Cell- And Polymerase-Selective Metabolic Labeling of Cellular RNA with 2′-Azidocytidine

Article abstract of DOI:10.1021/jacs.0c04566

Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies can be used to study individual RNA subpopulations within complex systems. Here, we describe a strategy for cell- and polymerase-selective RNA labeling with 2′-azidocytidine (2′-AzCyd), a modified nucleoside amenable to bioorthogonal labeling with SPAAC chemistry. In contrast to 2′-OH-containing pyrimidine ribonucleosides, which rely upon uridine-cytidine kinase 2 (UCK2) for activation, 2′-AzCyd is phosphorylated by deoxycytidine kinase (dCK), and we find that expression of dCK mediates cell-selective 2′-AzCyd labeling. Further, 2′-AzCyd is primarily incorporated into rRNA and displays low cytotoxicity and high labeling efficiency. We apply our system to analyze the turnover of rRNA during ribophagy induced by oxidative stress or mTOR inhibition to show that 28S and 18S rRNAs undergo accelerated degradation. Taken together, our work provides a general approach for studying dynamic RNA behavior with cell and polymerase specificity and reveals fundamental insights into nucleotide and nucleic acid metabolism.

Full text of DOI:10.1021/jacs.0c04566

Subscriber access provided by CARLETON UNIVERSITY
Communication
Cell- and polymerase-selective metabolic
labeling of cellular RNA with 2’-azidocytidine
Danyang Wang, Yu Zhang, and Ralph E. Kleiner
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c04566 • Publication Date (Web): 07 Aug 2020
Downloaded from pubs.acs.org on August 7, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Cell- and polymerase-selective metabolic labeling of cellular RNA
with 2’-azidocytidine
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Danyang Wang, Yu Zhang, & Ralph E. Kleiner*
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
Supporting Information Placeholder
cellular RNA7-10, but the most commonly used
ABSTRACT: Metabolic labeling of cellular RNA is a
strategy rely on cellular uptake and activation of
modified ribonucleosides. While labeling with
ribonucleosides such as 4-thiouridine (4-SU)4, 11 or
5-ethynyluridine (5-EU)1 proceeds efficiently, the
scope of modified nucleotides that can be accessed
through nucleoside feeding has remained limited.
Further, we lack approaches that can applied to cell-
specific10 or RNA polymerase-specific labeling in
vivo with minimal cytotoxicity12.
powerful approach to investigate RNA biology. In
addition to revealing whole transcriptome
dynamics, targeted labeling strategies can be used to
study individual RNA subpopulations within
complex systems. Here, we describe a strategy for
cell- and polymerase-selective RNA labeling with 2’-
azidocytidine (2’-AzCyd), a modified nucleoside
amenable to bioorthogonal labeling with SPAAC
chemistry. In contrast to 2’-OH-containing
pyrimidine ribonucleosides, which rely upon
uridine-cytidine kinase 2 (UCK2) for activation, 2’-
AzCyd is phosphorylated by deoxycytidine kinase
(dCK), and we find that expression of dCK mediates
cell-selective 2’-AzCyd labeling. Further, 2’-AzCyd is
primarily incorporated into ribosomal RNA and
displays low cytotoxicity and high labeling
efficiency. We apply our system to analyze the
turnover of ribosomal RNA during ribophagy
induced by oxidative stress or mTOR inhibition to
show that 28S and 18S rRNAs undergo accelerated
degradation. Taken together, our work provides a
general approach for studying dynamic RNA
behavior with cell and polymerase specificity and
reveals fundamental insights into nucleotide and
nucleic acid metabolism.
Previously, we applied protein engineering to
UCK2 in order to facilitate cell-specific labeling with
5-azidomethyluridine13. Here, we explore metabolic
labeling with 2’-azidocytidine (2’-AzCyd)14-19, a
modified nucleoside compatible with bioorthogonal
SPAAC chemistry20. We show that in contrast to
base-modified pyrimidine ribonucleosides, which
rely on UCK2, incorporation of 2’-AzCyd is primarily
mediated by deoxycytidine kinase (dCK)21 and
overexpression of this enzyme in diverse cell lines
can facilitate cell-specific RNA labeling. Further, we
find that 2’-AzCyd is well tolerated in cell culture and
is mainly incorporated into ribosomal RNA in an
RNA Pol I-dependent manner, providing a strategy
for polymerase-selective RNA labeling which we
exploit to study rRNA turnover during oxidative
stress and drug treatment.
The incorporation of modified nucleotides into
cellular RNA is a powerful approach for probing RNA
biology. In particular, nucleotides that serve as
2’-azido RNA nucleotides have emerged as a
promising scaffold for biorthogonal RNA labeling22-
23
and 2’-azidoadenosine (2’-AzAd) can be
biorthogonal tags1-3
,
photoaffinity labels4, or
metabolically incorporated into mRNA primarily
through polyA polymerases3. In contrast, less is
known about the potential of 2’-azidopyridimine
nucleosides for RNA labeling applications. While an
early study by Åkerblom15 found incorporation of 2’-
generate characteristic mutational signatures5-6 can
be used as chemical probes for studying
transcriptional
dynamics
and
protein-RNA
interactions in living cells. Several approaches have
been developed for incorporating modifications into
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
azidocytidine into cellular RNA, he concluded that
Page 2 of 8
1
2
3
4
5
6
only “minute” amounts were present. Further,
recent work has indicated that 2’-azidouridine is a
poor substrate for metabolic labeling but can be
utilized for cell-specific labeling together with UCK2
overexpression24.
To study metabolic 2’-
azidonucleoside labeling further, we began by
culturing HeLa cells with 1 mM 2’-AzAd or the
related pyrimidines 2’-azidouridine (2’-AzUrd) or 2’-
AzCyd (Figure 1a), and evaluated incorporation by
CuAAC and fluorescence microscopy. Consistent
with prior reports3, we found efficient uptake of 2’-
AzAd (Figure 1b). We could also detect uptake of 2’-
AzCyd, as has been reported15, 24, however labeling
was less intense as compared to 2’-AzAd, and we
were unable to observe incorporation by 2’-AzUrd
(Figure 1b). The labeling pattern with 2’-AzCyd was
both nuclear and cytosolic with strong staining in the
nucleoli, consistent with incorporation into RNA. To
confirm RNA labeling we performed feeding in the
presence of actinomycin D, an RNA polymerase
inhibitor, or hydroxyurea, an inhibitor of DNA
synthesis. While 2’-AzCyd labeling was unaffected by
hydroxyurea, we observed a strong reduction in
signal upon treatment with actinomycin D,
consistent with RNA incorporation (Figure 1c).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure
1.
Metabolic
incorporation
of
2’azidopyrimidine nucleosides into cellular RNA. (A)
Pyrimidine salvage pathway and 2’-azidopyrimidines.
(B) Labeling of HeLa cells with 1 mM 2’-AzCyd or 2’-
AzUrd for 6 hr. (C) 2’-AzCyd labeling in the presence of
2 µM ActD or 10 mM HU.
Next, we explored 2’-AzCyd metabolism (Figure
1a). Phosphorylation to NMPs has generally been
proposed as rate limiting for nucleoside salvage
therefore we focused on the known pyrimidine
ribonucleoside and deoxyribonucleoside kinases
UCK2 and dCK21, 25; indeed dCK has been proposed
as the primary enzyme responsible for 2’-AzCyd
phosphorylation although direct evidence is
lacking15. We purified recombinant enzymes
following literature precedent25-26 and used an
HPLC-based assay to analyze nucleoside
phosphorylation13. Both kinases were active as
judged by their ability to phosphorylate
cytidine/deoxycytidine (Supplementary Figures 1 &
2). We next tested the ability of UCK2 and dCK to
phosphorylate 2’-AzCyd and 2’-AzUrd. After 12 hr
incubation of 2’-AzCyd with dCK and excess ATP, we
2
ACS Paragon Plus Environment

Page 3 of 8
Journal of the American Chemical Society
observed near complete consumption of starting
hydrogen bond network formed between UCK2 and
the substrate 2’-hydroxyl group (Figure 2f). We also
tested the activity of both enzymes on 2’-AzUrd and
were unable to detect the formation of a new
product or observe consumption of 2’-azidouridine
during the course of the reaction (Figure 2b,
Supplementary Figure 1). We did however measure
a small amount of ADP production in both UCK2 and
dCK reactions with 2’-AzUrd, suggesting that low
amounts of phosphorylation may occur with this
1
2
3
4
5
6
7
8
material and the emergence of two new peaks – one
consistent with ADP, and the other that we
confirmed by mass spectrometric analysis as 2’-
AzCyd monophosphate (Figures 2a and 2b).
Incubation of 2’-AzCyd with UCK2 under the same
conditions produced only 4% conversion to the
monophosphate. To further characterize dCK and
UCK2 activity on 2’-AzCyd, we performed a time
course assay, which clearly demonstrates that dCK
exhibits significantly higher activity on 2’-AzCyd
than does UCK2 (Figure 2c). Interestingly, under our
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
conditions
we
found
that
2’-AzCyd
is
phosphorylated by dCK more rapidly than
deoxycytidine, the native substrate of the enzyme
(Figure 2c), and that cytidine is the best substrate
out of all pyrimidine analogs tested with dCK.
Analysis of dCK26 and
Figure 2. Phosphorylation of 2’-azidonucleosides by
UCK2 or dCK. (A) HPLC chromatagrah of 2’-AzCyd
phosphorylation by recombinant UCK2 or dCK
enzymes. (B) Quantification of 2’-azidonucleoside
phosphorylation. Data represent mean ± s.d. (n=3). (C)
Kinetic analysis of cytidine, deoxycytidine, or 2’-AzCyd
phosphorylation. (D) Structural model of 2′-AzCyd
bound to dCK. (E) Crystal structure of dCK–deoxyCyd
complex (PDB: 1P61) (F) Crystal structure of UCK2
with UTP (PDB: 1UEI).
Figure 3. dCK mediates 2’-AzCyd incorporation. (A)
Workflow for 2’-azidopyrimidine incorporation. (B)
and (C) HeLa cells transfected with plasmids encoding
FLAG-tagged UCK2 or dCK were treated with 1 mM 2’-
AzCyd or 1 mM 2’-AzUrd for 6 hr.
UCK227 x-ray crystal structures suggests that 2’-
azido or 2’-hydroxyl modifications can be readily
accommodated in the dCK active site (where these
functional groups could engage in additional polar
interactions) (Figures 2d and 2e), while a 2’-azido
group is likely unable to fully participate in the
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 8
substrate. Taken together, our results indicate that
upon dCK expression and a very minimal increase in
2’-AzUrd labeling with UCK2 (Figure 3c and
Supplementary Figure 4), in-line with our inability to
detect in vitro nucleoside phosphorylation.
1
2
3
4
5
6
7
8
dCK can efficiently phosphorylate 2’-AzCyd and is
likely to be the major cellular kinase mediating RNA
incorporation of this modified nucleoside. Further,
the activity of dCK on cytidine implies a potential
role for this kinase in ribonucleoside salvage.
To provide additional insight into 2’-
azidopyrimidine incorporation, we measured
modification levels in total RNA using quantitative
nucleoside LC-MS/MS. In brief, cells were fed 2’-
AzCyd or 2’-AzUrd for 12 hrs, total RNA was isolated
and digested to nucleosides, and the individual
levels were quantitated using LC-QQQ-MS with
appropriate standard curves. Using this assay and
consistent with our imaging results, 2’-AzCyd
incorporation was dramatically increased by dCK
overexpression (4.3-fold increase over control cells)
resulting in a final 2’-AzCyd level of 0.3% relative to
unmodified cytidine (Figure 4, Supplementary Table
3). UCK2 overexpression also increased 2’-AzCyd
labeling but by a smaller magnitude compared to
control cells. Additionally, we investigated 2’-AzUrd
incorporation. In control cells and dCK-transfected
cells, we were unable to detect 2’-AzUrd in total RNA.
However, upon UCK2 transfection we could measure
a small amount of cellular labeling resulting in
0.03% incorporation (Figure 4, Supplementary
Table 4). While detectable, this is roughly 10-fold
lower than 2’AzCyd labeling using the dCK
overexpression system.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Incorporation of 2-’azidopyrimidines into
HEK293T cellular RNA. Digested total RNA was
analyzed by LC-MS/MS. Data represent mean ±ꢀs.d.
(n=ꢀ3); **p <ꢀ0.01; N.D. = not detected.
The ability of dCK to phosphorylate 2’-AzCyd led
us to speculate that overexpression of this enzyme
could increase incorporation and provide an avenue
for cell-specific labeling. To probe this, we
transfected HeLa cells with dCK plasmid and treated
with 1 mM 2’-AzCyd. In parallel, we treated
untransfected cells and cells transfected with UCK2
plasmid. After treatment, cells were imaged using
CuAAC chemistry to detect the incorporation of 2’-
AzCyd. Gratifyingly, overexpression of dCK
produced a large increase in 2’-AzCyd labeling
(Figure 3b). We were able to observe a similar effect
using a lower concentration of 2’-AzCyd as well
(Supplementary Figure 3). Interestingly, despite its
poor ability to phosphorylate 2’-AzCyd in vitro, UCK2
overexpression also increased labeling relative to
control cells, although by a smaller degree than dCK
overexpression (Figure 3b) and only at the 1 mM
treatment concentration (Supplementary Figure 3).
Due to the finding that even low in vitro activity
could enhance cellular accumulation (presumably
due to high-level protein expression and low
endogenous protein levels), we also investigated
dCK and UCK2 overexpression with 2’-AzUrd. In this
case, we observed no increase in 2’-AzUrd labeling
Having validated the dCK-2’-AzCyd pair as a robust
metabolic
labeling
strategy,
we
further
characterized 2’-AzCyd labeling in cellular RNA
using a stable HeLa cell line containing inducible dCK
(Supplementary Fig. 6). First we investigated the
effect of long-term 2’-AzCyd treatment on cell
viability. Notably, we observed a negligible change in
viability compared to untreated cells after 3-day
treatment with concentrations ranging from 10-200
µM 2’-AzCyd (Supplementary Fig. 11). In contrast,
the commonly used ribonucleoside analog 4-SU
demonstrated negative effects on HeLa cell viability
at concentrations as low as 10 µM (Supplementary
Fig. 11). Next we investigated the distribution of 2’-
AzCyd in different cellular RNAs. We isolated total
RNA and polyA RNA and analyzed the distribution of
2’-AzCyd containing RNA using SPAAC chemistry
and gel electrophoresis. Interestingly, we observed
2’-AzCyd accumulation primarily in the 28S and 18S
large rRNA species (Figure 5a, Supplementary Fig. 7
& 10) as well as in the small RNA fraction that likely
consists of 5S RNA, tRNA, and other small non-
coding RNAs (Figure 5a, Supplementary Fig. 7);
minimal accumulation was observed in the polyA
4
ACS Paragon Plus Environment

Page 5 of 8
Journal of the American Chemical Society
rapamycin, and (F) 50 µM NaAsO2 (EtBr stained
RNA fraction (Supplementary Fig. 10). This is in
stark contrast to the labeling pattern observed upon
2’-AzAd feeding (Figure 5a, Supplementary Fig. 10),
which demonstrates a ‘smear’ of large transcripts
consistent with its incorporation into cellular mRNA
through polyA polymerases3. Previous studies with
base-modified pyrimidine ribonucleosides have also
observed accumulation primarily in mRNA1, 13. To
further probe this finding, we investigated 2’-AzCyd
labeling in the presence of small-molecule RNA
polymerase inhibitors with different specificities.
Consistent with our gel electrophoresis results, we
found 2’-AzCyd labeling to be strongly inhibited by
actinomycin D, an RNA pol I-specific inhibitor28, but
largely unchanged in the presence of α-amanitin, an
RNA pol II-specific inhibitor29, as measured by
cellular microscopy and
1
2
3
4
5
6
7
8
samples can be found in Supplementary Fig. 12).
in-gel fluorescence of total RNA (Figure 5b and
Supplementary Fig. 8).
Finally, to demonstrate the utility of an RNA pol I-
specific label, we performed pulse-chase
experiments with 2’-AzCyd in our dCK
overexpressing cell line to study rRNA turnover
under different conditions. After pulse labeling, we
observed a decrease of 2’-AzCyd labeling 12-24 hr
into the chase (Figure 5c and 5d). As this time point
is similar to the doubling time of HeLa cells we
conclude that the decrease in signal is primarily due
to dilution upon cell division rather than
degradation of rRNA, consistent with prior reports
of long rRNA half-life30. In contrast, when cells were
treated with the mTOR inhibitor rapamycin or
subjected to sodium arsenite, two conditions known
to induce ribosomal protein degradation
(‘ribophagy’)31-33, we found a much more rapid
decrease in signal starting around 3-6 hr (Figure 5e
and 5f), indicating accelerated degradation of rRNA.
Interestingly, rRNA degradation appeared to stop at
the 6 hr time point into the chase suggesting that
degradation during ribophagy only occurs on a
subset of rRNAs.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this work, we develop the 2’-AzCyd-dCK pair as
a robust strategy for cell- and polymerase-specific
RNA labeling. Compared to other nucleotides for
RNA labeling, this system has several advantages.
First,
azido-nucleotides
are
amenable
to
biorthogonal chemistry using SPAAC, which does not
result in RNA degradation. Second, 2’-AzCyd is less
toxic than the commonly used ribonucleoside analog
4-SU12, and can be incorporated at high levels into
cellular RNA (0.3% of C) allowing us to perform
functional assays on labeled RNA without
enrichment. Notably, Spitale and co-workers
recently reported cell-specific labeling using 2’-
AzUrd and WT UCK224. While we were able to
observe low level cellular incorporation of 2’-AzUrd
upon UCK2 overexpression (Figure 3 and Figure 4),
we find the 2’-AzCyd-dCK pair to be superior as it
facilitates RNA labeling with 10-fold higher
efficiency (Figure 4). Additionally, we show that 2’-
AzCyd is primarily utilized by RNA Pol I, providing a
distinct labeling profile compared to other available
probes. We exploit this property to develop a non-
radioactivity-based rRNA turnover assay and study
the lifetime of 28S and 18S rRNA during mTOR
inhibition and arsenite treatment. These conditions
Figure 5. 2’-AzCyd predominantly labels rRNA. (A) Gel
analysis of 2’-AzAd and 2’-AzCyd labeling. (B) Labeling
of dCK-expressing HeLa cells with 2’AzCyd in the
presence of ActD or -amanitin. (C) Turnover of 2’-
AzCyd labeled RNA in HeLa cells. Cells were pulsed
with 1 mM 2’-AzCyd for 12 h and chased with complete
medium. (D), (E) and (F) Turnover of 2’-AzCyd RNA
during ribophagy: (D) untreated, (E) 500 nM
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 8
5.
Schofield, J. A.; Duffy, E. E.; Kiefer, L.; Sullivan, M. C.;
have been previously shown to promote ribophagic
flux32 and our work demonstrates concomitant
degradation of rRNA, and provides a tool for
dissecting the mechanism of this process in
mammalian systems. Finally, while most approaches
for functionalizing cellular RNA have focused on
nucleobase modifications, our work demonstrates a
unique synergy between deoxynucleoside salvage
and RNA polymerases that can be exploited to
incorporate modifications in place of the ribose 2’-
OH, presenting new opportunities for labeling
cellular RNA with diverse chemical groups for
probing biological processes.
Simon, M. D., TimeLapse-seq: adding a temporal dimension to
RNA sequencing through nucleoside recoding. Nat Methods
2018, 15 (3), 221-225.
1
2
3
4
5
6
7
8
6.
Herzog, V. A.; Reichholf, B.; Neumann, T.; Rescheneder,
P.; Bhat, P.; Burkard, T. R.; Wlotzka, W.; von Haeseler, A.; Zuber,
J.; Ameres, S. L., Thiol-linked alkylation of RNA to assess
expression dynamics. Nat Methods 2017, 14 (12), 1198-1204.
7.
Sawant, A.; Tanpure, A.; Mukherjee, P.; Athavale, S.;
Kelkar, A.; Galande, S.; Srivatsan, S., A versatile toolbox for
posttranscriptional chemical labeling and imaging of RNA.
Nucleic Acids Res 2016, 44 (2), e16.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8.
Mehellou, Y.; Balzarini, J.; McGuigan, C., Aryloxy
Technology for Delivering
Phosphoramidate Triesters:
Monophosphorylated Nucleosides and Sugars into Cells.
Chemmedchem 2009, 4 (11), 1779-1791.
9.
a
Feldman, A. W.; Fischer, E. C.; Ledbetter, M. P.; Liao, J.
Y.; Chaput, J. C.; Romesberg, F. E., A Tool for the Import of Natural
and Unnatural Nucleoside Triphosphates into Bacteria. Journal of
the American Chemical Society 2018, 140 (4), 1447-1454.
ASSOCIATED CONTENT
Supporting Information. Experimental methods,
supplementary tables, and supplementary figures are
available free of charge on the ACS Publications
website.
10.
Miller, M. R.; Robinson, K. J.; Cleary, M. D.; Doe, C. Q., TU-
tagging: cell type-specific RNA isolation from intact complex
tissues. Nat Methods 2009, 6 (6), 439-41.
11.
Favre, A.; Moreno, G.; Blondel, M. O.; Kliber, J.; Vinzens,
F.; Salet, C., 4-Thiouridine photosensitized RNA-protein
crosslinking in mammalian cells. Biochem Biophys Res Commun
1986, 141 (2), 847-54.
AUTHOR INFORMATION
12.
Burger, K.; Muhl, B.; Kellner, M.; Rohrmoser, M.;
Corresponding Author
Gruber-Eber, A.; Windhager, L.; Friedel, C. C.; Dolken, L.; Eick, D.,
4-thiouridine inhibits rRNA synthesis and causes a nucleolar
stress response. RNA Biol 2013, 10 (10), 1623-30.
rkleiner@princeton.edu
Funding Sources
13.
Zhang, Y.; Kleiner, R. E., A Metabolic Engineering
Approach to Incorporate Modified Pyrimidine Nucleosides into
Cellular RNA. J Am Chem Soc 2019, 141 (8), 3347-3351.
No competing financial interests have been declared.
14.
Akerblom, L.; Reichard, P., Azidocytidine is a specific
ACKNOWLEDGMENT
inhibitor of deoxyribonucleotide synthesis in 3T6 cells. J Biol
Chem 1985, 260 (16), 9197-202.
This research was supported by the Damon Runyon
Cancer Research Foundation (DFS #21-16 to R.E.K.),
Sidney Kimmel Foundation (Sidney Kimmel Scholar
Award to R.E.K.), and the Alfred P. Sloan Foundation
(Sloan Foundation Research Fellowship to R.E.K.). Y.Z.
was supported by a generous gift from the Edward C.
Taylor 3rd Year Graduate Fellowship in Chemistry. All
authors thank Princeton University for financial
support.
15.
3T6 mouse fibroblasts. FEBS Lett 1985, 193 (2), 203-7.
16. Kruger, K.; Grabowski, P. J.; Zaug, A. J.; Sands, J.;
Akerblom, L., Azidocytidine is incorporated into RNA of
Gottschling, D. E.; Cech, T. R., Self-splicing RNA: autoexcision and
autocyclization of the ribosomal RNA intervening sequence of
Tetrahymena. Cell 1982, 31 (1), 147-57.
17.
Akerblom, L.; Pontis, E.; Reichard, P., Effects of
azidocytidine on DNA synthesis and deoxynucleotide pools of
mouse fibroblast cell lines. J Biol Chem 1982, 257 (12), 6776-82.
18.
Eliasson, R.; Pontis, E.; Reichard, P.; Eckstein, F.,
Replication of polyoma DNA in nuclei isolated from
azidocytidine-inhibited fibroblasts. J Biol Chem 1981, 256 (17),
9044-50.
REFERENCES
1.
Jao, C. Y.; Salic, A., Exploring RNA transcription and
19.
Skoog, L.; Bjursell, G.; Thelander, L.; Hagerstrom, T.;
turnover in vivo by using click chemistry. Proc Natl Acad Sci U S
A 2008, 105 (41), 15779-84.
Hobbs, J.; Eckstein, F., 2'-Deoxy-2'-azidocytidine, a new inhibitor
of DNA replication in mammalian cells. Eur J Biochem 1977, 72
(2), 371-8.
2.
Curanovic, D.; Cohen, M.; Singh, I.; Slagle, C. E.; Leslie, C.
S.; Jaffrey, S. R., Global profiling of stimulus-induced
polyadenylation in cells using a poly(A) trap. Nat Chem Biol
2013, 9 (11), 671-3.
20.
Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.;
Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R.,
Copper-free click chemistry for dynamic in vivo imaging. Proc
Natl Acad Sci U S A 2007, 104 (43), 16793-7.
3.
Nainar, S.; Beasley, S.; Fazio, M.; Kubota, M.; Dai, N.;
Correa, I. R., Jr.; Spitale, R. C., Metabolic Incorporation of Azide
Functionality into Cellular RNA. Chembiochem 2016, 17 (22),
2149-2152.
21.
Eriksson, S.; Munch-Petersen, B.; Johansson, K.; Eklund,
H., Structure and function of cellular deoxyribonucleoside
kinases. Cell Mol Life Sci 2002, 59 (8), 1327-46.
4.
Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.;
22.
Fauster, K.; Hartl, M.; Santner, T.; Aigner, M.; Kreutz, C.;
Hausser, J.; Berninger, P.; Rothballer, A.; Ascano, M., Jr.;
Jungkamp, A. C.; Munschauer, M.; Ulrich, A.; Wardle, G. S.; Dewell,
S.; Zavolan, M.; Tuschl, T., Transcriptome-wide identification of
RNA-binding protein and microRNA target sites by PAR-CLIP.
Cell 2010, 141 (1), 129-41.
Bister, K.; Ennifar, E.; Micura, R., 2'-Azido RNA, a versatile tool for
chemical biology: synthesis, X-ray structure, siRNA applications,
click labeling. ACS Chem Biol 2012, 7 (3), 581-9.
23.
Aigner, M.; Hartl, M.; Fauster, K.; Steger, J.; Bister, K.;
Micura, R., Chemical synthesis of site-specifically 2'-azido-
6
ACS Paragon Plus Environment

Page 7 of 8
Journal of the American Chemical Society
modified RNA and potential applications for bioconjugation and
RNA interference. Chembiochem 2011, 12 (1), 47-51.
24. Nainar, S.; Cuthbert, B. J.; Lim, N. M.; England, W. E.; Ke,
K.; Sophal, K.; Quechol, R.; Mobley, D. L.; Goulding, C. W.; Spitale,
R. C., An optimized chemical-genetic method for cell-specific
metabolic labeling of RNA. Nat Methods 2020, 17 (3), 311-318.
29.
Lindell, T. J.; Weinberg, F.; Morris, P. W.; Roeder, R. G.;
Rutter, W. J., Specific inhibition of nuclear RNA polymerase II by
alpha-amanitin. Science 1970, 170 (3956), 447-9.
30.
Borel, J. P., Protein synthesis in collagen lattice-cultured
fibroblasts is controlled at the ribosomal level. FEBS Lett 1995,
357 (3), 287-9.
1
2
3
4
5
6
7
8
Gillery, P.; Georges, N.; Wegrowski, J.; Randoux, A.;
25.
Van Rompay, A. R.; Norda, A.; Linden, K.; Johansson, M.;
Karlsson, A., Phosphorylation of uridine and cytidine nucleoside
analogs by two human uridine-cytidine kinases. Mol Pharmacol
2001, 59 (5), 1181-6.
31.
Wyant, G. A.; Abu-Remaileh, M.; Frenkel, E. M.; Laqtom,
N. N.; Dharamdasani, V.; Lewis, C. A.; Chan, S. H.; Heinze, I.; Ori, A.;
Sabatini, D. M., NUFIP1 is a ribosome receptor for starvation-
induced ribophagy. Science 2018, 360 (6390), 751-758.
26.
Sabini, E.; Ort, S.; Monnerjahn, C.; Konrad, M.; Lavie, A.,
9
Structure of human dCK suggests strategies to improve
anticancer and antiviral therapy. Nat Struct Biol 2003, 10 (7),
513-9.
32.
An, H.; Harper, J. W., Systematic analysis of ribophagy
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
in human cells reveals bystander flux during selective autophagy.
Nat Cell Biol 2018, 20 (2), 135-143.
27.
Suzuki, N. N.; Koizumi, K.; Fukushima, M.; Matsuda, A.;
33.
Pestov, D. G.; Shcherbik, N., Rapid cytoplasmic turnover
Inagaki, F., Structural basis for the specificity, catalysis, and
regulation of human uridine-cytidine kinase. Structure 2004, 12
(5), 751-64.
of yeast ribosomes in response to rapamycin inhibition of TOR.
Mol Cell Biol 2012, 32 (11), 2135-44.
28.
Perry, R. P.; Kelley, D. E., Inhibition of RNA synthesis by
actinomycin D: characteristic dose-response of different RNA
species. J Cell Physiol 1970, 76 (2), 127-39.
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 8
TOC graphic
1
2
3
4
5
NH2
N
NH2
N
NH2
N
O
P
dCK
N
O
HO
N
O
HO
O
O
N
O
O
O
O
O
deoxycytidine
kinase (dCK)
OH N3
OH N3
OH N3
2'-AzCyd
6
7
N3
8
9
NH2
RNA Pol I
N
O
O
P
O
N
O
N3
N3
3
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
rRNA
Ribosome
OH N3
8
ACS Paragon Plus Environment