RNA Control by Photoreversible Acylation

Article abstract of DOI:10.1021/jacs.7b12408

External photocontrol over RNA function has emerged as a useful tool for studying nucleic acid biology. Most current methods rely on fully synthetic nucleic acids with photocaged nucleobases, limiting application to relatively short synthetic RNAs. Here we report a method to gain photocontrol over RNA by postsynthetic acylation of 2′-hydroxyls with photoprotecting groups. One-step introduction of these groups efficiently blocks hybridization, which is restored after light exposure. Polyacylation (termed cloaking) enables control over a hammerhead ribozyme, illustrating optical control of RNA catalytic function. Use of the new approach on a transcribed 237 nt RNA aptamer demonstrates the utility of this method to switch on RNA folding in a cellular context, and underlines the potential for application in biological studies.

Full text of DOI:10.1021/jacs.7b12408

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Communication
RNA Control by Photoreversible Acylation
Willem Arend Velema, Anna M Kietrys, and Eric T. Kool
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12408 • Publication Date (Web): 23 Feb 2018
Downloaded from http://pubs.acs.org on February 23, 2018
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.
Journal of the American Chemical Society 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 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
RNA Control by Photoreversible Acylation
Willem A. Velema, Anna M. Kietrys and Eric T. Kool*
10
11
12
13
14
Department of Chemistry, Stanford University, California 94305, United States
*To whom correspondence should be addressed: kool@stanford.edu
Supporting Information Placeholder
ABSTRACT: External photocontrol over RNA function has
emerged as a useful tool for studying nucleic acid biology. Most
current methods rely on fully synthetic nucleic acids with
photocaged nucleobases, limiting application to relatively short
synthetic RNAs. Here we report a method to gain photocontrol over
RNA by postꢀsynthetic acylation of 2’ hydroxyls with
photoprotecting groups. Oneꢀstep introduction of these groups
efficiently blocks hybridization, which is restored after light
exposure. Polyacylation (termed cloaking) enables control over a
hammerhead ribozyme, illustrating optical control of RNA catalytic
function. Use of the new approach on a transcribed 237nt RNA
aptamer demonstrates the utility of this method to switch on RNA
folding in a cellular context, and underlines the potential for
application in biological studies.
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
RNA is a highly versatile biological macromolecule, carrying out a
diverse range of cellular functions including gene expression,
catalysis, and cell signaling among other functions.1 In recent years,
research has uncovered many additional properties and applications
of RNA,2 including modulation of signaling pathways by circular
RNAs,3 RNA epigenetics,2b and RNA nanobiology.4 Due to the
complexity of RNA biology, there is a high demand for chemical
tools to study functions and properties of RNA.5
Figure 1. Modular design of photocloaking agents and cloaking
strategy. A) Schematic of the photocloaking strategy; polyacylation
of 2’ꢀOH groups in an existing RNA blocks RNA structure and
function, while photoexposure (365 nm) removes these blocking
groups. B) Structures of PCA reagents 1,2. C) Gel electrophoretic
analysis of untreated, cloaked and uncloaked RNA.
One important way to gather insight into RNA’s
biological role is by exerting external control over its function,6
allowing researchers to study downstream effects. External control
can be obtained by introducing labels that perturb its natural state
and function and that can be removed on demand. This has been
achieved in multiple laboratories by introducing photoreleasable
protecting groups (PPGs or photocaging groups) into RNA that can
be removed by light exposure.7 For example, timeꢀresolved RNA
folding kinetics were analyzed in detail by employing an RNA
strand that contained photoprotected guanosine,8 and embryonic
development has been studied using RNAs with photocleavable
backbones.9
To address these obstacles, we set out to develop a
general and accessible method that can be applied postꢀ
synthetically to synthetic and native RNAs regardless of length.
Inspired by previous studies showing that 2’ꢀOH groups of RNA
can be selectively acylated in aqueous buffers with activated acyl
compounds in structure mapping experiments,13,14 we imagined a
strategy in which PPGs are reacted with 2’ hydroxyls
postsynthetically to block structure and interactions, but are
rendered photoresponsive by including photocleavable bonds.
Addition of several such blocking groups to the RNA (“cloaking”)
would in principle cover and protect it from folding and interacting
with other molecules. Subsequently, the acylation could be reversed
by exposing RNA to light, removing the blocking groups and
switching on activity.
The chief method for obtaining caged RNA is by use of
phosphoramidite reagents that are incorporated by solidꢀphase
synthesis.7b,c,k,m,10 While useful in the hands of chemists, synthesis
of the modified nucleotides is specialized, and requires DNA
synthesizers for their incorporation into RNA; thus nonꢀspecialist
labs often cannot access this technology easily or cheaply. Further,
limitations of solidꢀphase synthesis require that the PPGs be
applied in relatively short RNAs, precluding their use with most
biological RNAs, which are commonly much longer. One approach
to overcoming this limitation involves diazoketones that react with
RNA phosphodiesters.11 However, the resulting phosphotriester
adducts can hydrolyze to cause RNA strand cleavage.12
Design of our photocloaking agents (PCAs) began with
the knowledge that the 2’ꢀOH groups of RNA exhibit relatively
high nucleophilicity due to their low pKa.13,14 For reaction with
these groups, we envisioned the combination of an active acyl
group with an orthoꢀnitroveratryl photoreactive group15 (Fig. 1).
We chose unmethylated and methylated veratryl groups to test
(PCA 1, 2) (Fig. 1B); PCA 1 is less sterically hindered, while 2 is
expected to have higher efficiency in photocleavage.16 We also
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 5
included a dimethylaminoethyl group to enhance solubility. Finally,
of QSTAR reactions incubated for 1 h at 25 °C with RNA template
testing a range of leaving groups (imidazoles and triazoles (see
Supporting Information (SI)), led to the choice of 2ꢀ
chloroimidazole as having the ideal level of reactivity.
1 that was untreated, cloaked and uncloaked. Error bars represent
the standard deviation of triplicate experiments.
1
2
3
4
5
6
7
8
To test if prototypical PCA reagent 1 is effective in
Next, we explored the ability of this photocloaking
strategy to control the more complex function of a ribozyme RNA,
which must not only hybridize, but also bind metals and form
proper tertiary structure to function.18 Several examples of
photocontrolled ribozymes have been reported previously;7e,19
however, all prior studies have been performed with modifications
incorporated into RNAs during solidꢀphase synthesis. Thus, we
tested whether PCA 1 could be used to block a ribozyme in a single
reacting with RNA, it was incubated for 4 h at 100 mM at room
temperature with a 12 nucleotide (nt) RNA strand (10 ꢁM) in water
(see SI). Analysis by denaturing polyacrylamide gel electrophoresis
(PAGE) revealed up to five new mobilityꢀshifted bands, suggestive
of several adducts per strand, with little unreacted RNA remaining
(Fig. 1C). The polyacylation was further confirmed by mass
spectrometry (Figs. S1ꢀ3), although fewer labels were observed,
likely due to photodeprotection caused by the UV laser during the
mass spectrometric analysis.
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
postꢀsynthetic step, and subsequently phototrigger its activity.
A
34 nt hammerhead ribozyme RNA19a was cloaked under standard
conditions at room temperature and isolated by precipitation, and
was then incubated with Cy5ꢀlabeled substrate in 50 mM Tris pH =
6.0, 10 mM MgCl2 and analyzed by PAGE (Fig. 3B and C).
Control ribozyme showed almost full cleavage of the substrate
under these conditions as expected, but the cloaked ribozyme
exhibited strong abrogation of activity. Importantly, after light
exposure, ribozyme function was almost completely restored.
Analysis of ribozyme reaction rates (Fig. 3D and Fig. S9) revealed
that cloaking suppressed the rate by 370ꢀfold, showing strong
blocking of ribozyme function. This was readily restored in RNA
exposed to light, which exhibited a rate the same as that of the
Next, uncloaking of RNA by photoremoval of the PCA
labels was investigated. Photocloaked RNA in water (1.0 ꢁM) was
exposed to 365 nm light with a UV transilluminator analyzed with
denaturing PAGE gels. The results showed that the majority of
shifted bands resolved back to a band having the mobility of
untreated RNA. Successful photoꢀuncloaking was subsequently
confirmed by MALDIꢀTOF, which documented restoration of the
mass of the original RNA (Fig. S3). As a control, the analogous
DNA strand was incubated with PCA 1, and little (<5%) reaction
was observed (Fig. S4), supporting the conclusion that PCA 1
reacts with the 2’ꢀOH groups of RNA.
Encouraged by the initial results, we next assessed how
untreated
ribozyme.
these multiple carbonate groups affect RNA helix formation and
interactions. A 16 nt singleꢀstranded RNA (untreated or cloaked)
was hybridized with a DNA complement and analyzed by native
gel electrophoresis and UV melting (Fig. S5 and S6). The gel
analysis confirmed nearꢀcomplete blocking of hybridization, and
thermal denaturation showed a 10 °C drop in melting temperature
after polyacylation of the RNA (Fig. S5). To further test the effect
on hybridization, a nucleic acidꢀtemplated reaction was employed
with complementary fluorogenic DNA (QꢀSTAR) probes.17 Fig 2B
shows that the cloaked RNA was very inefficient in templating the
fluorogenic reaction, suppressing signal to 9% of untreated RNA
(Fig. 2). After exposure to light, a strong restoration of signal (82%
of original) was observed (Fig. 2BꢀC), consistent with uncloaking
at relatively high efficiency.
Figure 3. Use of photocloaking to control hammerhead ribozyme
activity. A) Sequence of the ribozyme and Cyꢀ5 labeled substrate.
B) Denaturing PAGE analysis of substrate conversion (1 h) after
incubation with untreated, cloaked and photouncloaked ribozyme.
C) Time course of substrate conversion when incubated with the
differently treated ribozymes. D) Plot showing initial rates of
substrate cleavage. Error bars represent the standard deviation of
triplicate experiments.
We proceeded to test the ability of PCAs to control
longer RNAs produced by transcription. Photocontrolled aptamers
have recently gained interest due to their potential for therapeutic
and imaging applications.21 We hypothesized that the current postꢀ
synthetic polyacylation strategy would offer a convenient and
simpler method for preparing photocontrolled aptamers of arbitrary
length. To test this, the transcribed 105 nt F30 Broccoli aptamer22
was chosen as a substrate. Broccoli RNA folds into a compact
tertiary structure, forming a binding site for the DFHBI dye,
resulting in a strong increase in fluorescence.22 After cloaking with
PCA 1, initial studies revealed strong suppression of fluorescence
in Broccoli RNA (Fig. S10), but a relatively poor recovery after
Figure 2. Control of RNA hybridization by photocloaking, as
measured by nucleicꢀacid templated probes. A) Schematic of the
RNAꢀtemplated QSTAR reaction. B) Time course of fluorescence
when RNA template has been treated with DMSO (untreated), PCA
1
(cloaked), and exposed to light after PCA 1 treatment
(uncloaked). The templated reaction is severely inhibited upon
cloaking and restored after photoꢀuncloaking. C) Total fluorescence
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
light exposure. Our hypothesis was that this poor recovery was due
formation of folded Broccoli structure. In contrast, cloaked RNA
showed no measurable signal above background, confirming that
cloaking strongly inhibits function in this 237 nt RNA. Finally,
experiments with subsequent light exposure revealed the
emergence of a visible green fluorescence signal attributable to
restored Broccoli RNA (Fig. 4C).
to low quantum yields for uncaging of nitrobenzyl carbonates,16
which may be problematic on longer RNAs containing greater
numbers of acyl groups. To address this, we prepared PCA 2 (Fig.
1B), which bears a methyl group at the benzylic position to
improve photocleavage efficiency.16
1
2
3
4
5
6
7
8
F30 Broccoli RNA (4 µg) was incubated with PCA 2
An overall assessment of the efficiency of the current
(100 mM) in water for 4 h and then isolated by precipitation (Fig.
S11). Untreated and cloaked Broccoli (500 nM) were dissolved in
folding buffer and DFHBI (1 µM) was added and the aptamer was
refolded. The cloaked sample exhibited very dim fluorescence,
whereas the untreated sample showed pronounced emission (Fig.
4). When the cloaked aptamer was exposed to light and incubated
with DFHBI, fluorescence was completely restored to the level of
the untreated sample. These experiments emphasized the strength
of the photocloaking approach, which is carried out in a single step
on RNAs considerably longer than are conveniently synthesized by
chemical methods. Based on these results, we conclude that both
PCA1 and PCA2 can be used to gain photocontrol of short RNAs,
RNA cloaking and photoꢀuncloaking strategy suggests that the new
approach compares favorably in performance to previous RNA
caging designs. The 10 °C difference in melting temperature upon
cloaking is comparable to previously reported values using
photocaged nucleobase phosphoramidites.7b,24 The present
methodology performed similarly in interfering with hybridization
compared to other caging methods.7l,25 Using the current
methodology on the Broccoli aptamer, we attained marked
OFF/ON behavior, comparable or better than previous studies with
light controlled DNA aptamers developed using photocaged
phosphoramidites.21b However, to the best of our knowledge our
data represent the first example of a photocaged RNA aptamer,
likely due to size limitations with prior methods.
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
and
for longer strands PCA2
is more
efficient.
In summary, our new photocloaking approach is a
convenient method for obtaining photoprotected RNA, representing
significant advantages over existing technologies. One potential
drawback of this new approach is its stochastic nature, which
generates RNAs with varying levels of acylation and does not
direct them to specific positions. However, our data show that
sufficient levels of acylation can be readily achieved to strongly
suppress RNA activity. Importantly, a great advantage of this postꢀ
synthetic labeling is that it allows for oneꢀstep photoprotection of
long biologically relevant RNAs that are difficult or impossible to
synthesize via solidꢀstate oligonucleotide synthesis. Since
photocloaking can be achieved without specialized equipment, the
new method could potentially find widespread use for study of the
biology of RNAs.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website. Further experimental details and spectra,
Figure S1 – S9.
AUTHOR INFORMATION
Corresponding Author
Figure 4. Cloaking effects on 105ꢀ237 nt Broccoli aptamer RNAs
prepared by in vitro transcription. A) Fluorescence emission spectra
of untreated, cloaked and uncloaked Broccoli 105mer incubated
with DFHBI. B) Emission of differently treated Broccoli aptamers
with DFHBI. The PCA 2 cloaked aptamer exhibits a substantially
decreased fluorescence which is fully restored after light exposure.
Error bars represent the standard deviation of triplicate
experiments. λex = 450 nm. C) Epifluorescence microscopy images
of HeLa cells transfected with untreated or cloaked 237nt Broccoli
aptamer.
*kool@stanford.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We are grateful for support from the U.S. National Institutes of
Health (GM068122 and GM127295) and the Netherlands
Organization for Scientific Research (RUBICON) and EMBO
ALTF 934ꢀ2014 to WAV.
Finally, the capability to use this photocloaking strategy
to switch on RNA function in cells was assessed by transfecting a
cloaked Broccoli RNA construct into human cells and then
exposing cells to light. For these experiments, we employed a
larger 237 nt RNA construct encoding dimeric Broccoli aptamers,
which is known to be stable in the intracellular medium.23 In vitro
cloaking of this longer RNA was carried out with PCA 2 under
standard conditions. The RNA was then transfected into HeLa
cells, and after incubating for 5 h, cells were illuminated with 365
nm light under an epifluorescence microscope. As a control, we
used the same RNA mockꢀtreated with DMSO alone. The
experiments revealed that the untreated RNA showed a positive
fluorescence signal after transfection (Fig. 4C), consistent with the
REFERENCES
(1) Armitage, B. A. Curr. Opin. Chem. Biol. 2011, 15 (6) 807.
(2) a) Cech, T. R.; Steitz, J. A. Cell. 2014, 157 (1), 77; b) He, C. Nat Chem Biol
2010, 6 (12), 863.
(3) Chen, L. Nat. Rev. Mol. Cell Biol. 2016, 17 (4), 205.
(4) Delebecque, C. J.; Lindner, A. B.; Silver, P. A.; Aldaye, F. A. Science 2011,
333 (6041), 470.
(5) Li, F.; Dong, J.; Hu, X.; Gong, W.; Li, J.; Shen, J.; Tian, H.; Wang, J.
Angew. Chemie Int. Ed. 2015, 54 (15), 4597.
(6) a) Lubbe, A. S.; Szymanski, W.; Feringa, B. L. Chem. Soc. Rev. 2017, 46
(4), 1052; b) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A.
Angew. Chem. Int. Ed. Engl. 2012, 51, 8446.
(7) a) Ankenbruck, N.; Courtney, T.; Naro, Y.; Deiters, A. Angew. Chemie Int.
Ed. 2017, DOI: 10.1002/anie.201700171; b) Govan, J. M.; Young, D. D.;
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 5
Lusic, H.; Liu, Q.; Lively, M. O.; Deiters, A. Nucleic Acids Res. 2013, 41
(22), 10518; c) Mikat, V.; Heckel, A. RNA 2007, 13 (12), 2341; d) Lucas, T.;
Schäfer, F.; Müller, P.; Eming, S. A.; Heckel, A.; Dimmeler, S. Nat.
Commun. 2017, 8 (May), 15162; e) Chaulk, S. G.; MacMillan, A. M. Nat.
Protoc. 2007, 2 (5), 1052; f) MatsushitaꢀIshiodori, Y.; Ohtsuki, T. Acc.
Chem. Res. 2012, 45 (7), 1039; g) Shah, S.; Rangarajan, S.; Friedman, S. H.
Angew. Chemie 2005, 117 (9), 1352; h) Meyer, A.; Mokhir, A. Angew.
Chemie - Int. Ed. 2014, 53 (47), 12840; j) Lu, J.; Koo, S. C.; Li, N.ꢀS.;
Piccirilli, J. A. Nucleosides, Nucleotides and Nucleic Acids 2015, 34 (2),
114; k) Resendiz, M. J. E.; Schön, A.; Freire, E.; Greenberg, M. M. J. Am.
Chem. Soc. 2012, 134 (30), 12478; l) Panja, S.; Paul, R.; Greenberg, M. M.;
Woodson, S. A. Angew. Chemie 2015, 54 (25), 7281; m) Höbartner, C.;
Silverman, S. K. Angew. Chemie - Int. Ed. 2005, 44 (44), 7305; n) Gautier,
A.; Gauron, C.; Volovitch, M.; Bensimon, D.; Jullien, L.; Vriz, S. Nat.
Chem. Biol. 2014, 10 (7), 533.
1
2
3
4
5
6
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
(8) a) Wenter, P.; Fürtig, B.; Hainard, A.; Schwalbe, H.; Pitsch, S. Angew.
Chemie Int. Ed. 2005, 44 (17), 2600; b) Fürtig, B.; Wenter, P.; Pitsch, S.;
Schwalbe, H. ACS Chem. Biol. 2010, 5 (8), 753.
(9) a) Shestopalov, I. A.; Sinha, S.; Chen, J. K. Nat. Chem. Biol. 2007, 3 (10),
650; b) Tang, X.; Maegawa, S.; Weinberg, E. S.; Dmochowski, I. J. J. Am.
Chem. Soc. 2007, 129 (36), 11000.
(10) Liu, Q.; Deiters, A. Acc. Chem. Res. 2014, 47 (1), 45.
(11) Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat Genet 2001, 28 (4),
317.
(12) Blidner, R. A.; Svoboda, K. R.; Hammer, R. P.; Monroe, W. T. Mol.
Biosyst. 2008, 4 (5), 431.
(13) a) Spitale, R. C.; Flynn, R. A.; Zhang, Q. C.; Crisalli, P.; Lee, B.; Jung, J.ꢀ
W.; Kuchelmeister, H. Y.; Batista, P. J.; Torre, E. A.; Kool, E. T.; Chang, H.
Y. Nature 2015, 519 (7544), 486; b) Spitale, R. C.; Crisalli, P.; Flynn, R. A.;
Torre, E. A.; Kool, E. T.; Chang, H. Y. Nat Chem Biol 2013, 9, 18.
(14) a) Merino, E. J.; Wilkinson, K. A.; Coughlan, J. L.; Weeks, K. M. J. Am.
Chem. Soc. 2005, 127 (12), 4223; b) Low, J. T.; Weeks, K. M. Methods
2010, 52, (2), 150.
(15) a) Matsuo, K.; Kioi, Y.; Yasui, R.; Takaoka, Y.; Miki, T.; Fujishima, S.;
Hamachi, I. Chem. Sci. 2013, 4 (6), 2573; b) Knežević, N. Ž.; Trewyn, B.
G.; Lin, V. S. Y. Chem. - A Eur. J. 2011, 17 (12), 3338.
(16) Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.;
Popik, V.; Kostikov, A.; Wirz, J. Chem. Rev. 2013, 113 (1), 119.
(17) a) Franzini, R. M.; Kool, E. T. J. Am. Chem. Soc. 2009, 131, 16021; b)
Velema, W. A.; Kool, E. T. J. Am. Chem. Soc. 2017, 139 (15), 5405.
(18) Yijin Liu, T. J. W. & D. M. J. L. Nat. Chem. Biol. 2017, 13, 508.
(19) a) Chaulk, S. G.; MacMillan, A. M. Nucleic Acids Res. 1998, 26 (13), 3173;
b) Nierth, A.; Singer, M.; Jäschke, A. Chem. Commun. (Camb). 2010, 46
(0), 7975; c) Young, D. D.; Deiters, A. Bioorganic Med. Chem. Lett. 2006,
16 (10), 2658.
(20) Scott, W. G.; Horan, L. H.; Martick, M. Prog. Mol. Biol. Transl. Sci. 2013,
120, 1.
(21) a) Buff, M. C. R.; Schäfer, F.; Wulffen, B.; Müller, J.; Pötzsch, B.; Heckel,
A.; Mayer, G. Nucleic Acids Res. 2009, 38 (6), 2111; b) Li, L.; Tong, R.;
Chu, H.; Wang, W.; Langer, R.; Kohane, D. S. Proc. Natl. Acad. Sci. 2014,
111 (48), 17099; c) Qiu, L.; Wu, C.; You, M.; Han, D.; Chen, T.; Zhu, G.;
Jiang, J.; Yu, R.; Tan, W. J. Am. Chem. Soc. 2013, 135 (35), 12952; d)
Heckel, A.; Mayer, G. J. Am. Chem. Soc. 2005, 127 (3), 822; e) Pinto, A.;
Lennarz, S.; RodriguesꢀCorreia, A.; Heckel, A.; O’Sullivan, C. K.; Mayer,
G. ACS Chem. Biol. 2012, 7 (2), 360.
(22) Filonov, G. S.; Moon, J. D.; Svensen, N.; Jaffrey, S. R. J. Am. Chem. Soc.
2014, 136 (46), 16299.
(23) Filonov, G. S.; Kam, C. W.; Song, W.; Jaffrey, S. R. Chem. Biol. 2015, 22
(5), 649.
(24) Deiters, A.; Garner, R. A.; Lusic, H.; Govan, J. M.; Dush, M.; Nasconeꢀ
Yoder, N. M.; Yoder, J. A. J. Am. Chem. Soc. 2010, 132 (44), 15644.
(25) Wu, L.; Wang, Y.; Wu, J.; Lv, C.; Wang, J.; Tang, X. Nucleic Acids Res.
2013, 41 (1), 677.
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
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
ACS Paragon Plus Environment