Chemical Profiling of A-to-I RNA Editing Using a Click-Compatible Phenylacrylamide

Article abstract of DOI:10.1002/chem.202001667

Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is “click”-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.

Full text of DOI:10.1002/chem.202001667

Accepted Article
Accepted Article
Title: Chemical Profiling of A-to-I RNA Editing Using a Click-
Compatible Phenylacrylamide
Authors: Steve D Knutson, Megan M. Korn, Ryan P. Johnson, Leanna
R. Monteleone, Deanna M. Dailey, Colin S. Swenson, Peter
A. Beal, and Jennifer Heemstra
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
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
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202001667
Link to VoR: https://doi.org/10.1002/chem.202001667
01/2020

10.1002/chem.202001667
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Chemical Profiling of A-to-I RNA Editing Using a Click-Compatible
Phenylacrylamide
Steve D. Knutsona, Megan M. Korna, Ryan P. Johnson, Leanna R. Monteleoneb, Deanna M. Daileya,
Colin S. Swensona, Peter A. Bealb and Jennifer M. Heemstraa
[a]
S. D. Knutson, M. Korn, R. Johnson, C. S. Swenson, D. Dailey, and J. M. Heemstra
Department of Chemistry, Emory University
1515 Dickey Dr., Atlanta GA 30322 (USA)
E-mail: jen.heemstra@emory.edu
[b]
L. R. Monteleone, and P.A. Beal
Department of Chemistry, University of California, Davis
One Shields Avenue, Davis, CA 95616 (USA)
Supporting information for this article is given via a link at the end of the document.
Abstract: Straightforward methods for detecting adenosine-to-
inosine (A-to-I) RNA editing are key to better understanding its
regulation, function, and connection with disease. We address this
need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide
(EPhAA), and illustrating its ability to selectively label inosine in RNA.
EPhAA is synthesized in a single step, reacts rapidly with inosine, and
is “click”-compatible, enabling flexible attachment of fluorescent
probes at editing sites. We first validate EPhAA reactivity and
selectivity for inosine in both ribonucleosides and RNA substrates,
and then apply our approach to directly monitor in vitro A-to-I RNA
editing activity using recombinant ADAR enzymes. This method
improves upon existing inosine chemical labeling techniques and
provides a cost-effective, rapid, and non-radioactive approach for
detecting inosine formation in RNA. We envision this method will
improve study of A-to-I editing and enable better characterization of
RNA modification patterns in different settings.
Scheme 1. Formation and detection of inosine in RNA. a) A-to-I editing is
catalyzed by adenosine deaminases acting on RNA (ADAR, pink). b) Inosine
nucleobases can be detected by reacting with Michael acceptors to yield N1
addition products.
RNA is chemically modified by a number of enzymes after
transcription, in turn influencing RNA stability, localization and
activity within the cell. Adenosine-to-inosine (A-to-I) RNA editing
is one of the most widespread modifications, and is performed by
adenosine deaminases acting on RNA (ADARs) (Scheme 1a).1
Adenosine deamination changes the molecular structure and
hydrogen bonding pattern of the nucleobase, and resulting
inosines instead base pair with cytidine to effectively recode these
sites as guanosine. Editing sites within protein-coding mRNAs
directly alter amino acid sequences and produce different protein
isoforms. Non-coding RNAs also undergo extensive editing,
including microRNAs and small-interfering RNAs, significantly
altering their biosynthesis, localization, and gene regulation
properties.2-3 A-to-I editing is essential for a number of biological
processes including tissue development,4-5 neurological function,6
and immune system activation.7 Dysfunctional editing is also
directly linked with autoimmune diseases,8-9 neurological
disorders,10 and several types of cancer.11-12
therapeutic site-directed RNA editing strategies,19 as both the
design and precise implementation of this machinery is reliant on
a thorough understanding of ADAR regulation.
Detecting inosine formation in RNA is of central importance
for characterizing editing mechanisms. While high-throughput
RNA sequencing (RNA-seq) is commonly employed for large
scale detection and mapping of A-to-I sites,20 this method is also
costly, prone to random sampling errors, and requires complex
bioinformatic analyses.21-22 Alternatively, model reactions using
ADAR enzymes with small RNA substrates (~20-50 nt) have
yielded substantial insights into how certain RNA sequences and
structural motifs are recognized and edited.17, 23-26 Although A-to-
I sites are “visible” as A-G transitions in Sanger sequencing,27-28
these methods require relatively large RNA substrates (>300-400
nt), and are incompatible with the small RNA strands that are ideal
for these experiments. To detect inosine in smaller substrates,
adenosines within chimeric RNA strands are often internally
radiolabeled with 32P. After ADAR editing, RNA substrates are
then digested with nuclease P1 and A-to-I nucleotide changes are
detected with autoradiographic thin layer chromatography.23, 25-26
While this method is effective, it is also time-consuming to
construct each RNA substrate, and assays using these
radioactive materials require specialized training, instrumentation,
and waste disposal protocols. Alternatively, deamination can be
Despite this importance, our overall understanding of A-to-I
editing regulation is limited. In particular, while many sites have
been identified (> 5 million),13-14 it is unclear why certain sites are
edited at higher frequency than others and what precise function
they each serve.15 Efforts to map A-to-I locations and ADAR
binding sites have revealed that editing patterns are highly
16-18
complex and variable in humans,7,
and the precise
mechanisms by which ADAR enzymes bind to and edit specific
RNA sequences remain unclear. This gap is also significant for
1
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Figure 1. a) Acrylamide derivatives were evaluated for reactivity with inosine by
b) monitoring product formation via HPLC. Values represent mean with S.D.
error bars (n = 3). c) Structural similarities (yellow) between N-phenylacrylamide
and acrylamidofluorescein.
detected by incorporating alkyne-modified or fluorogenic
thiolated-adenosine analogues into RNA substrates,29-30 but
these approaches can introduce structural alterations into RNA
targets that impact ADAR targeting, and they require lengthy
phosphoramidite monomer synthesis.
Figure 2. Reactivity assessment of EPhAA with ribonucleosides. a) Reaction
scheme of EPhAA with inosine. b) Representative HPLC traces depicting
formation of EPhAE1I over 24 hours. c) Dependence of pH on reaction rate
constants for EPhAA addition on inosine. d) EPhAA reactivity with each of the
major ribonucleosides over 24 hours. Values represent mean with S.D. error
bars (n = 3).
Direct chemical detection of A-to-I editing would circumvent
these assay limitations, and inosine has been shown to react with
Michael acceptors to yield N1 addition products (scheme 1b).31 In
particular, a recent approach utilized acrylonitrile to alkylate
inosines for reverse-transcription termination sequencing.
Termed “inosine chemical erasing sequencing” (ICE-seq), this
technique improved the accuracy of detecting A-to-I sites using
Sanger sequencing, but also suffered from significant limitations
in sensitivity, and requires matched DNA and RNA samples for
each assay.32 Subsequent work derivatized acrylonitrile for use in
“clickable”-biotinylation and enrichment of A-to-I edited
transcripts.33 While acrylonitrile is a promising scaffold for
chemical detection of inosine, derivatizing these reagents is
difficult and requires several synthetic and purification steps.
Alternatively, we recently reported an acrylamidofluorescein
reagent that enables fluorescent detection and enrichment of
inosine in RNA.34 While our initial study demonstrated feasibility,
acrylamidofluorescein also displayed poor solubility and was
restricted to fluorescein addition. However, acrylamide scaffolds
are simple to modify, and we were interested in elaborating upon
this architecture to develop an improved and more generalizable
inosine probe. Toward this goal, we first screened potential
acrylamide scaffolds for inosine reactivity using our previously
established
reaction
conditions
(50:50
EtOH:1M
triethylammonium acetate pH 8.6, 70 ºC) (Fig. 1). Acrylamide and
N-phenylacrylamide were both highly reactive toward inosine,
whereas alkylacrylamide scaffolds (mPEG acrylamide and N-
hydroxyethylacrylamide) gave little to no product formation (Figs.
1b, S1). Interestingly, N-phenylacrylamide is structurally similar to
acrylamidofluorescein (Fig. 1c), and it is likely this moiety exhibits
sufficient electron-withdrawing properties consistent in other
Michael acceptors.
We next sought to derivatize N-phenylacrylamide to enable
secondary functionalization with fluorescent probes using copper-
catalyzed azide-alkyne cycloaddition (CuAAC), or “click”
chemistry. We quickly identified 4-ethynylaniline as
a
commercially available, alkyne-functionalized intermediate,
enabling us to employ a one-step coupling with acrylic acid to
yield N-(4-ethynylphenyl)acrylamide (EPhAA, scheme 2). After
verifying product identity (Figs. S3-S5), we tested EPhAA for
reactivity with inosine and confirmed appearance of the expected
addition product N1-ethynylphenylamidoethylinosine (EPhAE1I)
by HPLC and ESI-MS analysis (Figs 2a-b, S6a, S7).
Deprotonation of N1 on inosine is known to mechanistically drive
this reaction,31 and so to further confirm that EpHAA undergoes
addition at this position, we tested our labeling reaction across
different pH values (Fig. 2c). As expected, we observed a steep
increase in reaction rates consistent with the known pKa value of
inosine N1 (~8.7).35 In assembling reaction mixtures, we also
Scheme 2. One-step synthesis of EPhAA.
2
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Figure 3. Validating chemical detection of inosine in RNA. a) Sequence and
structure of the model HER1 RNA hairpin substrate with target A (red) and the
orphan C base (black). b) EPhAA labelling and PAGE analysis of different
simulated RNA editing rates and c) densitometric quantification of signal
(arbitrary units, A.U.) across reactions. Values represent mean with S.D. error
bars (n = 2).
Figure 4. Chemical detection of ADAR1-mediated A-to-I RNA editing. a) ADAR1
amino acids interact with the orphan C base, with the E1008Q point mutation
providing increased stability and overall catalytic efficiency. b) Overall workflow
for detecting A-to-I editing with EPhAA labeling and CuAAC. c) EPhAA labelling
and PAGE analysis of in vitro A-to-I RNA editing reactions. d) Densitometric
quantification of signal across different RNA editing reactions using wild type
(WT, black) and E1008Q mutant (red) ADAR enzymes. Values represent mean
with S.D. error bars (n = 2).
noted
improved
solubility of EPhAA
compared
to
acrylamidofluorescein, and we were able to double our normal
working concentrations to ~500 mM. As expected, this resulted in
more rapid overall reaction kinetics, and when compared to our
previous reagent, we observed a ~2-3-fold increase in conversion
percentages at similar reaction times (Fig. 2d).34 Next, we
incubated EPhAA with the remaining ribonucleosides uridine (U),
guanosine (G), adenosine (A), and cytidine (C) (Figs. 2d, S6). In
this test, we observed robust selectivity towards I, and while U
and G have similar acidic nitrogens that can be labeled with
Michael acceptors,36-37 N1 on inosine displays much higher
nucleophilicity and reactivity with these reagents,31 and off-target
labeling was only observed at extended reaction times.
Acrylonitrile and acrylamide reagents are also known to react with
pseudouridine (Ψ),31 and we determined that our reagent
exhibited similar reactivity characteristics, as we observed the
expected N1 addition product (Figure S6f, S7, S9). While this off-
target reactivity may seem problematic, the primary application
we envision for our EPhAA reagent is detecting inosine in model
RNA strands to monitor ADAR activity, and Ψ can be omitted from
these substrates. Additionally, if assays necessitate the use of
cellular RNA or require Ψ content, existing carbodiimide reagents
can be employed to selectively block and deplete Ψ sites.38-39
Lastly, we assessed general EPhAA stability by incubating the
reagent in the absence of ribonucleoside, and while we observed
some degradation of the reagent in our labeling conditions, this
effect was minimal and only seen in extended reaction times (Fig.
S6g).
With our validated reagent in hand and given our ultimate
goal of detecting ADAR-mediated A-to-I editing, we next sought
to label inosine in RNA oligonucleotides. ADARs commonly
deaminate double-stranded RNA substrates at A:C mismatches,
“flipping out” the target A into the active site to leave behind an
“orphan C” base.1-2, 40 To mimic this, we synthesized a target
strand inspired by an mRNA hairpin (HER1) that undergoes
editing by human ADAR1 (hADAR1) at a defined site (Fig. 3a).23
To detect inosine, we planned to use CuAAC to install a
fluorophore after EPhAA labeling. We first wanted to verify that
our click-labeling conditions were optimal, so we subjected an
alkyne-functionalized DNA oligonucleotide to a standard CuAAC
protocol with a picolyl azide-functionalized Cyanine5 fluorophore
(Cy5-N3).41 As shown in Fig. S10, we observed complete labeling
of the alkyne-modified strand, indicating these conditions would
be compatible with our workflow. Given our previous data showing
background labeling of U and G nucleotides at very long reaction
times (Fig. 2d), we were next interested in optimizing EPhAA
labeling time to minimize off-target attachment. To test this, we
reacted HER1 RNA A and I substrates with EPhAA for increasing
amounts of time in independent duplicate trials followed by
3
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CuAAC labeling (Fig. S11). In RNA I samples, we expectedly saw
a rapid appearance of a major product band, indicative of inosine
fluorescent chemical labeling. RNA A did not produce significant
signal, but did exhibit a “smear” in longer reaction times, likely
indicating a mixture of products resulting from off-target labeling
at U and G residues. This distribution was also observed in RNA
I reactions with extended labeling times, further corroborating this
hypothesis. We also stained all RNA species in gels using SYBR
gold to assess overall labeling efficiency (Fig. S11). Although we
did not achieve full conversion of the RNA I strand, we identified
6 hours as an optimal EPhAA reaction time to achieve robust
selectivity (~60-fold I vs A labeling, Fig. S11). In particular, when
compared to our previous acrylamidofluorescein reagent, we
achieved significantly better selectivity (~60-fold vs ~8-fold) and
with much shorter reaction times (6 h vs 24 h).34 Lastly, given our
ultimate goal of detecting RNA editing, we were also interested in
assessing the linearity of our method for measuring different A-to-
I editing “rates.” To test this, we performed a series of duplicate
labeling reactions using varying ratios of A and I substrate while
keeping the total amount of RNA constant. As shown in Fig. 3b-c,
inosine content was highly proportional to fluorescent intensity
and we observed linearity between these variables (R2 = 0.95, r =
0.98), providing additional confidence that our method could
accurately measure A-to-I editing activity.
editing signatures in a variety of contexts. In particular, we view
EPhAA labeling as a cost-effective and rapid method to elucidate
the effects of RNA sequence and structure on ADAR editing
activity in vitro, better assess the biochemical impact of disease-
relevant ADAR mutations on pathological A-to-I editing, and
accurately measure the activity of engineered recombinant
enzymes for site-directed RNA editing.
Acknowledgements
This work was supported by the National Institutes of Health
(R01GM116991 to J.M.H.). P.A.B acknowledges support from the
National Institutes of Health in the form of R01GM061115. The
authors also thank Tewoderos Ayele and Travis Loya for helpful
discussions and advice.
Keywords: RNA • A-to-I editing • ADAR • click-chemistry •
bioconjugation • RNA chemistry
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Entry for the Table of Contents
Detecting Adenosine-to-Inosine (A-to-I) RNA editing is vital to improving our understanding of its regulation and function in humans.
Here we develop a new “click”-compatible reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), to selectively functionalize inosine in
RNA. EPhAA offers a cost-effective and rapid approach for detecting enzymatic A-to-I RNA editing activity in vitro.
Institute and/or researcher Twitter usernames: @MrSteveKnutson, @jenheemstra, @LeannaMonteleo1, @EmoryChem,
6
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