Temporal Labeling of Nascent RNA Using Photoclick Chemistry in Live Cells

Article abstract of DOI:10.1021/jacs.7b03121

We report the first cellular application of a photoclick SPAAC reagent to label azide-functionalized RNA. 350 nm irradiation of a cyclopropenone caged oxo-dibenzocyclooctyne (photo-ODIBO) biotin yields formation of the SPAAC reactive species, which rapidly forms adducts with RNA containing 2′-azidoadenosine (2′N₃-A). Photo-ODIBO was found to be highly stable in the presence of thiols, conferring greater stability relative to ODIBO. Light activated photo-ODIBO enabled tagging of cellular RNA, in addition to fluorescent imaging as well as enrichment of RNA in cell subpopulations via selective irradiation.

Full text of DOI:10.1021/jacs.7b03121

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Communication
Temporal Labeling of Nascent RNA Using Photo-click Chemistry in Live Cells
Sarah Nainar, Miles Kubota, Christopher D. McNitt, Catherine Tran, Vladimir V. Popik, and Robert C. Spitale
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 31 May 2017
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Temporal Labeling of Nascent RNA Using Photo-click Chemistry in
Live Cells
Sarah Nainar,† Miles Kubota,† Christopher McNitt,‡ Catherine Tran,† Vladimir V. Popik‡ and
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†
§
Robert C. Spitale *
†Department of Pharmaceutical Sciences and §Department of Chemistry, University of California, Irvine, Irvine, California
92697, United States. ‡Department of Chemistry, University of Georgia, 140 Cedar St., Athens, GA 30602, United States.
Supporting Information Placeholder
While these methods are highly useful, they do not enable
cell-specific tagging, which would endow experimenters
with kinetic control of RNA trapping.
ABSTRACT: We report the first cellular application of a
photo-click SPAAC reagent to label azide-functionalized
RNA. 350 nm irradiation of a cyclopropenone caged
oxo-dibenzocyclooctyne (photo-ODIBO) biotin yields
formation of the SPAAC reactive species, which rapidly
forms adducts with RNA containing 2’-azidoadenosine
(2’N3-A). Photo-ODIBO was found to be highly stable
in the presence of thiols, conferring greater stability rela-
tive to ODIBO. Light activated photo-ODIBO enabled
tagging of cellular RNA, in addition to fluorescent imag-
ing as well as enrichment of RNA in cell subpopulations
via selective irradiation.
It has been reported that short irradiation at 350 nm of
cyclopropenone-caged cyclooctyne causes decarbonylation
to yield a reactive triple bond.12 Notably, the products of
such decarbonylation reactions can serve as SPAAC rea-
gents with faster reaction kinetics.13-16 These light-activated
photo-click SPAAC reagents have thus far been used as
cross-linkers for surface immobilization and probes for cell
surface labeling.17,18 However, their intracellular stability
and subsequent applications for the selective labeling of
many biomolecules have yet to be fully explored. This has
limited the use of photo-click SPAAC reactions in cells,
and specifically their utility for selective RNA tagging.
In this Communication, we report the first intracellular
use of a photo-click SPAAC reagent for tagging nascent
azide-modified RNA. We demonstrate greater in vitro sta-
Recent gene expression analysis has revealed that the
majority of cellular RNA is non-coding. It has further been
elucidated that RNA controls or regulates nearly every step
of gene expression, from transcription to translation.1 The
RNA transcriptome is thus highly dynamic and can vary
amongst different cell types, even within the same tissue.
Despite the critical importance of RNA and its specificity
of expression, there is a deficit of chemical methods to
probe the transcriptome of unique cell types with precise
kinetic control.
Bioorthogonal chemistries have, however, provided av-
enues toward these analyses, as there is currently a robust
array of “click” reactions that enable tagging of biomole-
cules of interest.2 The chemical reporter strategy has has-
tened the development of such reagents, which have found
widespread use in studying glycosylation, protein function
and localization and DNA dynamics.3-5
The most widely utilized click-reaction for RNA label-
ing is the azide-alkyne cycloaddition, of which there are
two classes.6 While the copper catalyzed azide-alkyne cy-
cloaddition (CuAAC) has been used extensively in many
biological contexts, the use of a cytotoxic copper catalyst
limits the use of terminal alkynes in living systems.7,8 Con-
versely, azide functionality is more versatile, providing
additional utility in metal-free strain promoted azide-alkyne
cycloaddition (SPAAC) reactions.9 Our lab has recently
reported that azide-modified nucleosides can metabolically
incorporate into nascent RNA, thus enabling SPAAC reac-
tivity.10 SPAAC reactions have been widely used for many
live cell applications in addition to in vivo imaging.4,11
bility
of
the
cyclopropenone-caged
oxa-
dibenzocyclooctyne versus the uncaged molecule in the
presence of thiols, which most importantly confers greater
reactivity of tagging in living cells. We also demonstrate
the ability to use this reagent for light-activated tagging of
RNA in live cells, in addition to tagging the RNA of specif-
ic cell subpopulations via selective irradiation and RNA
fluorescence imaging or enrichment.
With chemically synthesized cyclopropenone-caged
oxa-dibenzocyclooctyne (photo-ODIBO 1) and ODIBO 1a
in hand (synthesis in Supporting Information), we set out to
establish their reactivity with the nucleoside analog
2’azidoadenosine (2’N3-A). Upon 2 min of irradiation at
350 nm, photo-ODIBO 1 decarbonylates to form ODIBO
1a, which reacts rapidly with 2’N3-A to form the resulting
triazole product (Figure 1A). We were able to fully observe
this transformation via UV spectroscopy, where the spectra
before irradiation displays a combination of absorbances
from both 1 at 350 nm and 2’N3-A, with a characteristic
absorbance at 260 nm. Following decarbonylation, the for-
mation of ODIBO 1a results in a notable blue shift to 340
nm. Finally, as the 2’N3-A reacts with 1a, this band under-
goes bleaching as the triazole product is formed. It was
seen that after only 15 min, the reaction had gone to near
completion (Figure 1B and S1). We also measured the rate
constant of ODIBO 1a with 2’N3-A via UV spectroscopy.
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Using pseudo-first order conditions in excess of 1a, we
found an exceptionally fast rate constant of 40 ± 2 M-1 s-1
(Figure 1C).
for 15, 30 and 60 min. Control RNA was extracted from
HeLa cells treated with DMSO.
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Having demonstrated light-triggered labeling of RNA in
vitro, we next explored the stability of photo-click SPAAC
reagents in the presence of thiols. While cyclooctyne rea-
gents have ample utility for performing ligations in live
cells, their robustness can be hampered by thio-yne addi-
tion from cellular species such as cysteines and glutathi-
one.19 Moreover, the cyclopropenone is potentially suscep-
tible to ring opening in the presence of nucleophiles.12 In
order to establish the feasibility of using this reagent intra-
cellularly, we performed in vitro competition assays in the
presence of titrated thiol. We used β-mercaptoethanol
(BME), as its redox potential is comparable to that of gluta-
thione (GSH), the most abundant source of thiol in the
cell.20
Upon pre-incubating 1 and 1a with increasing amounts
of thiol, we saw changes in the efficiency of labeling azido-
RNA at BME concentrations of 10 and 100 mM. While the
signal from adducts formed by photo-ODIBO 1 was re-
duced at 100 mM, this greatly exceeds physiological condi-
tions and is not likely to be encountered by the probe. Im-
portantly, labeling by ODIBO 1a was abolished at the
physiologically relevant concentration of 10 mM and great-
er (Figure 3A and 3C). We also observed no changes in
signal when pre-incubating azido-RNA with BME, ruling
out thiol-mediated azide reduction (Figure S3).
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Figure 1. Establishing photo-click reactivity. A)
Scheme of photo-click reaction between photo-ODIBO 1
and 2’azidoadenosine. B) UV spectra of 1:1 500 µM solu-
tion of 1 and 2’N3-A before and after irradiation (solid, and
dashed), and subsequent formation of the triazole product
after 15, 30 and 60 min (green, red, blue respectively). C)
Reactivity of ODIBO with azide in PBS, 5% MeOH at 25
°C displaying the linear dependence of the observed rates
on 2’N3-A concentration.
After establishing the kinetics of this reaction, we
sought to test the in vitro reactivity of photo-ODIBO 1 with
extracted cellular RNA that has been metabolically labeled
with azide (Figure 2A). HeLa cells were treated with 500
µM 2’N3-A for 5 h, after which the total RNA was extract-
ed and purified. The azido-RNA was then incubated in
vitro with 50 µM of photo-ODIBO 1 or ODIBO 1a and
analyzed via a streptavidin Northern blot (Figure 2B and
S2). Following a 2 min irradiation with 350 nm light, total
RNA was robustly labeled with 1 after only 15 min. Non-
irradiated samples showed no labeling even after 60 min of
incubation with azido-RNA. DMSO-treated control RNA
also showed no labeling.
Figure 3. Photo-ODIBO 1 is refractory to thiol addi-
tion. A & C) Streptavidin Northern blot of 50 µM 1 or 1a
pre-incubated with a titration of BME (0, 10 µM, 100 µM,
1 mM, 10 mM, 100 mM) for 30 min at 37 °C, and then
reacted with RNA-N3 for 1 h. B & D) 500 µM of 1 or 1a
(red) incubated with 100 mM BME (orange) at 37 °C after
0, 30 and 60 min (blue, green and black, respectively).
To further investigate this, we monitored the reaction of
1 and 1a with BME via UV spectroscopy. We saw that the
characteristic peak of photo-ODIBO 1 at 350 nm is unaf-
fected after 1 h, while the blue shifted peak of ODIBO 1a
becomes fully bleached at 30 min (Figure 3B and 3D).
From this, we observe that photo-ODIBO 1 is more refrac-
tory to thiol addition, demonstrating greater stability of the
cyclopropenone-caged cyclooctyne species. We also de-
termined the second order rate constant of ODIBO 1a with
GSH, which was found to be 3.5 ± 0.2 × 10-4 M-1 s-1, while
Figure 2. In vitro photo-click SPAAC with azido-RNA.
A) Schematic of reaction of azido-RNA with photo-
ODIBO 1. B) Streptavidin Northern blot time titration
analysis of RNA-N3 treated with 50 µM 1, reacted at 37 °C
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there was no measurable reactivity of photo-ODIBO 1 cDNA successfully synthesized the small non-coding RNA
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(Figure S4). This is of great significance, as free thiol con-
centrations in cells can exceed 10 mM, concentrations at
which the de-caged cyclooctyne species become suscepti-
ble to addition before they are able to react.21 This data
concurs with previous findings that report cyclopropenone
stability in aqueous buffers and its ability to be conjugated
to thiol ligands.22,23 Lastly, this is the first direct demonstra-
tion that cyclopropenones with extended conjugation are
stable to thiol degradation, further supporting their utility as
SPAAC precursors for intracellular ligations.
Encouraged by the observed stability of photo-ODIBO
1a in the presence of high thiol concentrations, we moved
to investigate our ability to tag azido-RNA inside living
cells. After treating HeLa cells with 2’N3-A to install azide
functionality (or DMSO control), we allowed them to react
with 50 µM of compounds 1 or 1a for 2 h (Figure 4A).
Cells treated with photo-ODIBO 1 were pre-incubated for
30 min to allow the probe to diffuse into and distribute
throughout cells, after which they were irradiated for 2 min.
Following RNA extraction and Northern blot analysis, we
found that we were again able to light-dependently label
azido-RNA (Figure 4B). Interestingly, we saw that the effi-
ciency of labeling with 1 was reproducibly greater than that
of 1a, supporting our earlier findings of the cyclopropenone
conferring chemical inertness to nucleophilic milieu.
Using chemical probes to label RNA is of special inter-
est to the RNA community because it introduces methods
to enrich populations of the transcriptome that can be sub-
jected to gene expression analysis. To demonstrate this, we
performed semi-quantitative RT-PCR of azido-RNA from
cells treated with photo-ODIBO 1 for only 30 min. After
extraction and cDNA synthesis, we performed enrichment
over streptavidin beads followed by PCR. We were able to
successfully recover and enrich two chosen targets: 7SK
RNA, a non-coding nuclear transcript, and GAPDH, a cy-
tosolic mRNA. This labeling and subsequent enrichment
was also irradiation dependent. RNA from cells not treated
with 2’N3-A (DMSO control) did not enrich for these tar-
gets (Figure S5). Herein, we conclude that labeling with 1
is sensitive enough to be used for selective enrichment of
transcripts labeled on short timescales.
7SK and the mRNA GAPDH.
Bioorthogonal labeling is also useful for imaging of bi-
omolecules. Thus, we examined our ability to use photo-
ODIBO to visualize cellular RNA via fluorescent imaging.
To do this, we synthesized photo-ODIBO appended to the
fluorophore Rhodamine B (photo-ODIBO-RhB, 2) (Figure
S6). After installing azido-functionality, we incubated 2 in
cells, after which we were able to visualize strong signal
from nascent RNA. In irradiated cells, signal was seen in
the nucleolus as well as the cytoplasm, which concurs with
our previous findings that 2’N3-A is in both nuclear ribo-
somal RNA and cytosolic mRNA transcripts.10 DMSO con-
trol treated cells showed no fluorescent labeling with 2,
regardless of light irradiation (Figure S6). Treatment with
RNase A significantly reduced the fluorescent signal from
2, supporting our findings of RNA labeling (Figure S7).
A photo-click SPAAC reagent presents the unique op-
portunity to label the RNA of specific cells. We posited
that the selective irradiation of a targeted region of cells
would yield spatio-temporally labeled RNA only in the
light-irradiated region of interest. Thus, we created a sim-
ple rubber mask through which a window was cut to allow
light exposure of half of a cover glass on which HeLa cells
are grown (Figure S8). Cells treated with 2’N3-A were in-
cubated with 2 and one half of the cover glass was irradiat-
ed through the exposure window (Figure S8). Following
incubation, we were able to visualize clear regions of fluo-
rescently labeled and non-labeled cells on opposing sides
of the cover glass. These cells also demonstrated the same
staining pattern expected from 2’N3-A.
We envisioned our approach could be used to selective-
ly label, enrich, and profile the RNA content from unique
cells, as current bioorthogonal techniques do not enable this
type of application. To further test this, we grew EGFP-
expressing cells that were restricted to one half of a tissue
culture dish in co-culture with wild-type (WT) HeLa cells
(Figure S9). After incubation with 2’N3-A and pre-
incubation with 1, small regions of the cells were irradiated
to specifically tag either EGFP-expressing or WT cells.
After labeling for 30 min, RNA was extracted from the
whole plate and subjected to enrichment and RT-PCR
analysis.
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We again observed that we were able to selectively la-
bel the RNA of cells based on the location of irradiation.
Enrichment of the EGFP gene only occurred when a region
of this subpopulation was exposed to light, while irradia-
tion of the WT population did not enrich for EGFP.
GAPDH served as a positive control that could be enriched
from both populations. This experiment provides proof of
concept for spatiotemporally labeling and enriching the
transcriptomes of targeted cell-types, while eliminating the
need for specific isolation of these cells prior to RNA ex-
traction.
Figure 4. Photo-click labeling of RNA in living cells.
A) Schematic of live cell labeling of HeLa cells with com-
pounds 1 and 1a. B) Streptavidin Northern blot of 2’N3-A
or DMSO-treated cells labeled with 50 µM 1 (plus or mi-
nus 350 nm light) or 1a for 2 h. C) RT-PCR of azido-RNA
from cells labeled with photo-ODIBO 1 for 30 min. PCR of
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ported by the UC, Irvine, and the NIH (1DP2GM119164
RCS) and 1RO1MH109588 (RCS). Popik lab is supported
by NSF (CHE-1565646).
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dibenzocyclooctyne remains inert to high concentrations of
thiols, while at the same time conferring temporal control
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RNA in living cells, and purify various transcripts to be
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on
the ACS Publications website. Experimental methods, syn-
thetics schemes and spectra for all compounds.
(23) Luo, W.; Gobbo, P.; McNitt, C. D.; Sutton, D. A.; Popik, V. V.;
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AUTHOR INFORMATION
Corresponding Author
*rspitale@uci.edu
ACKNOWLEDGMENT
We thank members of the Spitale Lab for discussions. S.N.
is supported as a BEST-IGERT fellow. Spitale lab is sup-
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