RNA Cloaking by Reversible Acylation

Article abstract of DOI:10.1002/anie.201708696

We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100–200 mm) yields several 2′-OH acylations per RNA strand in as little as 10 min. This poly-acylated (“cloaked”) RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups (“uncloaking”). This fully restores RNA folding and biochemical activity.

Full text of DOI:10.1002/anie.201708696

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Accepted Article
Title: RNA Cloaking by Reversible Acylation
Authors: Anastasia Kadina, Anna Maria Kietrys, and Eric T. Kool
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201708696
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10.1002/anie.201708696
Angewandte Chemie International Edition
COMMUNICATION
RNA Cloaking by Reversible Acylation
Anastasia Kadina, Anna M. Kietrys, and Eric T. Kool*[a]
Abstract: We describe a selective and mild chemical approach to
controlling RNA hybridization, folding, and enzyme interactions.
Reaction of RNAs in aqueous buffer with an azide-substituted
acylating agent (100-200 mM) yields several 2’-OH acylations per
RNA strand in as little as 10 min. This poly-acylated (“cloaked”) RNA
is strongly blocked from hybridization with complementary nucleic
acids, from cleavage by RNA-processing enzymes, and from folding
into active aptamer structures. Importantly, treatment with a water-
soluble phosphine triggers a Staudinger reduction of the azide
groups, resulting in spontaneous loss of acyl groups (“uncloaking”).
This fully restores RNA folding and biochemical activity.
limits applications to short RNA constructs that are not readily
accessible to many biology laboratories, while longer transcribed
or native RNAs cannot be studied. One strategy for photocaging
of longer transcribed RNAs was reported in 2001 by Ando et
al.;[11b] the method uses a diazo compound to react with
backbone phosphate groups in RNAs via carbene chemistry. A
significant drawback of the method is the instability of the
phosphotriester products, leading to RNA fragmentation.[16] The
method has not been demonstrated to block hybridization or
folding, possibly due to the low numbers of modifications that
can be appended.
Two chemical blocking strategies for RNA and DNA have
been previously reported.[17] In this methods, one or several
guanine bases are caged with a trichloroethyl (TCE)[17a] or 4-
nitrobenzyl (NB)[17b] groups and incorporated into a nucleic acid
strand during solid-phase synthesis. The cage can be released
upon reduction of TCE group with Zn/AcOH, or NB group by
treatment with sodium thiosulfate. These methods, however, can
only be used on relatively short, synthetic nucleic acids.
The great complexity of RNA biology presents challenges to
chemical and biological analysis. Beyond the better-studied
messenger RNAs, the largest fraction of cellular RNA consists of
noncoding species, including not only transfer RNAs (tRNAs)
and ribosomal RNAs (rRNAs), but also a growing range of other
long and short noncoding species,[1] including small nuclear
RNAs (snRNA),[2] microRNAs (miRNA),[3] small nucleolar RNAs
(snoRNA),[4] circular RNAs (circRNA),[5] and tRNA fragments
(tRF).[6] The biological functions of a major fraction of these
species remain to be characterized, and their interactions with
other RNAs, proteins, and small molecules remain elusive.
Recent advancements in RNA sequencing[7] and in structure
mapping[8] have been important in characterizing RNAs, but the
field will benefit greatly from new methods as well. For example,
studies of interactions and changes that occur with RNA in time
and space could benefit from methods to control structure and
interactions in a chemically selective way. Moreover, the rapid
rise in use of RNA as a diagnostic biomarker might also benefit
from methods of controlling its properties in vitro.
RNA function can be studied with methods that temporarily
suspend its activity. For example, light-based blocking
(photocaging) strategies have been used recently in temporal
control of RNA activity.[9] In this approach, a photolabile caging
group is added onto one or multiple RNA bases,[10] phosphate
groups[11] or 2’-OH groups of synthetic oligoribonucleotides.[12]
Upon photoirradiation, the cage is released, restoring RNA
function. Such photocaging strategies have been used to control
and study gene expression[10,11b,13] and ribozyme function.[12b,14]
While photodeblocking offers the potential advantage of spatially
localized RNA activation, it carries the limitation of needing to
protect the RNA from light during preparation and handling. A
more significant issue is toxicity of intense short-wavelength light
exposure in cell-based experiments.[15] Moreover, standard
strategies require the application of caging only in chemically
synthesized RNAs, with synthetically modified components. This
Figure 1. Structure of RNA acylating reagent NAI-N3 and mechanism of RNA
cloaking/uncloaking. (a) Structure of acylating agent NAI-N3 (1) (b) Multi-
acylation of RNA 2’-OH groups (red) with 1. Polyacylation blocks hybridization
and folding. Subsequent removal of those groups (“uncloaking”) by phosphine
treatment restores RNA biophysical and biochemical activity. The byproduct is
a lactam 2 (shown) and a phosphine oxide. (c) Mechanism of deacylation.
We describe here a novel and versatile strategy for covalent
acylation and de-acylation of RNA. Our approach is distinct from
prior photocaging approaches in two important ways: it is carried
out post-synthetically in a single step; and it is reversed
chemically rather than by light. Different from previous RNA
acylation reactions[8c,8d,18] we now show that the reaction of RNA
with acylating reagent NAI-N3 (Figure 1a) can be carried out in
high yields, with multiple acyl blocking groups appended per
strand. We further show that this polyacylation (“cloaking”) can
block RNA folding, small molecule binding, hybridization, and
enzyme recognition. At a subsequent time, the unmodified RNA
with native activity can be restored by bioorthogonal deacylation
with a water-soluble phosphine (Figures 1b-c). Thus, this new
cloaking and uncloaking strategy enables chemical control of
[a]
Dr. A. Kadina, Dr. Eng. A. M. Kietrys, Prof. Dr. E. T. Kool
Department of Chemistry, Stanford University
Stanford, CA 94305 (USA)
E-mail: kool@stanford.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201708696
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COMMUNICATION
RNA. The methodology is exceptionally simple and mild, and
functions both with chemically synthesized or transcribed RNAs.
Design of a new de-acylation strategy. To achieve this
blocking and unblocking, we adopted our recently developed
water-soluble nicotinyl acylating agent NAI-N3,[18] but we
completely changed its subsequent reactivity to enable de-
acylation. Previously, the azide group in this reagent was used
as a site for a “click” reaction with a biotin group, and the ester
remained linked to the RNA. In the new work, we recognized
that the azide group is ideally positioned to provide a handle for
self-removal of the group from the RNA 2’-OH. Staudinger
reduction of the azide could result in cyclization, releasing
restored RNA and lactam 2 (Figure 1b-c). Azide reduction with
phosphines has been studied for multiple biological and
biotechnological applications: for example, for reduction of
azidophenylalanine in proteins,[19] and as a reversible terminator
in sequencing chemistry.[7] O-azidomethylbenzoyl groups were
previously used in organic synthesis to protect alcohols and
amines, and were released during the deprotection step with aryl
phosphines.[20]
yielded high levels of uncloaking (Figure S4); we selected 2-
(diphenylphosphino)benzoic acid[21] (DPBA 3), one of the best
performers, for further studies. Incubation of cloaked RNA with
20 mM DPBA (Figure 3) at 37 ˚C in Tris buffer (with 20% DMSO
to support DPBA solubility) resulted in removal of AMN groups in
a time-dependent manner (Figure S5). We confirmed almost
complete RNA uncloaking by MALDI-TOF mass spectrometry
after 6 hours of incubation (Figure S5-f), however, a significant
To test our proposed cloaking-uncloaking strategy with NAI-
N3, we chose to study reactions initially with short transfer RNA-
derived RNA fragments (tRFs).[6] tRFs represent an ideal
research target due to their small size, diversity, and biological
significance.[6] Initial reactions were carried out with 18
nucleotide (nt) tRNA fragments tRF-3005 and tRF-3004.[6a] In
addition, to determine whether secondary structure influences
possibility of RNA labeling, we designed tRF-3005 analogs (tRF-
3005/8 and tRF-3005/4) incorporating hairpin architecture with 8
nt and 4 nt loops, respectively (see sequences in the Supporting
Information (Table S1)). RNAs were labeled at their 5’-ends with
Cy5 to facilitate analysis.
Prior acylation mapping studies were carried out at a level of
<1 acyl group per RNA strand.[18] In the new cloaking strategy,
much higher degrees of acylation were required, in order to
block hybridization, folding, and intermolecular interactions. To
test this, we prepared a high concentration (2 M) NAI-N3 stock
solution in DMSO.[18] We incubated RNAs with 200 mM NAI-N3
at 37 ˚C for 2 h in HEPES buffer. Successful reaction was
confirmed by denaturing polyacrylamide gel electrophoresis
(PAGE) in (Figure 2a, lanes 1 and 2) and quantified by MALDI-
TOF MS (Figure 2b). Gratifyingly, we observed a range of 8 to
16 azidomethylnicotinyl (AMN) groups per 18 nucleotide-long
tRF-3005 RNA (Figure 2b), order of magnitude higher acylation
than achieved before with mapping experiments.[8,18] We found
that the labeling extent depended on the number of accessible
single-stranded 2’-OH groups (Figures S1-S2).[8c,8d,18] Hairpin
Figure 2. Cloaking and uncloaking of tRF-3005 RNA. (a) PAGE gel showing
untreated (lane 1), cloaked (lane 2), and uncloaked (DPBA, 1 h, 37 ˚C) (lane
3) tRF-3005. (b) MALDI-TOF mass spectrum of cloaked tRF-3005; numerals
in red indicate numbers of adducts. See Figure S4 for details.
Figure 3. Structures of phosphines used in this study.
level of RNA restoration was already observed after 1 h (Figure
2a (lane 3), and Figure S5c). Incubation with DPBA results in
rapid reduction of azides; after 30 min incubation, no azides
remained, and masses corresponding to the reduced amino
adducts were seen. The limiting step is the intramolecular
nucleophilic attack of the intermediate, but extended incubation
resulted in loss of the majority of those intermediates (Figure
S5). Importantly, complete loss of these groups is not required
for restoration of activity (vide infra). As expected for the new
designed self-deprotection mechanism, lactam 2 is a byproduct,
as confirmed by LC-MS (Figure S7).
tRF-3005/8 having
8
single-stranded nucleotides yielded
acylated RNA with 4 to 10 labels (Figure S2 a-c), and reducing
the loop size to 4 nt in tRF-3005/4 reduced this number to 2-7
AMN groups per strand (Figure S2 d-f). Importantly, the DNA
analog of tRF-3005 showed very little acylation (<0.2 groups per
strand, as estimated by MALDI-MS (Figure S3)), which is likely
the result of minor reaction with a terminal hydroxyl group or a
hydroxyl group of Cy5. Thus, the NAI-N3 reagent is highly
selective for RNA over DNA, due to the reactive 2’-OH
nucleophiles in the former. The data show that RNA acylation
with NAI-N3 can be carried out conveniently in a single step,
yielding high loading of acyl groups.
Blocking and triggering hybridization. The next important
question to address was whether polyacylation of tRF RNAs
could block their biophysical and biochemical properties.
Although acyl groups have been shown to block progress of a
reverse transcriptase enzyme,[8] they have not been tested for
their effect on duplex stability. First, we sought to see if cloaking
can be used to control hybridization to a complementary
Next, we tested whether our proposed Staudinger reduction
strategy could proceed efficiently for uncloaking of polyacylated
RNA. Initial testing of a range of phosphines showed that most
oligonucleotide. To quantify hybridization, we designed
a
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10.1002/anie.201708696
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molecular beacon (MB) assay,[22] employing
a
MB
30 min is only about a half that of control RNA (Figure 5b, Table
S4). Finally, uncloaking tRF-3005 with DPBA completely
restored the RNase H cleavage of RNA (Figure 5b, Table S4
and Figure S10). Overall, we find that acylation-based cloaking
complementary to tRF-3005 (Table S1). For untreated RNA, we
observed a large increase in fluorescence upon addition of tRF-
3005 (Figure 4, green line and bar). Cloaking tRF-3005 with
NAI-N3 largely abolished the MB signal (>95% blocked; Figure 4,
red line and bar), establishing that polyacylation strongly impairs
hybridization. Significantly, uncloaking the RNA with DPBA for 1
h at 37 ˚C completely restored molecular beacon fluorescence
(Figure 4, blue line and bar). Thus, the results show that the
acylation cloaking – uncloaking strategy provides a high degree
of control over RNA hybridization.
can act as
a strong mechanism for blocking enzymatic
recognition and cleavage of RNA, and that uncloaking efficiently
restores biochemical activity.
Figure 5. Cloaking protects RNA from cleavage by enzymes that require
hybridization, while uncloaking restores enzymatic cleavage. (a) Mechanism of
NAI-N3 mediated RNA protection from cleavage by DNAzyme 10-23. (b) NAI-
N3-treated RNA slows cleavage by the 10-23 DNAzyme: bar chart represents
tRF-3004 fraction cleaved after 30 min incubation with 25 nM DNAzyme 10-
23: green, untreated tRF-3004 RNA, red, cloaked RNA, blue, uncloaked RNA
(20 mM DPBA, 3 h, 37 ˚C). (c) Acylated RNA is a poor substrate for HIV-1
RNase H: bar chart represents tRF-3005 fraction cleaved after 30 min
incubation with RNase H as determined by PAGE: green, untreated tRF-3005
RNA, red, cloaked RNA, blue, uncloaked RNA (20 mM DPBA, 3 h, 37 ˚C).
Error bars: ±s.d. and p-values: ** p<0.01, *** p<0.001, ns. - not significant.
Figure 4. Cloaking and uncloaking of RNA can be used to control its
hybridization, as measured by molecular beacon assay. (a) Mechanism of
NAI-N3 enabled hybridization control. (b) Plot of effect of cloaking/uncloaking
on MB fluorescence. Green line: Increase of fluorescence of MB upon its
incubation with untreated tRF-3005 RNA; red, cloaked tRF-3005; blue, signal
after uncloaking (20 mM DPBA (3), 1h, 37 ˚C). Data are representative of 3
experiments; (c) Molecular beacon (MB) hybridization to tRF-3005 RNA at 37
˚C is strongly suppressed by cloaking as measured by MB fluorescence
(background subtracted). Green bar, untreated tRF-3005; red, cloaked tRF-
3005; blue, uncloaked tRF-3005 (20 mM DPBA, 1h, 37 ˚C). Error bars
represent ±s.d. and p-values: *** p<0.001, * p<0.05.
Control of folding of RNA aptamers. The above
experiments were carried out primarily with short, synthetic,
single-stranded oligoribonucleotides. However, many biologically
relevant RNAs of interest are considerably longer than this, and
they often fold into secondary and tertiary structures that confer
their biophysical and biochemical properties. Thus, we aimed to
test whether a longer transcribed RNA could be efficiently
cloaked and controlled by polyacylation with NAI-N3. The
Spinach RNA aptamer was selected as an ideal test case, as it
is ~100 nt in length and folds into its active structure, binding the
DFHBI chromophore and yielding an intense green fluorescence
signal in its folded state.[25] We transcribed and purified a 102 nt
Spinach construct (Table S1), and confirmed that the Spinach
RNA yields a strong fluorescence light-up signal (250 fold) with
DFHBI in vitro in folding buffer (37 ˚C, 1 h) (Figure S11a).
Comparable results were obtained with F-30 Broccoli
aptamers[26] as well (Figure S11b).
Control of RNA cleavage by enzymes. We proceeded to
test whether cloaking of an RNA could hinder its recognition and
cleavage by nucleic acid or protein enzymes. In DNAzyme 10-
23,[23] two segments of DNA complementary to the RNA of
interest flank a 15 nt core that catalyzes cleavage of a 5’-Pu-Py-
3’ linkage in RNA. To measure DNAzyme activity, we chose tRF-
3004 RNA (Table S1) which conveniently has a 5’-A-U-3’
cleavage site in the center. 10-23-promoted cleavage of RNA
was analyzed by PAGE. For untreated RNA, we found that the
DNAzyme cleaves ≈15% tRF-3004 in 30 min (Figure 5a and
Figure S8). Cloaking RNA with NAI-N3 almost completely
abolishes RNA cleavage, indicating failure of the DNAzyme to
recognize the RNA. Uncloaking tRF-3004 by incubation with 20
mM DPBA (3 h, 37 ˚C) nearly completely restored the amount of
RNA cleaved in 30 min (Table S3). Similar results were also
seen with a second DNAzyme with a different target sequence
(Figure S9). We also found that cloaking tRF-3005 partially
protects it from degradation by Ribonuclease H (RNase H). This
enzyme recognizes and degrades RNA when it is hybridized to
its DNA complement.[24] Data showed that RNase H almost
completely degrades non-cloaked RNA in the presence of its
DNA complement after 30 min (Figure 5b and Figure S10). The
experiments show that the amount of cloaked RNA cleaved after
Next, we treated the RNA with 0.1 M NAI-N3 for 10 min - 1 h
(pH 7.5 MOPS buffer). This yielded cloaked RNA with a loss of
85% of the original signal (see spectra in Figure S11). NAI-N3
was originally used as a structure-specific reagent that is
selective for single-stranded nucleotides.[8c,8d,18] Since in
principle this reaction would yield little acylation in folded
regions, we next tested acylation in low ionic strength solution,
to destabilize folding and enable reaction in previously folded
regions.[25a,27] The resulting RNA cloaked under these low-salt
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conditions yielded almost complete loss of DFHBI signal (98.6%
loss, Figure 6), indicating virtually complete disruption of folded
aptamer structure and/or ligand binding ability. We observed that
acylation for only 10 min yielded this strong disruption under
these reaction conditions. Mock treatments with DMSO alone
yielded no disruption of signal. Next, we proceeded to test
uncloaking of the Spinach RNA, treating it with several
phosphines for varied times (Figure S12) and concentrations
(Figure S13). Interestingly, the folded RNA showed more
selectivity among phosphines in uncloaking (Figure S14).
Treatment with DPPEA for as little as 10 min completely
restored fluorescence of the Spinach RNA with DFHBI (100 % of
untreated RNA, Figure S12). Notably, PAGE analysis of the 102
nt RNA after the uncloaking procedure (Figure S15) confirms no
degradation of the RNA. Thus, we confirm that our
cloaking/uncloaking strategy can be used successfully to control
function of a transcribed RNA that relies on folding for its activity.
with “hot start” polymerase enzymes.[28] In addition, the
selectivity of the current acylation chemistry might enable
selective control of RNA in the presence of DNA, which can
otherwise be difficult to achieve. Further, since acylation blocks
hybridization, it could be used to reversibly block RNA structure
formation and trigger folding temporally. More studies are
planned to test some of these applications. Further in the future,
it is possible that such chemical blocking and unblocking of
RNAs could be carried out in living cells, to trigger biological
activity upon addition of
a chemical reductant. While an
intriguing possibility, this will require the development of cell-
permeable, low-toxicity reductants that can reduce the azide of
NAI-N3 (or related reagents) and achieve de-acylation efficiently
without adverse effects on cells.
Acknowledgements
We thank the U.S. National Institutes of Health (GM110050,
GM106067, and CA217809) for support.
Keywords: cloaking • RNA • caging • acylation • Staudinger
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Figure 6. Acylation-based control of RNA folding. (a) Mechanism of NAI-N3
driven control of RNA folding using Spinach 2 aptamer. (b) Spinach RNA (102
nt) was treated with 100 mM NAI-N3 in buffer, resulting in
a loss of
fluorescence signal (orange bar). Treatment with NAI-N3 under low ionic
strength conditions yielded greater loss of signal (red). Subsequent treatment
with a phosphine (DPPEA, 5 mM, 1 h), yielded 100% recovery of signal (dark
blue). Control treatment of RNA by DMSO in either buffer or water shows no
significant changes. Error bars represent ±s.d. and p-values: *** p<0.001, ns. -
not significant; (c) Fluorescence of Spinach cloaking reactions, from left to
right: untreated RNA, cloaked, and uncloaked RNA (DPPEA, 5 mM, 1 h).
Our experiments establish the use of NAI-N3 acylating agent
combined with phosphines to reversibly block interactions of
RNA with other molecules. Due to the controlled reactivity and
aqueous solubility of NAI-N3, high-density loading of RNAs is
possible, at least to a level of >50% of the 2’-OH groups. The
resulting AMN groups on the RNA destabilize duplex structures
involving the RNA, and thus block hybridization effectively.
Further, the acyl groups can block enzymatic recognition of the
RNA and significantly change rates of reaction, including RNase
H activity and DNAzyme cleavage. Importantly, AMN groups that
cloak RNA activity can be removed under mild conditions that
restore free RNA and its biochemical activity.
The current chemical cloaking/uncloaking strategy suggests
multiple possible future in vitro applications. Examples include
orthogonal RNA activities, in which some RNAs are temporarily
inactivated while others are not, or designating specific timing of
RNA activity in an assay. An analogous temporary biomolecular
inactivation is currently used for proteins in PCR amplification,
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10.1002/anie.201708696
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Anastasia Kadina, Anna M. Kietrys, and
Eric T. Kool*
Page No. – Page No.
RNA Cloaking by Reversible
Acylation
Chemical switch for RNA: We describe simple selective chemical approach for
control of RNA function. Polyacylation of 2’-OH groups with a nicotinyl-imidazole
reagent (cloaking) switches off structure and biomolecular recognition. RNA is
switched on again (uncloaking) by Staudinger reductions that remove the acyl
groups.
This article is protected by copyright. All rights reserved.