5-Oxyacetic Acid Modification Destabilizes Double Helical Stem Structures and Favors Anionic Watson–Crick like cmo5U-G Base Pairs

Article abstract of DOI:10.1002/chem.201805077

Watson–Crick like G-U mismatches with tautomeric G^enol or U^enol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5-oxyacetic acid uridine (cmo⁵U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson–Crick like cmo⁵U^enol-G mismatches. A detailed investigation on the influence of the cmo⁵U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo⁵U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson–Crick like cmo⁵U^enol-G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson–Crick like cmo⁵U^?-G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson–Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus—the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns.

Full text of DOI:10.1002/chem.201805077

A Journal of
Accepted Article
Title: The 5-oxyacetic acid modification destabilizes double helical
stem structures and favors anionic Watson-Crick like cmo5U-G
base pairs
Authors: Elisabeth Strebitzer, Atul Rangadurai, Raphael Plangger,
Johannes Kremser, Michael Andreas Juen, Martin Tollinger,
Hashim Al-Hashimi, and Christoph Kreutz
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
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201805077
Link to VoR: http://dx.doi.org/10.1002/chem.201805077
Supported by

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COMMUNICATION
The 5-oxyacetic acid modification destabilizes double helical
5
stem structures and favors anionic Watson-Crick like cmo U-G
base pairs
Elisabeth Strebitzer$[a], Atul Rangadurai$[b], Raphael Plangger , Johannes Kremser , Michael Andreas
[a]
[a]
[
a]
[a]
[b]
[a]
Juen , Martin Tollinger , Hashim M. Al-Hashimi* , and Christoph Kreutz*
enol
Abstract: Watson-Crick like G-U mismatches with tautomeric G or
crystallography the precise nature of the Watson-Crick like
species could not be unambiguously determined.
enol
U
bases can evade fidelity checkpoints and thereby contribute to
5
Here, we have synthesized a 15N isotopically labeled version of
translational errors. The 5-oxyacetic acid uridine (cmo U) modification
is a base modification at the wobble position on tRNAs and is
presumed to expand the decoding capability of tRNA at this position
5
the cmo U modification that allows for the application of relaxation
dispersion NMR to probe the formation of Watson-Crick like base
pairs. Surprisingly, we find that the modification promotes the
5
enol
by forming Watson-Crick like cmo U -G mismatches. A detailed
5
5
–
investigation on the influence of the cmo U modification on structural
transient formation of anionic cmo U -G base pairs via decreasing
and dynamic features of RNA was carried out using solution NMR
spectroscopy and UV melting curve analysis. The introduction of a
the pK
a
of the nucleoside. Our results provide key mechanistic
5
insights into how cmo U influences base pairing properties and
modulates decoding, and lay out a framework for studying the
5
stable isotope labeled variant of the cmo U modifier allowed the
impacts of other RNA modifications.[
1]
application of relaxation dispersion NMR to probe the potentially
5
enol
Our first efforts focused on the synthesis of a 15N
5
formed Watson-Crick like cmo U -G base pair. Surprisingly, we find
2
-cmo U
that at neutral pH, the modification promotes transient formation of
phosphoramidite. The synthetic access is loosely based on
published syntheses but represents the first comprehensive
5
–
anionic Watson-Crick like cmo U -G and not enolic base pairs. Our
results suggest that recoding is mediated by an anionic Watson-Crick
like species as well as bring an interesting aspect of naturally
occurring RNA modifications into focus – the fine tuning of nucleobase
properties leading to modulation of the RNA structural landscape via
adoption of alternative base pairing patterns.
5
description of the chemical synthesis of the cmo U RNA building
block. The synthesis started from 15N
-uridine, which was
prepared according to published procedures using 15N -urea.[6] In
2
2
the next steps the 5-hydroxyl group was installed [7], followed by
protection of the sugar hydroxyls and installation of the 5-
oxyacetic acid side chain as an ester functionality.[8] Removal of
the 2’,3’-ketal protecting group was followed by the regioselective
incorporation of the 2’-tert.-butyl-dimethylsilyl group using a
transient 3’,5’-di-tert.-butylsilyl protection. Then, after removal of
the methyl ester functionality the 4-nitrophenylethylester was
obtained via a Steglich esterification. The remaining steps were
the introduction of the 5’-(4,4’-dimethoxy)-trityl group and
phosphitylation of the 3’-hydroxyl group yielding the desired
The fidelity of translation relies on the ability of the rigid ribosomal
decoding center to recognize the shape of Watson-Crick base
pairs.[1] Mismatches can evade these fidelity mechanisms via
spatial mimicry of the Watson-Crick like shape through
[
2]
tautomerization and ionization. The propensity to adopt such
Watson-Crick like base pairs can be tuned by modifications of the
[
3]
canonical bases at the wobble position, in order to rewire and
5
stable isotope modified cmo U RNA building block. A detailed
[
4]
change the efficiency of translational decoding. Uridine 5-
synthetic scheme and further experimental details on the
synthetic steps can be found in the supporting information.
5
oxyacetic acid (cmo U) is one such modification that based on X-
ray crystallographic and biochemical data has been proposed to
increase the efficiency of translation of G-ending codons by
5
Then, the cmo U modification was introduced into 20 nt RNA
model system to probe changes in the overall thermodynamic
5
enol
promoting formation of Watson-Crick like cmo U -G base
pairs.[5] However, as hydrogen atoms could not be visualized by
5
stability. The cmo U-modified RNAs were characterized by anion
exchange chromatography and mass spectrometry (Supporting
Figure 1 and 2). Four RNA constructs differing at the central U5-
A16 base pair were synthesized and subjected to UV melting
analysis to give the melting temperatures and thermodynamic
parameters of the 20 nt hairpins - the U-A hp, the U•G hp, the
[
[
[
a]
b]
$]
Mag. E. Strebitzer, Mag. R. Plangger, Mag. J. Kremser, Dr. M.
Tollinger, Dr. C. Kreutz
Institute of Organic Chemistry and Center for Molecular Biosciences
Innsbruck (CMBI)
University of Innsbruck
Innrain 80/82, 6020 Innsbruck, Austria
E-mail: christoph.kreutz@uibk.ac.at
5
5
cmo U-A hp and the cmo U•G hp (Figure 1a-c, Supporting
Figure 3). As expected, the U-A hairpin comprising only canonical
base pairs showed the highest melting transition at 85°C and the
-1
most negative free energy of -12.2 kcal mol , closely followed by
A. Rangadurai, Dr. HM. Al-Hashimi
the U•G hp (T
m
= 83.3 °C, G2 = -11.3 kcal mol ). The cmo U-
98K
-1
5
Department of Biochemistry and Department of Chemistry
Duke University School of Medicine, Durham, NC, USA
Nanaline H. Duke Building, 307 Research Drive
Durham NC 27710, US
modified hairpins showed slightly decreased stabilities in-line with
previously conducted free energy calculations.[9] The cmo5U-A hp,
although forming a canonical base pair exhibits an even lower
E-mail: hashim.al.hashimi@duke.edu
U•G/cmo5U-A
thermodynamic stability than the U•G hp (T
m
= 1.3 °C,
G2
98K, U•G/cmo5U-A
= 0.5 kcal mol ). The UV melting experiment
-1
These authors contributed equally.
gives information on the global thermodynamic stabilities of the
stem-loop folds, but no structural features and local residue-
specific dynamics can be delineated from the UV data.
Supporting information for this article is given via a link at the end of
the document.
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Chemistry - A European Journal
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COMMUNICATION
5
Solution NMR spectroscopy, however, is perfectly suited to study
structure and dynamics in biomolecules at atomic resolution.[6, 10]
Based on NMR data, the U-A hp forms a standard A-form RNA
stem capped with an extra-stable UUCG loop. The U•G hp was
extensively investigated earlier and in the major populated ground
state a U•G wobble base pair was observed.[11] Basically, the
The water proton exchange NMR experiment of cmo U-A hp gave
slightly to strongly elevated imino proton water exchange rates
compared to its unmodified U-A counterpart. The cmo U
modification strongly influences the stability of the 5’-preceding
base pair – in this case the G4-C17 base pair. We observed an
almost ten-fold increase in the water exchange rate of the G4-NH
5
1
5
5
same base pairing properties were observed for the cmo U-
proton. A structural model of the cmo U-A hairpin gives a potential
5
modified stem-loop RNAs (Figure 2). The cmo U-A hp showed
explanation for the destabilization of the 5’-preceding base pair.
The model reveals that the negatively charged 5-oxyacetate side
chain gets in proximity to the likewise negatively charged
phosphate group of G4 (Figure 2c). The repulsion effect induced
by this unfavorable electrostatic interaction gives a rational for the
10-fold weakening of the G4-C17 base pair and the concomitant
global fold destabilization as seen in the UV-melting curve
analysis. In the thermodynamically most unstable hairpin the
highly dynamic hotspot at the cmo U•G mismatch site was directly
reflected in the imino proton water exchange rates. We found
strongly elevated exchange rates at the mismatch site. Most
prominently, the cmo U5-N -H resonance was strongly
broadened at 25°C preventing the determination of the exchange
rate with water protons. The G16 NH water exchange is 6-fold
slightly shifted imino proton resonances compared to the U-A
wildtype sequence, which could be assigned using a 1H-
homonuclear NOESY experiment (Figure 2a, upper trace).
Noteworthy, the N3H of resonance is shifted from 13.6 ppm (U-A
5
base pair) to 14.1 ppm (cmo U-A base pair) pointing toward a
more de-shielded magnetic environment. We further
demonstrated the Watson-Crick like base pairing via an HNN-
COSY with residue-specific 15N3 and N1 labeling of cmo U5 and
15
5
5
5
A16, respectively (Figure 2a). The cmo U•G wobble base pair
1
15
was confirmed by H- N-correlation NMR spectroscopy making
use of 15N3 and 15N1 labeling of cmo U5 and G16 (Figure 2b).
5
5
3
5
Again, a 0.5 ppm de-shielding effect for the cmo U imino proton
resonance (U•G hp U5 H3 11.6 ppm; cmo U•G hp U5 H3 12.1
5
1
ppm) was induced by the 5-oxyacetic acid modification.
enhanced compared to the U•G wobble base pair (59.88 vs. 10.14
-1
5
s ). Again, the destabilization effect introduced by the cmo U•G
wobble is highly localized as other imino proton water exchange
rates are only marginally affected.
5
Figure 1. Thermodynamic stability of the unmodified and cmo U-modified RNAs.
a) Secondary structures of the four RNAs under investigation. The inset shows
5
the structure of the cmo U modification. The nitrogen atoms highlighted in blue
15
are N-labeled. b) Bar graph of the melting temperatures of the hairpin RNAs.
c) Bar graph of the free energies at 298 K of the hairpin RNAs.
We then addressed individual base pair exchange kinetics in all
four hairpins by the application of CLEANEX-PM experiments
5
5
Supporting Table 1 and Supporting Figure 4).[12] The slow to
Figure 2. Structural features of cmo U-modified hairpins. a) A canonical cmo U-
(
1
15
A base pair is observed in an HNN-COSY experiment at 25 °C. b) A H- N-
HSQC showing chemical shift signatures for the formation of a cmo U•G wobble
base pair (at 5 °C). c) Structural model of the G4-C17 and cmo U5-A16 base
pairs in the cmo U-A hp. Interfering negative charges of the 5-oxyacetate and
-1
very slow imino proton exchange rates between 0.8 to 10 s for
the U-A hp are in-line with the high thermodynamic stability of the
stem loop structure and reveal no dynamic hotspot. In the U•G hp
the wobble base pair represents a dynamic base pair opening hot
spot with the U5-NH displaying the highest exchange rate with
bulk water protons (22.15 s ) followed by its interaction partner
5
5
5
the phosphate backbone are shown in dot representation.
3
-1
3
-1
Finally, we addressed the postulated stabilization of the rare
enolic uridine state by the cmo U modification via 15N-R1ρ
relaxation dispersion NMR experiments. We previously showed
in the hpU•G sequence context (Figure 1a) that the U•G wobble
mismatch exists in dynamic equilibrium with two equally
G16-NH (10.14 s ). The destabilization effect introduced by the
U•G wobble appears to be only localized as other imino proton
exchange rates are only marginally affected (e.g. U18).
5
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populated and rapidly exchanging U-Genol and Uenol-G base pairs,
with the anionic species falling below detection limits at neutral
relaxation dispersion NMR spectroscopy. We found that an
5
–
anionic Watson-Crick like cmo U -G base pair represents the
major species in the excited state. Thus, we have evidence that
pH (p
B
B
< 0.05 %) and only becoming detectable (p ~ 0.3%) at
high pH > 7.9.[
11]
in a previous work the cmo U -G WC-like base pair was
misinterpreted and is in fact an anionic cmo U-G base pair [5a].
5
enol
5
We examined how replacement of the U•G mismatch with a
5
cmo U•G base pair affects these dynamics. Strikingly, we find that
at neutral pH, the modification significantly increased 15N RD at
5
both cmo U5-N3 and G16-N1 indicating that it promotes the
exchange process to WC-like mismatches (by ~5-fold, Figure 3a,
Supplementary Table 2). As a negative control, no RD was
5
observed for cmo U-A hp (Supporting Figure 5). Unlike the data
for unmodified U•G hp which could be interpreted in terms of 2-
state exchange between the wobble and two rapidly exchanging
U-Genol and Uenol-G base pairs, the RD data for the cmo U•G hp
called for a 3-state fit yielding two ESs (Supplementary Table 2).
5
GS-B
-1
ES1 (p
(
B
= 0.16 ± 0.03 % and kex
= 5949 ± 709 s ) has 
5
cmo U5-N3) = 16.0 ± 4.8 ppm and  (G16-N1) = 40.4 ± 4.1 ppm
consistent with a mixture of rapidly exchanging cmo U-G and
5
enol
5
enol
cmo U -G and species in a 70:30 ratio. ES2 (p
c
= 0.24 ± 0.03 %
= 976 ± 683 s ) has (cmo U5-N3) = 55.4 ± 2.9 and
 (G16-N1) = -0.02 ± 3.5 consistent with the formation of a
GS-C
-1
5
and kex

5
–
cmo U -G anionic base pair. This was confirmed through
measurement of RD profiles at higher pH 8.0 (Supplementary
Table 2, Supporting Figure 6). Therefore, the modification
increases the population of the anionic species by > 5-fold such
that it becomes the dominant species in the ES at neutral pH
(
~60%) while minimally impacting the population of the tautomeric
species (~2 fold) or tautomeric equilibrium (from 50/50 to 70/30
cmo U-G /cmo Uenol-G).These findings are in-line with a recent
computational study, in which no evidence for the stabilization of
the enolic uridine state by the oxyacetic acid modification was
found.[ They also mirror the effects of 5-bromo-2’-deoxyuridine
5
enol
5
13]
(
5BrdU), which is a mutagenic agent in DNA that causes
increased mis-incorporation via an anionic species.[14] We got
further support for the preference of the anionic Watson-Crick like
5
base pairing from the pK
a
values determined for the cmo U
nucleoside (Supporting Figure 7 and 8). Using pH-dependent
13C-NMR data the pK
of the 5-oxyacetic acid side chain was
a
found to be 2.8. Thus, at physiological pH the 5-oxyacetic acid is
negatively charged in-line with the observed destabilization effect
5
found for the cmo U-A RNA. The pK
a
of the imino proton H3 in
5
cmo U is 8.7 – i.e. the proton is more acidic than in the unmodified
a
uridine nucleoside (pK 9.2-9.6) by almost a factor of ten. This
higher acidity favors the anionic Watson-Crick like base pair state
as seen in the relaxation dispersion data. Further, the higher
acidity of the N3 bound proton rationalizes the observed de-
shielding effect by the change in its electronic environment.
To conclude, we successfully synthesized a 15N stable isotope
5
Figure 3. Probing the excited states in cmo
5
U-modified RNA. a) 15N-R1ρ
labeled variant of a cmo U RNA building block that allowed to
3
5
1
relaxation dispersion experiments for N of cmo U5 and N of G16 with a three-
state global fit at pH 6.9 and 10° C. b) Summary on ground/excited state
equilibria for the unmodified U•G and the cmo U•G wobble base pairs.
obtain unprecedented insights into the structure and dynamics of
RNA carrying this naturally occurring modification via solution
NMR spectroscopy. We found a generally destabilizing effect for
5
5
double helical RNA stem structures by the cmo U modification
very likely due to the additional negative charge introduced by the
oxyacetic acid sidechain. The destabilization is reflected in a
lowered melting temperature and higher free energy values, and
also slightly enhanced imino water proton exchange rates. We
further probed the presumed stabilizing effect of the 5-oxyacetic
acid modification on the U-enol tautomeric form by 15N-R1ρ
This anionic Watson-Crick base pair between the mRNA and the
tRNA anticodon loop might be further stabilized by the controlled
conditions regarding pH or metal ion positioning within the
ribosome, thus giving an explanation for the expansion of the
5
decoding capacity via the cmo U modification by a lowered N-H3
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COMMUNICATION
value.10 The electron density map in the previous work of
pK
a
[1]
A. Rozov, N. Demeshkina, E. Westhof, M. Yusupov, G. Yusupova,
Trends in Biochemical Sciences 2016, 41, 798-814.
Weixlbaumer et. al. is also compatible with the anionic base pair
[
2] aA. Rozov, E. Westhof, M. Yusupov, G. Yusupova, Nucleic Acids
5
formed by N3 and O2 of cmo U and N1-H and N2-H of G (Figure
Research 2016, 44, 6434-6441; bA. Rozov, P. Wolff, H. Grosjean, M.
Yusupov, G. Yusupova, E. Westhof, Nucleic Acids Research 2018, gky547-
gky547.
[
5a]
3b).
5
Further, the effects of cmo U in RNA are reminiscent to that of 5-
[3]
aW. A. Cantara, F. V. Murphy, H. Demirci, P. F. Agris, Proceedings of
the National Academy of Sciences 2013, 110, 10964; bT. Muramatsu, K.
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Yokogawa, T. Ogata, T. Wada, T. Suzuki, T. Suzuki, Nature Chemical Biology
BrdU in DNA, which also promotes formation of an anionic base
.[14] Thus, cmo U can be regarded as
5
pair by lowering the pK
a
nature’s version of 5-BrdU. Whereas 5-BrdU is a well-known
2010, 6, 277.
chemotherapeutic agent that acts by promoting G point mutations,
[
4] aS. Kurata, A. Weixlbaumer, T. Ohtsuki, T. Shimazaki, T. Wada, Y.
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Biological Chemistry 2008, 283, 18801-18811; bF. A. P. Vendeix, F. V.
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Nature Communications 2016, 7, 10457.
the third codon position by the formation of an anionic base pair.
To summarize, the results highlight an important aspect of the
function of naturally occurring RNA modifications – the alteration
of the folding landscape of RNA by fine-tuning nucleobases
properties leading to alternative base pairing patterns. We are
further confident that solution NMR spectroscopy will give
important insights into the function of naturally occurring RNA
modifications, which is especially important in the light of the
recent finding that RNA epigenetics plays an important role in
many cellular processes.
[5]
aA. Weixlbaumer, F. V. Murphy Iv, A. Dziergowska, A. Malkiewicz, F. A.
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Acknowledgements
[
1
[
This work was supported by the Austrian Science Fund (FWF,
project P28725 and P30370 to CK). H.M.A. acknowledges
funding from NIH (RO1 GM089846).
[
Keywords: RNA modifications • NMR • relaxation dispersion
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Chemistry - A European Journal
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COMMUNICATION
COMMUNICATION
Changing the acidity. A
detailed NMR spectroscopic
investigation revealed the
influence of the 5-oxyacetic
acid uridine modification on
the RNA folding landscape.
The modifier enhances the
acidity of the uridine N3-proton
leading to the formation of
transient anionic Watson-Crick
like U-G base pairs even at
neutral pH.
Elisabeth Strebitzer, Atul
Rangadurai, Raphael Plangger,
Johannes Kremser, Michael
Andreas Juen, Martin Tollinger,
Hashim M. Al-Hashimi*, and
Christoph Kreutz*
Page No. – Page No.
Title
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