this work.
[**] We gratefully acknowledge the Major State Basic Research Program
of China (2015CB856203), the CAS grant (KJZD-EW-L01), and the
National Science Foundation of China (91440116, 21325211,
91313301, 31370016, 21102172). We thank Yan Teng for help with
fluorescence imaging.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410433.
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Figure 1. A) In the presence of ATP, Tias catalyzes the conjugation of
agmatine to C34 of tRNAIle2, thereby resulting in the formation of
agmatidine. B) Labeling of an RNA of interest (ROI). Tias facilitates
the site-specific transfer of an agmatine analogue bearing an azide
group to the ROI-tRNAIle2 fusion RNA. Subsequent strain-promoted
azide–alkyne cycloaddition allows the conjuration of biophysical
probes.
Scheme 1. Structure of agmatine (AGM) and agmatine analogues.
Figure 2. A) Acid–urea PAGE with tRNAIle2 modified by using Tias and
AGM analogues. The gel was stained by ethidium bromide (EB).
B) While Tias catalyzes the efficient conjugation of AGM (1) and AGN
(2) to Af tRNAIle2, the reaction does not occur for the Af tRNAIle2 C34U
mutant, thus indicating that the small-molecule probes are transferred
specifically to the C34 position. The reaction mixtures were analyzed
by acid–urea PAGE. MALDI-TOF analyses are shown for unlabeled Af
tRNAIle2 (C), Af tRNAIle2 modified with 1 (D), Af tRNAIle2 modified with
2 (E), and Af tRNAIle2 modified with 3 (F).
Supporting Information).[13,14] Since neither Tias nor tRNA
bearing this set of identity elements is present in mammalian
cells (Figure S13),[16] we envisioned that the introduction of
Tias and archaeal tRNAIle2 into mammalian cells could
facilitate the sequence- and site-specific labeling of RNA if
Tias can transfer small molecule probes bearing azide or
alkyne functional groups to an RNA of interest.
As a first step to developing a covalent method for
labeling RNAs, we synthesized three AGM analogues: N-(4-
aminobutyl)-2-azidoacetamide (AGN, 2), 2-propynylamine
compound 4. The much bulkier carbamate and alkyne
functional groups may result in inefficient recognition of
compound 4 by Tias. We therefore choose compounds 2 and 3
for further investigation.
(3),
and
(but-3-yn-1-yl)(4-aminobutyl)carbamate
(4;
Scheme 1). Modification of Af tRNAIle2 was performed with
Tias (8 mm), Af tRNAIle2 (1 mm), Tris-HCl (100 mm), pH 8.0,
KCl (10 mm), MgCl2 (5 mm), DTT (5 mm), ATP (1 mm), and
substrates 1, 2, 3, or 4 (1 mm). AGM (1) was used as a positive
control. The modified and unmodified RNAs were then
separated by 6.5% acid–urea polyacrylamide gel electro-
phoresis (PAGE), as previously described.[17] When tRNAIle2
was modified, the modified RNA exhibited lower mobility
(Figure 2A). To our delight, we found that in the presence of
substrates 2, 3, or 4, tRNAIle2 was covalently modified, thus
resulting in lower mobility in the acid–urea polyacrylamide
gel. Perhaps owing to the lower molecular weight of
compound 3, tRNAIle2 modified with compound 3 exhibited
higher mobility than tRNAIle2 modified with compound 2. In
the presence of compound 4, two major bands were present,
thus indicating that tRNAIle2 was only partially modified with
We then investigated whether Tias catalyzes the specific
modification of nucleotide C34 of Af tRNAIle2. While Tias
catalyzed the efficient ligation of compounds 1 and 2 to Af
tRNAIle2, this activity was abolished for the C34U mutant
(Figure 2B). These results indicate that Tias can target both
the natural substrate AGM and the unnatural AGM ana-
logues specifically to nucleotide C34 of Af tRNAIle2. To
further demonstrate that compounds 2 and 3 conjugate
precisely with nucleotide C34 of Af tRNAIle2, we digested
Af tRNAIle2 with RNase T1 (Figure S15)[18] and then analyzed
the RNA fragment containing the anticodon loop by
MALDI-TOF. The unmodified tRNAIle2 fragment gave an
observed average mass of 3184.08 Da, which closely matches
the calculated mass of 3184.4 Da (Figure 2). After AGM or 2-
propynylamine modification, the tRNAIle2 fragment gave
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observed average masses of 3295.3 and 3220.3 Da, respec-
tively, which match the calculated masses of 3295.5 and
3220.3 Da. After AGN modification, the tRNAIle2 fragment
gave an observed average mass of 3311.2 Da, while the
calculated mass is 3337.5 Da. This can be explained by the loss
of two nitrogen atoms and the addition of two hydrogen
atoms during the laser irradiation used for MALDI-TOF, as
previous observed.[19] Taken together, these results demon-
strate that Tias can facilitate the conjugation of azide/alkyne-
bearing AGM analogues 2 and 3 to nucleotide C34 in Af
tRNAIle2
.
To unravel the molecular basis for AGN recognition by
Tias, we solved the structure of Tias with AGN bound to its
active site. The main chain carbonyl group of Asp193 and the
side chain of Asn194 form two hydrogen bonds with the azide
group of AGN, while the guanidine group of Arg217 forms
a hydrogen bond with the carbonyl group of AGN (Figure 3).
Figure 4. A) Structure of BCN-FITC. B) AGN-modified tRNAIle2 was
visualized through strain-promoted click reaction with BCN-FITC. The
bottom panel (EB staining) indicates that equivalent amounts of
tRNAIle2 with and without AGN modification were loaded. C) The
structure of Sulfo-Cy5-azide. D) tRNAIle2 modified with 2-propynylamine
was visualized through CuI-catalyzed click reaction with Sulfo-Cy5-
azide. The bottom panel (EB staining) shows that equivalent amounts
of tRNAIle2 with and without 2-propynylamine modifications were
loaded.
These results indicate that BCN-FITC reacts specifically with
the azide group present on tRNAIle2. Under similar conditions
and in the presence of CuI catalyst (1 mm), Sulfo-Cy5-azide
reacts specifically with tRNAIle2 modified with 2-ropynyl-
amine (3), but not with tRNAIle2 without the modification
(Figure 4C and 4D).
Figure 3. A) Overall structure of Tias in complex with AGN. The overall
structure of Tias resembles a tennis racket. The AGN binding site is
highlighted by a red box. B) Structure of the AGN binding site of Tias.
The Fo-Fc electron density map of AGN was contoured at 2.7 s.
Nitrogen atoms are shown in blue, oxygen atoms in red, and carbon
atoms in yellow.
Once we had demonstrated that Tias can facilitate the
site-specific incorporation of azide and alkyne groups into
tRNAIle2, we then asked whether this strategy could be used to
label any RNA of interest (ROI). Through in vitro run-off
transcription, we synthesized tRNAIle2 harboring 3’ and 5’
extensions (termed tRNAIle2-3-5, Figure 5A). We then incu-
bated tRNAIle2-3-5 with Tias in the presence or absence of
AGN. After removing excess AGN, BCN-FITC was added.
Acid–urea PAGE was then used to analyze the reaction. Only
in the presence of AGN did BCN-FITC selectively label
tRNAIle2-3-5 (Figure 5B), thus indicating that Tias can
catalyze the conjugation of AGN to tRNAIle2-3-5 in the
presence of 3’ and 5’ extensions. This result is consistent with
previous reports that the agmatidine modification occurs
early in the tRNA maturation process to ensure high-fidelity
translation.[13–14]
We then asked whether our method can be used to label
a specific RNA in the mammalian cell transcriptome. 293T
cells expressing Tias with or without tRNAIle2-5S fusion RNA
expression were grown in medium containing AGN, followed
by cell lysis. Subsequently, total cellular RNA was incubated
with BCN-Cy5. In the absence of tRNAIle2-5S expression,
BCN-Cy5 did not label any RNA in the 293T transcriptome
(Figure 5C and 5D). When cells expressed tRNAIle2-5S,
Meanwhile, the side chain of Val203 stabilizes the carbon
chain of agmatine through hydrophobic interactions. Similar
hydrophobic interactions may also be needed to facilitate Tias
recognition of 2-propynylamine. Since Tias can also use
compounds 3 and 4 as substrates, although these are either
much smaller or much bulkier than compound 2, it appears
that Tias has a somewhat plastic binding pocket for primary
amine substrates. We are currently testing whether Tias can
also use primary amines bearing cyclopropene groups as
substrates, which may facilitate photoclick reactions or
inverse electron demand Diels–Alder reactions.[20]
We then tested whether Tias can facilitate RNA fluores-
cence labeling through bioorthogonal chemistry. tRNAIle2
with or without AGN (2) modification was incubated with
BCN-FITC and then analyzed by 6.5% acid–urea PAGE,
followed by fluorescent imaging. tRNAIle2 modified with
AGN reacted with BCN-FITC selectively, whereas tRNAIle2
without AGN modification did not react (Figure 4A and 4B).
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Figure 6. Fluorescent imaging of Af tRNAIle2-5S in U2OS cells. Upper
panel: Cy5 fluorescence (left), DAPI fluorescence (nuclear staining;
middle), and differential interference contrast (DIC) imaging (right) of
U2OS cells harboring the pCMV-Myc-Tias plasmid. Lower panel: Cy5
fluorescence (left), DAPI fluorescence (nuclear staining; middle), and
DIC imaging (right) of U2OS cells harboring the pCMV-Myc-5S-Tias
plasmid. DAPI=4’,6-diamidino-2-phenylindole.
the RNA modifying enzyme Tias to enable the highly specific
conjugation of biophysical probes to any RNA of interest in
mammalian cells. This new technique should be useful for
investigating RNA folding, RNA/protein interactions, RNA
transportation, and RNA modification, as well as for con-
structing RNA-based nanomaterials, in vitro and in mamma-
lian cells.[1]
In comparison to noncovalent RNA imaging methods
such as use of the Spinach aptamer,[2] the major advantages of
the Tias strategy are its specificity, highly affinity, and
versatility. The Tias labeling strategy relies on the selective
recognition of Af tRNAIle2 by Tias (which are both absent in
Figure 5. A) Sequence and secondary structure of tRNAIle2 in the
presence of 3’ and 5’ extensions (denoted tRNAIle2-3-5). B) AGN-
modified tRNAIle2-3-5 is visualized through strain-promoted click reac-
tion with BCN-FITC. The bottom panel (EB staining) shows that
equivalent amounts of tRNAIle2-3-5 with and without AGN modification
were loaded. C) Structure of BCN-Cy5. D) Tias specifically modify
tRNAIle2-5S with AGN in the 293T cells transcriptome, which allows
subsequent strain-promoted click reaction with BCN-Cy5. The bottom
panel (EB staining) shows that equivalent amounts of total RNA with
and without tRNAIle2-5S expression were loaded.
mammalian cells). Since
a covalent approach is then
employed after an azide or alkyne group is introduced,
a covalent bond is created between the fluorophore and target
RNA. This would potentially allow the imaging of low-
abundance RNA. By contrast, the noncovalent interaction
between the GFP chromophore analogue and the Spinach
aptamer has affinity only in the micromolar range.[2] Finally,
the Tias strategy enables versatile site-specific RNA labeling
with fluorescent/IR/NMR/EPR agent through click chemis-
try, without changing the Tias protein or tRNAIle2 sequence.
The challenge, however, is to develop fluorogenic cyclooctyne
dyes that are membrane permeable, reasonably hydrophobic,
and suitable for live cell RNA imaging. We are currently
working to achieve this goal.
however, BCN-Cy5 specifically labeled tRNAIle2-5S. Reverse
transcription polymerase chain reaction (RT PCR) experi-
ments confirmed that tRNAIle2-5S existed as a full-length
fusion RNA in the 293T cells (Figure S15). To further
demonstrate the utility of our method for RNA imaging in
mammalian cells, U2OS cells expressing Tias with or without
tRNAIle2-5S fusion RNA expression were grown in the
presence of 2-propynylamine and then fixed, permeabilized,
and incubated with Sulfo-Cy5-azide and a CuI catalyst. We
found that only the cells expressing tRNAIle2-5S fusion RNA
could be labeled and imaged with Sulfo-Cy5-azide (Figure 6).
These results demonstrate that in mammalian cells, Tias can
recognize the unique Af tRNAIle2 sequence in the tran-
scriptome, thus enabling the sequence- and site-specific
conjugation of an azide/alkyne group to an RNA of interest
bearing the tRNAIle2 tag. Strain-promoted or CuI-catalyzed
click chemistry then enables the specific labeling and imaging
of the RNA of interest in the cells.
Experimental Section
In vitro modification of Af tRNAIle2: Modification of Af tRNAIle2 was
performed with Tias (8 mm), Af tRNAIle2 (1 mm), Tris-HCl (100 mm),
pH 8.0, KCl (10 mm), MgCl2 (5 mm), DTT (5 mm), ATP (1 mm), and
substrates 1, 2, 3, or 4 (1 mm). AGM (1) was used as a positive control.
Each reaction mixture was incubated at 378C for 2 h. A 5 mL aliquot
from each reaction mixture was analyzed by 6.5 % acid–urea PAGE
(0.1m sodium acetate, pH 5.0, 8m urea). In-gel fluorescence images
were taken at a KF BIO-2800 gel imager and the gel was then stained
with ethidium bromide (EB).
A great deal of modern RNA biology and RNA biotech-
nology involves the site-specific incorporation of modified
nucleotides into RNA molecules through chemical
approaches.[1] However, the length of synthetic RNA is
typically less than 30–50 nucleotides owing to limitations in
coupling efficiency. Our new method relies on the RNA
transcription machinery and the high sequence specificity of
Specific fluorescent labeling of Af tRNAIle2 in vitro: The AGN
(2)-modified Af tRNAIle2 (1 mm) was mixed with BCN-FITC (100 mm)
4
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in the buffer (20 mm Tris, pH 7.4, 150 mm NaCl) and incubated for
30 min at room temperature. The 2-propynylamine (3)-modified Af
tRNAIle2(1 mm) was mixed with Sulfo-Cy5 azide (Lumiprobe, B3330)
(100 mm), and CuSO4 (1 mm), ascorbic acid (2 mm) and BTTPS
(2 mm) in the buffer (20 mm Tris, pH 7.4, 150 mm NaCl) and
incubated for 30 min at room temperature. The unmodified Af
tRNAIle2 was used as a negative control. The reaction mixtures were
analyzed by 6.5% TAE–urea PAGE (40 mm Tris, pH 8.0, 1 mm
EDTA, 8m urea), and in-gel fluorescence images were taken at
a Typhoon 9400 variable mode imager (Amersham Biosciences).
BCN-FITC: Excitation at 488 nm, emission at 520 nm (520 BP 40
filter); Sulfo-Cy5-azide: Excitation at 633 nm, emission at 670 (670
BP 30 filter).
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RNA imaging of tRNAIle2--5S in mammalian cells: The U2OS
cells were allowed to grow to 50–70% confluence on a 20 mm culture
dish in DMEM supplemented with 10% FBS, and transfected with
2.2 mg of the plasmids pCMV-Myc-Tias or pCMV-Myc-5S-Tias,
respectively, by using Lipofectamine 2000 Reagent (Invitrogen)
according to the manufacturerꢀs instructions. 36 hrs after transfection,
the cells were cultured in the presence of 1 mm 3 for 4 hrs. The cells
were then washed twice with PBS and fixed in PIPES (125 mm),
pH 6.8, EGTA (10 mm), MgCl2 (1 mm), Triton X-100 (0.2%), and
formaldehyde (3.7%) for 30 min at room temperature. The fixed cells
were rinsed with TBS and stained for 30 min at room temperature
with TBS buffer (pH 7.5) containing CuSO4 (1 mm), ascorbic acid
(100 mm), Sulfo-Cy5-azide (20 mm) and BTTPS (1 mm).[21] After
staining, the cells were washed three times with TBS with 0.5%
TritonX-100. The cells were then imaged by fluorescence microscopy
and differential interference contrast microscopy (DIC). DTT=
dithiothreitol, PBS = phosphate-buffered saline, DMEM = Dulbec-
coꢀs modified Eagle’s medium, FBS = fetal bovine serum, PIPES =
1,4-piperazinediethanesulfonic acid, EGTA = ethylene glycol tetra-
acetic acid, BTTPS = 3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)
methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]propyl hydrogen sulfate,
TBS = Tris-buffered saline.
Received: October 24, 2014
Revised: December 8, 2014
Published online: && &&, &&&&
Keywords: bioorthogonal chemistry · biotechnology ·
.
RNA labeling · RNA modification · tRNA
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These are not the final page numbers!

Angewandte
Chemie
Communications
RNA Labeling
Tag and click: The ability to specifically
label RNAs in vitro and in mammalian
cells would be highly significant for RNA
research, however, covalent RNA labeling
methods with scope and versatility com-
parable to those for protein labeling have
not been reported. A method was devel-
oped for the site- and sequence-specific
covalent labeling of RNAs in mammalian
F. H. Li, J. S. Dong, X. S. Hu, W. M. Gong,
J. S. Li, J. Shen, H. F. Tian,
J. Y. Wang*
&&&& — &&&&
A Covalent Approach for Site-Specific
RNA Labeling in Mammalian Cells
cells, based on the action of tRNAIle2
-
agmatidine synthetase (Tias) and click
chemistry.
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!