Stable Isotope-Labeled RNA Phosphoramidites to Facilitate Dynamics by NMR

Article abstract of DOI:10.1016/bs.mie.2015.06.013

Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes, methods to synthesize these RNAs for biophysical studies are much needed. Here, we showcase the applicability of 6-¹³C-pyrimidine phosphoramidites to introduce

Full text of DOI:10.1016/bs.mie.2015.06.013

CHAPTER EIGHTEEN
Stable Isotope-Labeled RNA
Phosphoramidites to Facilitate
Dynamics by NMR
Christoph H. Wunderlich*,1, Michael A. Juen*,1, Regan M. LeBlanc†,1,
Andrew P. Longhini†,1, T. Kwaku Dayie†,1,2, Christoph Kreutz*,1,2
*Institute of Organic Chemistry and Center for Biomolecular Sciences Innsbruck, University of Innsbruck,
Innsbruck, Austria
†Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University
of Maryland, College Park, Maryland, USA
1Disclosures: CH Wunderlich none, MA Juen none, C Kreutz, none; RM LeBlanc none, AP Longhini none,
TK Dayie, none.
2Corresponding authors: e-mail address: dayie@umd.edu; christoph.kreutz@uibk.ac.at
Contents
1. Theory
2. Equipment
3. Materials
3.1 Solutions and Buffers
4. Protocol
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463
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4.1 Duration
4.2 Preparation
4.3 Caution
5. Step 1: Synthesis of 6-13C-Uridine TOM Phosphoramidite
5.1 Overview
5.2 Duration
6. Step 2: Synthesis of 6-13C-Cytidine TOM Phosphoramidite
6.1 Overview
6.2 Duration
7. Step 3: Chemical RNA Synthesis
7.1 Overview
7.2 Duration
8. Step 4: Applications
8.1 Overview
8.2 Segmental Stable Isotope Labeling Using Chemically Synthesized RNAs
8.3 13C CPMG RD NMR Spectroscopy
8.4 13C ZZ Exchange NMR Spectroscopy
8.5 Chemical Exchange Saturation Transfer
#
Methods in Enzymology, Volume 565
ISSN 0076-6879
http://dx.doi.org/10.1016/bs.mie.2015.06.013
2015 Elsevier Inc.
All rights reserved.
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Christoph H. Wunderlich et al.
9. Conclusions
Acknowledgments
References
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491
492
Abstract
Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes,
methods to synthesize these RNAs for biophysical studies are much needed. Here,
we showcase the applicability of 6-13C-pyrimidine phosphoramidites to introduce iso-
lated 13C–1H spin pairs into RNAs up to 40 nucleotides long. The method allows the
incorporation of 6-13C-uridine and -cytidine residues at any desired position within a
target RNA. By site-specific positioning of the 13C-label using RNA solid phase synthesis,
these stable isotope-labeling patterns are especially well suited to resolve resonance
assignment ambiguities. Of even greater importance, the labeling pattern affords accu-
rate quantification of important functional transitions of biologically relevant RNAs (e.g.,
riboswitch aptamer domains, viral RNAs, or ribozymes) in the μs- to ms time regime and
beyond without complications of one bond carbon scalar couplings. We outline the
chemical synthesis of the 6-13C-pyrimidine building blocks and their use in RNA solid
phase synthesis and demonstrate their utility in Carr Purcell Meiboom Gill relaxation dis-
persion, ZZ exchange, and chemical exchange saturation transfer NMR experiments.
1. THEORY
Ribonucleic acid (RNA) is well recognized as a central player in key
biological processes such as signaling and gene regulation, catalysis, or viral
infections (Blount & Breaker, 2006; Breaker, 2011; Coppins, Hall, &
Groisman, 2007; Haller, Souliere, & Micura, 2011; Reining et al., 2013;
Serganov & Nudler, 2013). NMR spectroscopy has made significant
contributions in revealing molecular details underlying these processes
(Al-Hashimi, 2007; Al-Hashimi & Walter, 2008; Campbell, Bouchard,
Desjardins, & Legault, 2006; Dayie, 2012; Furtig, Buck, Richter, &
¨
Schwalbe, 2012; Hennig, Williamson, Brodsky, & Battiste, 2001;
Johnson & Hoogstraten, 2008; Kloiber, Spitzer, Tollinger, Konrat, &
Kreutz, 2011; Latham, Brown, McCallum, & Pardi, 2005; Mittermaier &
Kay, 2009; Rinnenthal et al., 2011; Wenter, Reymond, Auweter,
Allain, & Pitsch, 2006; Zhang, Sun, Watt, & Al-Hashimi, 2006). The first
mandatory step to study both structure and dynamics of biomacromolecules
is the introduction of a stable isotope-labeling pattern (Dayie, 2008; Hennig
et al., 2001; Lu, Miyazaki, & Summers, 2010). Currently, enzymatic
methods to introduce uniformly 13C- and/or 15N-labeled nucleotides into
RNA and DNA are state-of-the-art (Hennig et al., 2001; Milligan, Groebe,

Stable Isotope-Modified RNA Phosphoramidites
463
Figure 1 (A) All heteronuclei can be addressed for stable isotope labeling in uracil by
choosing the appropriate precursor compounds. (B) 6-13C-cytidine (left) and -uridine
(right) triisopropylsilyl-oxmethylprotected RNA phosphoramidites. Orange (gray in
the print version) circle: 13C.
Witherell, & Uhlenbeck, 1987; Milligan & Uhlenbeck, 1989). Using these
methods, nucleotide-specific-labeling patterns (i.e., only adenosines/uri-
dines or guanosines/cytidines) can be applied in a straightforward manner
by mixing labeled and unlabeled (deoxy-) ribonucleotide triphosphates
(rNTPs) in polymerase-catalyzed reactions. However, the nature of the sta-
ble isotope-labeling pattern can limit the type of NMR experiments that can
be run (Wunderlich et al., 2012). An isolated X-1H (X¼13C or 15N) spin
topology is particularly important for obtaining accurate parameters from
the very powerful relaxation dispersion (RD) and chemical exchange satu-
ration transfer (CEST) NMR experiments that address μs- to ms RNA
dynamics. Our labeling strategy eliminates the deleterious strong homonu-
clear scalar coupling and therefore provides artifact-free dispersion profiles.
Here, we outline a robust synthetic access to selectively labeled 6-13C-
pyrimidine RNA phosphoramidites using any combination of the follow-
ing: 15N1, 15N3, and 13C2 from 13C–15N-labeled urea; 13C4 and 13C5 from
13C-labeled bromoacetic acid; and 13C6 from K13CN (Fig. 1A and B).
2. EQUIPMENT
0.22-μm cellulose acetate filters
2-ml Eppendorf tubes
1
600/800 MHz NMR instrument equipped with H/13C/31P probe
with gradients
Analytical Dionex DNAPac PA-100 column 4ꢀ250mm
Balloons with a wall thickness of at least 0.3 mm
Bent adapters with NS-stopcocks
C18 SepPak cartridges (Waters)
Dewar
DNA/RNA synthesizer

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Christoph H. Wunderlich et al.
Freezer at ꢁ20°C
Glass chromatography columns
High-pressure liquid chromatography system with a column oven
(e.g., Thermo Fisher Ultimate 3000)
High vacuum rotary vane pump
Magnetic stirrer with heating and an oil bath or heat block
Magnetic stirring bar
¨
Medium pressure liquid chromatography system (e.g., AKTA start)
NMR tubes (5 or 3 mm) suitable for 600 and 800 MHz NMR
spectrometer
Parafilm
pH meter and electrode
Reflux condenser
Rotary evaporator with a diaphragm pump
Round-bottom flasks with volumes ranging from 25 to 1000 ml
Semipreparative Dionex DNAPac PA-100 column 9ꢀ250mm
Separatory funnel
Silica thin layer chromatography plates with fluorescence indicator
Suction filter
Syringes (1, 10, 20, and 50 ml volume)
UV/vis spectrophotometer
UV/vis cuvettes
3. MATERIALS
(4,40-Dimethoxytriphenyl)methyl chloride (DMT-Cl)
[(Triisopropylsilyl)oxy]methyl chloride (TOM-Cl)
1-O-Acetyl-(20,30,50-O-tribenzoyl)-β-D-ribofuranose (ATBR)
1,2-Dichloroethane
1H-benzylthiotetrazole
2-Cyanoethyl N,N-diisopropylchlorophosphoramidite
2,4,6-Triisopropylbenzenesulfonyl chloride (TPS-Cl)
28% Aqueous ammonia solution
4-(Dimethylamino)pyridine (DMAP)
Acetic acid
Acetic anhydride
Acetonitrile anhydrous
Acetonitrile DNA synthesis grade

Stable Isotope-Modified RNA Phosphoramidites
465
Argon
Bis(trimethylsilyl)acetamide
CDCl3
Celite
Citric acid
D2O
Dibutyltin dichloride (or alternatively: di-tert-butyltin dichloride)
Dichloroacetic acid
Dimethylformamide anhydrous
Ethanol
Ethyl acetate
Hexanes (mixture of isomers)
Iodine
Methanol
Methylamine solution in ethanol (8 M)
Methylamine solution in water (30%)
Methylene chloride
˚
Molecular sieve (4 A)
N-methyl-2-pyrrolidone
N,N-diisopropylethylamine (DIPEA)
N,N-dimethylethylamine (DMEA)
Pyridine anhydrous
RNA support for solid phase synthesis (e.g., Custom primer support
from GE healthcare)
˚
Silica gel for column chromatography pore size 60 A, ꢂ440 mesh
particle size
Sodium acetate trihydrate
Sodium bicarbonate
Sodium perchlorate hydrate
Sodium sulfate anhydrous
Sym-collidine
Tetrabutylammonium fluoride trihydrate (TBAF)
Tetrahydrofuran (containing butylhyroxytoluene (BHT) as stabilizer)
Toluene
Triethylamine
Trimethylsilyl trifluoromethylsulfonate (TMSOTf )
Trizma base
Unlabeled [(triisopropylsilyl)oxy] methyl (TOM) protected RNA pho-
sphoramidites (e.g., from ChemGenes)

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Christoph H. Wunderlich et al.
Uracils (unlabeled and in various stable isotope-labeled forms by chem-
ical synthesis)
Urea
3.1 Solutions and Buffers
Saturated sodium bicarbonate solution:
Add 200 g sodium bicarbonate to 1000 ml deionized water.
5% Citric acid
Dissolve 50 g citric acid in 1000 ml deionized water.
Cap A solution:
Dissolve 5 g DMAP in 50 ml acetonitrile.
Cap B solution:
Mix 25 ml acetonitrile with 10 ml acetic anhydride and 15 ml
sym-collidine.
Oxidation solution:
Dissolve 250 mg iodine in 25 ml tetrahydrofuran. Then add
10 ml pyridine and 5 ml deionized water.
Detritylation solution:
Mix 10 ml dichloroacetic acid with 240 ml 1,2-dichloroethane.
100 mM sodium acetate solution:
Dissolve 13.6 g sodium acetate trihydrate in 1000 ml HPLC
grade water.
Dionex column buffer A:
Component
Final concentration
25 mM
Stock
250 mM
-
Amount
100 ml
360 g
Tris–HCl buffer, pH 8.0
urea
6 M
Add water to 1-l. filter through 0.22-μm hydrophilic membrane.
Dionex column buffer B:
Component
Final concentration
25 mM
Stock
Amount
100 ml
360 g
Tris–HCl buffer, pH 8.0
urea
250 mM
6 M
-
-
Sodium perchlorate hydrate
500 mM
70.2 g
Add water to 1-l filter through 0.22-μm hydrophilic membrane.

Stable Isotope-Modified RNA Phosphoramidites
467
4. PROTOCOL
4.1 Duration
Preparation
Step 1
1–3 weeks
5–7 days
5–7 days
5–7 days
3 days
Step 2
Step 3
Step 4
4.2 Preparation
Uracils with various stable isotope-labeling patterns are accessible via chem-
ical synthesis as reported earlier (Alvarado, Longhini, et al., 2014). Starting
from potassium cyanide and 2-bromoacetic acid, the cyano acetylurea pre-
cursor is obtained. In the final step, uracil is formed under reductive reaction
conditions using palladium on barium sulfate under a hydrogen atmosphere.
Prepare this key intermediate using the previously reported methods to pro-
ceed directly with step 1 (Fig. 2).
Purchase the unlabeled TOM protected RNA and DNA pho-
sphoramidites along with the synthesis reagents (e.g., water free
(<5 ppm) acetonitrile, 1H-benzylthiotetrazole (BTT) from a commercial
supplier (e.g., ChemGenes, Glen Research). It is advisable to carry out test
oligonucleotide syntheses to optimize the coupling, oxidation, and capping
steps. For that purpose, DNA oligonucleotide synthesis should be used
before switching to the more expensive RNA phosphoramidite chemistry
for fine-tuning.
4.3 Caution
Please check the safety data sheets of the synthesis reagents before starting.
Some of these reagents are very hazardous and should be handled by expe-
rienced chemists with extreme care in a well-vented fume hood. Pho-
sphoramidites are rather sensitive chemical compounds and are degraded
in the presence of acids and water or elevated temperatures. Pho-
sphoramidite solutions (prepared using water-free acetonitrile) should be
stored over freshly activated molecular sieve and are stable for several weeks
at ꢁ20 °C.

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Christoph H. Wunderlich et al.
Figure 2 Workflow of the protocol. The part shown in dotted outline is an alternate
extension to chemoenzymatic labeling using the selectively stable isotope-modified
uracils (Alvarado, LeBlanc, et al., 2014; Alvarado, Longhini, et al., 2014).
5. STEP 1: SYNTHESIS OF 6-13C-URIDINE TOM
PHOSPHORAMIDITE
5.1 Overview
Transformationsa,b,andc(Fig.3)werereportedearlier(Alvarado,Longhini,
etal.,2014).Usingtheappropriateprecursorcomposition,anyheteronucleus
of the uracil can be specifically enriched. The stable isotope-modified uracil
key intermediate is coupled to a ribose building bock under Vorbru¨ggen
nucleosidation conditions (d) (Fig. 3). Subsequent removal of the benzyol
protecting groups (e) followed by tritylation (f ) gives the precursor for the
protection of the 20-hydroxyl group using the TOM group (g) (Fig. 3). In
the last step, the phosphoramidite is obtained (h) (Fig. 3).
5.2 Duration
5–7 days
1.1. Synthesis of 6-13C-20,30,50-O-tribenzoyluridine: In a 100 ml round-
bottom flask, suspend 6-13C-uracil (560 mg, 5 mmol) together with

Figure 3 Synthetic access to 6-13C-uridine labeled fully protected RNA phosphoramidite.

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Christoph H. Wunderlich et al.
ATBR (2.5 g, 5 mmol) in 20 ml dry acetonitrile and evaporated to
dryness. Repeat this procedure two times.
1.2. Add 20 ml dry acetonitrile and heat the suspension to 60 °C using a
reflux condenser and an oil bath or a heat block.
1.3. Then add bis(trimethylsilyl)acetamide (3.7 ml, 15 mmol) and stir the
reaction mixture for 30 min at 60 °C. The suspension should turn
into a clear solution.
1.4. Finally, add TMSOTf (3.2 ml, 17.5 mmol) and continue stirring for
another 30 min at 60 °C.
1.5. The reaction progress can be monitored using TLC (ethyl acetate/
hexanes 1/1, Rf (product)¼0.8).
1.6. After that the reaction mixture is evaporated to yield an orange brown
oil which is dissolved in methylene chloride and subsequently washed
with saturated sodium bicarbonate solution. The organic phase is
dried over sodium sulfate, filtered, and evaporated to dryness to give
a yellow solid. The compound can be directly used in the next step.
1.7. Synthesis of 6-13C-uridine: In a 100-ml round-bottom flask crude
6-13C-20,30,50-O-tribenzoyluridine (2.5 g, 4.5 mmol) is dissolved
in an ethanolic solution of methylamine (15 ml, 8 M) and an argon
atmosphere and stirred at room temperature for 16 h.
1.8. The reaction progress can be monitored using TLC (CH2Cl2/
methanol 9/1, Rf (product)¼0.1).
1.9. Then, the solution is evaporated to yield a yellow oil, which is dis-
solved in water and repeatedly extracted with methylene chloride to
remove the N-methylbenzamide from the aqueous phase.
1.10. The aqueous phase containing only 6-13C-uridine is then evaporated
to dryness resulting in a yellow foam.
1.11. To remove traces of impurities, recrystallization from ethanol is
advisable.
1.12. Synthesis of 50-O-(4,40-dimethoxytrityl)-6-13C-uridine: The prod-
uct from the previous step (1.0 g, 4.1 mmol) is coevaporated three
times with anhydrous pyridine in a 100-ml round-bottom flask.
1.13. The residual oil is dissolved in 10 ml anhydrous pyridine. The reac-
tion is carried out under an argon atmosphere. Add DMT (1.7 g,
5.0 mmol) in three portions within an hour.
1.14. After 4 h at room temperature, the solvent is evaporated and
coevaporated three times with toluene.

Stable Isotope-Modified RNA Phosphoramidites
471
1.15. The orange foam is dried in high vacuum for 30 min and then dis-
solved in methylene chloride and washed with 5% citric acid, water,
and saturated sodium bicarbonate solution.
1.16. The organic phase is dried over sodium sulfate, filtered, and evapo-
rated to give the crude product.
1.17. The crude product is further purified by silica column chromatogra-
phy using a gradient of methylene chloride and methanol (99/1
CH2Cl2/methanol to 95/5).
1.18. Synthesis of 50-O-(4,40-dimethoxytrityl)-20-O-[[(triisopropylsilyl)
oxy]methyl]-6-13C-uridine: The tritylated uridine (616 mg,
1.13 mmol) is dissolved a mixture of DIPEA(765 μl, 4.5 mmol)
and 1,2-dichloroethane (10 ml).
1.19. To the clear solution, dibutyltin dichloride (Bu2SnCl2; 616 mg,
2.02 mmol) is added, and the mixture is stirred for 1 h at room
temperature.
1.20. Then, the solution is heated to 80 °C using a reflux condenser, and an
oil bath or a heat block and TOM-Cl; 276 mg, 1.24 mmol) is added.
1.21. The reaction progress can be monitored using TLC (ethyl acetate/
hexanes 1/1, Rf (product)¼0.6).
1.22. After 30 min, the reaction mixture is diluted with methylene chlo-
ride and the organic phase is washed with saturated sodium bicarbon-
ate solution.
1.23. The organic phase is dried over sodium sulfate, filtered over Celite,
and then evaporated to dryness.
1.24. The crude product is further purified by silica column chromatogra-
phy using a gradient of hexanes and ethyl acetate (80/20 hexanes/
ethyl acetate to 40/60).
1.25. Synthesis of 50-O-(4,40-dimethoxytrityl)-20-O-[[(triisopropylsilyl)
oxy]methyl]-6-13C-uridine
30-O-(20-cyanoethyl
N,N-
diisopropylphoshphoramidite): Anhydrous methylene chloride
(5 ml) and DMEA (540 μl, 5.0 mmol) are mixed and then added
to the product of the previous step (720 mg, 0.98 mmol).
1.26. The clear solution is stirred at room temperature for 15 min.
1.27. Add 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (350 mg,
1.5 mmol) dropwise via a syringe and stir the reaction for 2 h.
1.28. The reaction progress can be monitored using TLC (ethyl acetate/
hexanes 7/3+1% triethylamine, Rf (product)¼0.5).

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Christoph H. Wunderlich et al.
1.29. The reaction mixture is then diluted with methylene chloride, and
the organic phase is washed with half saturated sodium bicarbonate
solution.
1.30. The organic phase is dried over sodium sulfate, filtered, and evapo-
rated to dryness.
1.31. The crude product is further purified by silica column chromatogra-
phy using a gradient of hexanes and ethyl acetate (50/50 hexanes/
ethyl acetate to 40/60+1% triethylamine).
1.32. The quality of the final product should be checked by NMR spec-
troscopy (1H, 13C, and 31P NMR in CDCl3). The 31P NMR spec-
trum consists of two peaks at 150.5 and 150.1 ppm (referenced to
external 85% H3PO4). A 31P peak at approximately 15 ppm indicates
a phosphonate impurity, which needs to be removed by silica column
chromatography to guarantee high coupling yields in the RNA solid
phase synthesis.
5.2.1 Tip
The products of the respective steps should be characterized using NMR
spectroscopy (1D-1H and 13C spectra) and mass spectrometry and compared
to data from literature before proceeding with the next step.
5.2.2 Tip
In the nucleosidation reaction (step 1.1), the temperature (60 °C) should be
checked several times. Overheating will result in the formation of the unde-
sired α-substituted uridine nucleoside.
5.2.3 Tip
In the nucleosidation reaction (step 1.3), let the solution cool to room tem-
perature before adding TMSOTf to avoid a too vigorous reaction.
5.2.4 Tip
The extraction of N-methylbenzamide should be checked using silica TLC
(CH2Cl2/MeOH 9/1). After each extraction step, spot the aqueous phase
and the organic phase on a silica TLC plate to check the composition of
the phases. Normally, after three extraction steps no N-methylbenzamide
is found in the aqueous phase.

Stable Isotope-Modified RNA Phosphoramidites
473
5.2.5 Tip
In the tomylation reaction (step 1.19), separation of organic and aqueous
phase can be retarded especially when carrying out the reaction on a larger
scale. In that case, the use of the bis-(tert-butyl)tin dichloride is
recommended.
5.2.6 Tip
The tomylation reaction gives a distribution of desired 20-O-regioisomer/
undesired 30-O-regioisomer/starting material of 1/1/1. To improve the
yield, collect the 30-O-regioisomer and remove the TOM protecting group
using 1 M tetrabutylammonium fluoride in acetonitrile (1 ml/250 mg) and
combine it with the starting material. This recovered material can then be
reused in the tomylation reaction.
5.2.7 Tip
When carrying out the silica column chromatography of phosphoramidites,
it is absolutely necessary to add 1% triethylamine to the eluents to avoid deg-
radation of the amidites on the column. Triethylamine should also be added
to the TLC solvent.
6. STEP 2: SYNTHESIS OF 6-13C-CYTIDINE TOM
PHOSPHORAMIDITE
6.1 Overview
Starting from 50-O-(4,40-dimethoxytrityl)-20-O-[[(triisopropylsilyl)oxy]
methyl]-6-13C-uridine, the 30-hydroxyl group is protected using acetic
anhydride (a) (Fig. 4). Using TPS-Cl, the O4 of uridine is activated as a leav-
ing group and can be replaced with an amino group using an ammonium
hydroxide solution (b) (Fig. 4). If desired, 15N-labeled ammonium chloride
in combination with a base (e.g., K2CO3) can be used to introduce a 15N
label at the exocyclic amino group at this stage. N-selective acetylation
(c) and finally phosphitylation (d) yield the desired 6-13C-cytidine pho-
sphoramidite (Fig. 4).
6.2 Duration
5–7 days
2.1. Synthesis
of
30-O-acetyl-50-O-(4,40-dimethoxytrityl)-20-O-
[[(triisopropylsilyl)oxy]-methyl]-6-13C-uridine: In a 50-ml round-

Figure 4 Synthetic access to 6-13C-cytidine labeled fully protected RNA phosphoramidite.

Stable Isotope-Modified RNA Phosphoramidites
475
bottom flask, 50-O-(4,40-dimethoxytrityl)-20-O-[[(triisopropylsilyl)
oxy]methyl]-6-13C-uridine (350 mg, 0.47 mmol) is dissolved in
1 ml anhydrous pyridine and is stirred at room temperature.
2.2. Add a catalytic amount of DMAP (5 mg, 0.05 mmol).
2.3. Cool the reaction mixture to 0 °C and add acetic anhydride (48 μl,
0.51 mmol) dropwise.
2.4. Remove the ice bath and continue stirring for 2 h.
2.5. The reaction progress can be monitored using TLC (ethyl acetate/
hexanes 1/1, Rf (product)¼0.7).
2.6. The solvent is evaporated, and the residue coevaporated twice with
toluene.
2.7. The residue is dissolved in methylene chloride, and the organic phase
is washed with 5% citric acid, water, and saturated sodium bicarbon-
ate solution.
2.8. Dry the organic phase over sodium sulfate, filter, and evaporate to
dryness.
2.9. The crude product can directly be used in the next step.
2.10. Synthesis of 50-O-(4,40-dimethoxytrityl)-20-O-[[(triisopropylsilyl)
oxy]-methyl]-6-13C-cytidine: Mix anhydrous methylene chloride
(4 ml) and triethylamine (NEt3, 528 μl, 3.8 mmol). To this mixture,
add the crude product from the previous step (300 mg, 0.38 mmol)
and DMAP (5 mg, 0.05 mmol).
2.11. After 10 min, add TPS-Cl; 176 mg, 0.58 mmol) in small portions
over a period of 1 h.
2.12. Stir the reaction for another 4 h at room temperature.
2.13. Dilute the reaction mixture with methylene chloride and wash the
organic phase with half saturated sodium bicarbonate solution.
2.14. Dry the organic phase over sodium sulfate, filter, and evaporate to
dryness.
2.15. Dry the resulting foam in high vacuum for 30 min.
2.16. Dissolve the crude product from the previous step in 4 ml tetrahy-
drofuran and add 4 ml 28% aqueous ammonia solution. Continue
stirring for 18 h at room temperature.
2.17. After 18 h, evaporate the reaction mixture to yield an oily residue.
Take up the residue in 4 ml ethanolic methylamine solution (8 M)
and stir for 1 h.
2.18. The reaction progress can be monitored using TLC (CH2Cl2/
methanol 96/4, Rf (product)¼0.45).

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Christoph H. Wunderlich et al.
2.19. Evaporate the solvent and dry the residual oil in high vacuum.
2.20. Dissolve the oil in methylene chloride and wash the organic phase
with saturated sodium bicarbonate solution.
2.21. Dry the organic phase over sodium sulfate, filter, and evaporate to
dryness.
2.22. Purify the resulting foam from the previous step using silica column
chromatography (CH2Cl2/methanol 99/1 to 95/5).
2.23. Synthesis
of
N4-acetyl-50-O-(4,40-dimethoxytrityl)-20-O-
[[(triisopropylsilyl)-oxy]methyl]-6-13C-cytidine: Dissolve the prod-
uct from the previous step (190 mg, 0.26 mmol) in 1 ml anhydrous
dimethylformamide. Then add acetic anhydride (25 μl, 0.26 mmol)
dropwise.
2.24. Stir the reaction for 22 h at room temperature.
2.25. The reaction progress can be monitored using TLC (ethyl acetate/
hexanes 7/3, Rf (product)¼0.38).
2.26. Quench the reaction by the addition of a few drops of methanol.
2.27. Evaporate the solvent and dissolve the oily residue in methylene
chloride.
2.28. Wash the organic phase with saturated sodium bicarbonate solution.
2.29. Dry the organic phase over sodium sulfate, filter, and evaporate to
dryness.
2.30. Purify the crude product using silica column chromatography
(CH2Cl2/methanol 99/1 to 98/2).
2.31. Synthesis
of
N4-acetyl-50-O-(4,40-dimethoxytrityl)-20-O-
[[(triisopropylsilyl)-oxy]methyl]-6-13C-cytidine 30-O-(20-Cyanoethyl
N,N-diisopropylphoshphoramidite): Anyhdrous methylene chloride
(5 ml) and DMEA (250 μl, 2.3 mmol) are mixed and then added to
the product of the previous step (130 mg, 0.98 mmol).
2.32. The clear solution is stirred at room temperature for 15 min.
2.33. Add 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (83 mg,
0.35 mmol) dropwise via a syringe and stir the reaction for 2 h.
2.34. The reaction progress can be monitored using TLC (ethyl acetate/
hexanes 7/3+1% triethylamine, Rf (product)¼0.46).
2.35. The reaction mixture is then diluted with methylene chloride, and
the organic phase is washed with half saturated sodium bicarbonate
solution.
2.36. The organic phase is dried over sodium sulfate, filtered, and evapo-
rated to dryness.

Stable Isotope-Modified RNA Phosphoramidites
477
2.37. The crude product is further purified by silica column chromatogra-
phy using a gradient of hexanes and ethyl acetate (70/30 hexanes/
ethyl acetate+1% trimethylamine to 30/70 hexanes/ethyl acetate
+1% triethylamine).
2.38. The quality of the final product should be checked by NMR spec-
troscopy (1H, 13C, and 31P NMR in CDCl3). The 31P NMR spec-
trum should show two peaks at 150.8 and 150.6 ppm (referenced to
external 85% H3PO4). A 31P peak at approximately 15 ppm indicates
a phosphonate impurity, which needs to be removed by silica column
chromatography to guarantee high coupling yields in the RNA solid
phase synthesis.
6.2.1 Tip
The products of the respective steps should be characterized using NMR
spectroscopy (1D-1H and 13C spectra) and mass spectrometry and compared
to data from literature before proceeding with the next step.
6.2.2 Tip
The crude product from step 2.15 is not stable and should not be stored for a
prolonged time at room temperature. It is best used directly after drying in
high vacuum.
6.2.3 Tip
When carrying out the silica column chromatography of phosphoramidites,
it is absolutely necessary to add 1% triethylamine to the eluents to avoid deg-
radation of the amidites on the column. Triethylamine should also be added
to the TLC solvent.
7. STEP 3: CHEMICAL RNA SYNTHESIS
7.1 Overview
Using an automated RNA/DNA synthesizer, the 6-13C-pyrimidine-
modified TOM-protected RNA phosphoramidites can be introduced as
site-specific stable isotope labels into a target RNA for NMR application.
The phosphoramidite chemistry is well established and high-quality syn-
thetic RNAs can be obtained (Fig. 5; Micura, 2002). The main advantage
of the chemical oligonucleotide synthesis is that no sequence requirements,

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Christoph H. Wunderlich et al.
Figure 5 Oligonucleotide solid phase synthesis cycle: After removal of the trityl
protecting group, the 50-OH group is attached to the next building block in the activa-
tion/coupling step using a weak acid (e.g., 1H-benzylthiotetrazole) and the respective
phosphoramidite. Unreacted 50-hydroxyls are capped using acetic anhydride. In the
final step, the P(III) species is oxidized to the more stable P(V) species before the next
synthesis cycle is entered.
as e.g., in the case of T7 RNA polymerase assisted in vitro transcription, are
encountered. Furthermore, the number and positioning of the stable mod-
ified phosphoramidites can be freely chosen. Several DNA/RNA synthe-
sizers are commercially available (e.g., from Applied Biosystems, GE
Healthcare, Bioautomation). As aforementioned in Section 4.2, the synthe-
sis cycle should be optimized before using stable isotope-modified RNA
phosphoramidites.
Optimized synthesis cycles for an ABI 391 PCRmate are available on
request from the authors. The assembled RNA sequence is then deprotected
in a two-step procedure: (i) alkaline deprotection: releases the oligonucle-
otide from the solid support, removes the nucleobase protecting groups
and the cyanoethyl groups; (ii) fluoride deprotection: using 1 M

Stable Isotope-Modified RNA Phosphoramidites
479
tetrabutylammonium fluoride in tetrahydrofuran the TOM group is cleaved
off. Finally, anion exchange chromatography is used to purify the stable
isotope-modified RNA.
7.2 Duration
5–7 days
3.1. Freshly prepare phosphoramidite solutions: dissolve 1 g of each
TOM protected phoshporamidite in 10 ml water-free acetonitrile
(<5 ppm water) and add freshly activated molecular sieve.
3.2. Freshly prepare activator solution: dissolve 1H-benzythiotetrazole
(1.44 g) in 30 ml water-free acetronitrile and add freshly activated
molecular sieve.
3.3. Fill a 1.3-μmol synthesis column with the appropriate amount of
solid support (e.g., 16 mg rA Custom Primer support, 80 μmol gꢁ1,
GE Healthcare).
3.4. Attach synthesis reagents (Cap A, Cap B, oxidation solution,
detritylation solution, and water-free acetonitrile) to the appropriate
ports on the RNA synthesizer. Attach phosphoramidite solutions
(rA, rG, rC, U, and stable isotope-modified amidites to the additional
ports X1, X2, etc.) to the appropriate ports on the RNA synthesizer.
3.5. Fill the reagent lines with the respective solution. Fill the pho-
sphoramidite lines with a minimal volume of respective amidite solu-
tion to reduce loss (especially for the stable isotope-labeled
phosphoramidites). Attach the synthesis column and wash the col-
umn with water-free acetonitrile to check for leaks.
3.6. Enter the target sequence and start the synthesis procedure.
3.7. Check the first detritylation step of the solid support. A deep red
color should be observable.
3.8. Check the coupling of the first phosphoramidite by observing the
detritylation step. Again a deep red color of the detritylation solution
should be observed. Let the synthesis proceed until the sequence
assembly is finished. If possible check the final detritylation step.
A red color of the solution hints at a successful sequence synthesis.
3.9. Dry the solid support in high vacuum for 30 min. Transfer the solid
support into a 1.5-ml Eppendorf© tube with a sealing.
3.10. Ethanolic methylamine/aqueous methylamine deprotection: Add
650 μl aqueous methylamine solution (30%) and 650 μl ethanolic
methylamine solution (8 M) to the solid support and let stand for

480
Christoph H. Wunderlich et al.
6 h at room temperature. Vortex 1/h. Or alternatively: aqueous
methylamine/aqueous ammonia (AMA) deprotection: Add 650 μl
aqueous methylamine solution (30%) and 650 μl ammonium
hydroxide solution (28%) and heat to 37 °C for 1.5 h.
3.11. Centrifuge to pellet the solid support on the bottom of the tube and
transfer the supernatant to a 10-mlround-bottom flask. Wash the solid
support three times with 1 ml tetrahydrofuran/water (1/1, v/v) and
add the washings to the 10-ml round-bottom flask. Evaporate the
solution in the flask to dryness and dry the residue in high vacuum
for 30 min to remove the bases.
3.12. Dissolve the residue in the 10-ml round-bottom flask in 1.6 ml 1 M
tetrabutylammonium fluoride solution in tetrahydrofuran. If the res-
idue does not readily dissolve, add 100 μl N-methyl-2-pyrrolidone to
obtain a clear solution. Stopper the 10-ml round-bottom flask tightly
with the aid of Parafilm© and keep the deprotection solution at 33 °
C for at least 14 h. Quench the 20-O-deprotection reaction with
1.6 ml 1 M triethylammonium acetate solution. Evaporate the
deprotection solution to an approximate volume of 1 ml.
3.13. Load the crude RNA on a HiPrep desalting 26/10 column (GE
Healthcare) using a MPLC system with UV and conductivity detec-
tion. Use HPLC grade water (18 MΩ cm) to elute the crude RNA.
The RNA elutes first and can be identified by its high UV absorbance
at 254 nm, whereas the later eluting salts lead to strong increase in
conductivity. Collect the RNA in a 50-ml round-bottom flask.
Evaporate the RNA containing fraction to dryness and redissolve
the oligonucleotide in 1 ml HPLC grade water. The solution of
the crude oligonucleotide product can be stored at ꢁ20 °C for sev-
eral weeks without degradation (Fig. 6).
3.14. To check the quality of the crude RNA, anion exchange chromatog-
raphy using an analytical Dionex DNAPac PA-100 (4ꢀ250 mm)
column and buffer A and B on a HPLC system should be carried
out. For RNAs comprising less than 20 nucleotides, a gradient from
0% to 40% B in A in 30 min is sufficient, and for larger RNAs, a gra-
dient from 0% to 60% B in A in 45 min needs to be applied. The
chromatographic analysis is carried out at 80 °C to fully denature
the RNA (Fig. 7).
3.15. To further purify the crude RNA semipreparative anion exchange
chromatography on a Dionex DNAPac PA-100 (9ꢀ250 mm) col-
umn using buffer A and B is carried out. The gradient needs to be

Stable Isotope-Modified RNA Phosphoramidites
481
Figure 6 Desalting of crude RNA on a ÄKTA start system (GE Healthcare) using a HiPrep
desalting column 26/10. 6-13C-pyrimidine labels are highlighted in orange (gray in the
print version).
Figure 7 Anion exchange chromatographic analysis of a crude RNA comprising
27 nucleotides. The analysis was carried out on a Dionex DNAPac PA-100 column
(4ꢀ250mm) using a gradient of 0–60% buffer B in buffer A in 45 min at 80 °C. The
UV absorbance trace at 260 nm is shown.
optimized for every target RNA and depends on its length and nucle-
otide composition. The chromatographic purification is carried out
at 80 °C to fully denature the RNA. Several runs (typically 5–6) load-
ing approximately 30 nmol crude RNA per run are needed to purify
the product of a RNA synthesis on a 1.3 μmol scale (Fig. 8).
3.16. Dilute the fractions containing desired RNA with an equal volume of
100 mM sodium acetate solution.

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Christoph H. Wunderlich et al.
Figure 8 Anion exchange chromatographic purification of a crude RNA comprising
27 nucleotides. A gradient of 30–40% buffer B in buffer A in 15 min at 80 °C was used.
The UV absorbance trace at 260 nm is shown.
3.17. Prepare a C18 SepPak cartridge (Waters): (i) 3 column volumes (CV)
acetonitrile, (ii) 3 CV acetonitrile/water (1/1, v/v), (iii) 3 CV water,
(iv) 3 CV 100 mM sodium acetate.
3.18. Then load the RNA containing fractions on the SepPak cartridge.
3.19. Elute the buffer salts by washing the RNA with 3 CV water.
3.20. Elute the RNA using 3 CV acetonitrile/water (1/1, v/v) into a
50-ml round-bottom flask.
3.21. Evaporate the solution containing the target RNA to dryness and
redissolve the RNA in 1 ml HPLC grade water.
3.22. To check the quality of the purified RNA, anion exchange
chromatography using an analytical Dionex DNAPac PA-100
(4ꢀ250 mm) column and buffer A and B on a HPLC system should
be carried out. For RNAs comprising less than 20 nucleotides, a
gradient from 0% to 40% B in A in 30 min should be used, for larger
RNAs a gradient from 0% to 60% B in A needs to be applied. The
chromatographic analysis is carried out at 80 °C to fully denature
the RNA (Fig. 9).
3.23. The yield of purified target RNA can be calculated by applying the
Beer–Lambert law:
À
Á
c ¼ A= ε260nm
l
with c concentration in mol lꢁ1, A absorption at 260 nm, ε260nm
extinction coefficient at 260 nm in l molꢁ1 cmꢁ1 and l length of solu-
tion light passes in cm. The extinction coefficient ε260nm of the RNA
can be estimated by adding the ε260nm values of the monomers.

Stable Isotope-Modified RNA Phosphoramidites
483
Figure 9 Anion exchange chromatographic analysis of a purified RNA comprising
27 nucleotides. The analysis was carried out on a Dionex DNAPac PA-100 column
(4ꢀ250mm) using a gradient of 0% to 60% buffer B in buffer A in 45 min at 80 °C.
The UV absorbance trace at 260 nm is shown. The purity is estimated by peak integra-
tion to be >95%.
Nucleotide
ε260nm [l mol21 cm21
]
rA
rC
rG
U
15,300
7400
11,700
9900
3.24. Lyophilize the desired amount of RNA for the NMR spectroscopic
application.
7.2.1 Tip
Do not overfill the synthesis column with solid support as the growing RNA
chain could clog the column.
7.2.2 Tip
If using the AMA procedure at elevated temperatures, allow cooling to
room temperature before opening the tube to avoid spilling of the
deprotection solution.
7.2.3 Tip
Use tetrahydrofuran containing the stabilizer BHT for the preparation of the
TBAF solution. Stabilizer-free tetrahydrofuran can form radicals leading to
irreproducible deprotection results.

484
Christoph H. Wunderlich et al.
7.2.4 Tip
The integrity and homogeneity of the purified RNA (from step 3.21) can
further be checked using LC-ESI mass spectrometry and denaturing and
native PAGE.
8. STEP 4: APPLICATIONS
8.1 Overview
Using these stable isotope-modified RNA phosphoramidites, an isolated
1H–13C-spin pair is introduced which allows the application of Carr Purcell
Meiboom Gill (CPMG) RD NMR experiments in a straightforward man-
ner (Wunderlich et al., 2012). Additionally, a significant side benefit of this
methodology is that the site selectively labeled nucleobase can be coupled
enzymatically with ribose to make labeled rNTPs (Alvarado, LeBlanc,
et al., 2014; Alvarado, Longhini, et al., 2014). Importantly, this very pow-
erful methodology can be exploited to detect and quantify important func-
tional dynamics occurring on the μs- to ms time scale. Thus, structural
transitions of RNA important for ligand binding or catalysis can be inves-
tigated readily. In this section, we discuss the potential of these chemically
synthesized RNAs in a segmental isotope-labeling protocol to address func-
tional dynamics in larger RNAs, as well as its incorporation using
chemoenzymatic tricks.
We present 13C CPMG RD data on a 27 nt RNA that mimics the bac-
terial 16S decoding site (A site), which is involved in discriminating cognate
and near-cognate tRNAs (Dethoff, Petzold, Chugh, Casiano-Negroni, &
Al-Hashimi, 2012). The labeling pattern is also well suited to study slower
RNA secondary structure refolding kinetics occurring at exchange rates at
1 sꢁ1. We illustrate the applicability of the stable isotope-labeled RNAs in a
13C ZZ exchange NMR experiment addressing the refolding kinetics
between two hairpin folds, one representing the terminator and the other
the antiterminator fold of the Fsu preQ1 riboswitch system (Rieder,
Kreutz, & Micura, 2010). Finally, we also showcase the usefulness to CEST
experiments on a 48 nt RNA labeled with 13C on the base chemically but
coupled to the ribose enzymatically (Alvarado, LeBlanc, et al., 2014;
Alvarado, Longhini, et al., 2014).
8.2 Segmental Stable Isotope Labeling Using Chemically
Synthesized RNAs
Segmental isotope labeling is the key methodology when studying high-
molecular weight nucleic acid systems where spectral overlap dominates

Stable Isotope-Modified RNA Phosphoramidites
485
(Duss, Maris, von Schroetter, & Allain, 2010; Lebars et al., 2014; Lu,
Miyazaki, & Summers, 2010; Nelissen et al., 2008; Tzakos, Easton, &
Lukavsky, 2007). In short, a stable isotope-labeled RNA produced either
by chemical or enzymatic methods is ligated to another (e.g., unlabeled)
RNA giving access to larger constructs with a few NMR active residues to
address structural and dynamic features. The procedure makes use of two
enzymes, the T4 RNA ligase and T4 DNA ligase, but also deoxyribozymes
were used to ligate two RNA strands (Dayie, 2008; Wawrzyniak-Turek &
Ho€bartner, 2014). When using T4 RNA or DNA ligase a 50-monophosphate
is required on the donor strand and a 30-hydroxyl group at the acceptor frag-
ment. The T4 DNA ligase further needs a DNA splint as the enzyme catalyzes
the joining of RNA to another RNA strand in a duplex molecule but will not
join single-stranded nucleic acids. In contrast, T4 RNA ligase accepts single-
stranded RNAs as substrates. Solid phase RNA synthesis is perfectly suited to
craft oligonucleotides with the desired 50-monophosphate (using a commer-
cially available phosphorylation reagent) and 30-OH, termini which are not
directly accessible via in vitro transcription. For in vitro synthesis, a ribozyme
sequence (e.g., hepatitis delta virus) must be appended at the 50- and 30-ends
of the donor fragment RNA (Dayie, 2008; Kieft & Batey, 2004), and the
50-diphosphate is cleaved enzymatically by RNA 50-polyphosphatase to
obtain the desired monophosphorylated group at the 50-termini (Chen,
Zuo, Wang, & Dayie 2012; Dayie, 2008).
We explored the applicability of chemically synthesized RNAs for
segmental stable isotope labeling. Here, we showcase the tremendous poten-
tial of the approach by the ligation of two RNAs comprising 13C-methyl
modifications naturally occurring in transfer RNAs (Fig. 10). Ligation of
a 39 nt acceptor and a 37 nt donor strand using T4 RNA ligase yielded a
13C-methyl-modified phenylalanine tRNA. NMR studies on the dynamics
of the 13C-methyl-modified residues are currently carried out in our
laboratories.
8.3 13C CPMG RD NMR Spectroscopy
The 27 nt A-site RNA was modified using 6-13C-uridine and 6-13C-
cytidine phosphoramidites. The successful incorporation of the six stable
isotope labels (five 6-13C-uridines and one 6-13C-cytidine) was confirmed
by NMR spectroscopy. A standard heteronuclear single quantum correla-
tion (HSQC) spectrum did show the expected six 1H–13C-correlation peaks
(Fig. 11A). We then addressed the previously reported conformational
dynamics of this RNA using 13C CPMG RD NMR spectroscopy

Figure 10 (A) One step T4 RNA ligase catalyzed joining of a 37 and 39 nt RNA comprising 13C-modified methyl residues (highlighted in red
(darkgray in the print version), ligation site highlighted in orange (gray in the print version)) yielding a full length 76 nt tRNAPhe. Ligation
progress can be readily followed using denaturing polyacrylamide gel electrophoresis (see inset). (B) Electrospray Ionization mass spectro-
metric analysis of tRNAPhe ligation product.

Stable Isotope-Modified RNA Phosphoramidites
487
Figure 11 (A) Standard heteronuclear single quantum correlation experiment confirm-
ing the successful incorporation of six 6-13C-pyrimidine labels into the 27 nt A-site
RNA. (B) Dispersion profiles of U6 and U95 at 212.5 (red) and 175 (blue) MHz carbon
larmor frequency. Dots represent the experimental data, and black x represent repeat
experiments. The dashed and dotted blue and red lines represent the individual and
global fits for a two-state equilibrium in the intermediate exchange regime.
(Fig. 11B) (Dethoff et al., 2012). For that purpose, we used a longitudinal
optimized transverse relaxation optimized spectroscopy (TROSY) selected
dispersion experiment (Weininger, Respondek, & Akke, 2012). This exper-
iment is especially useful for the fast relaxing aromatic RNA nucleobase 13C
atoms introduced by the presented labeling pattern.
Two residues could be identified that displayed a nonflat dispersion
profile—U6 and U95. Data were recorded at two field strengths (16.4 and
20 T at 288 K), and the dispersion profiles were fitted using an exact solution
for a two-site exchange process. The results are summarized (Table 1) and
are in good agreement with results obtained from R1ρ dispersion profiles at
298 K (Dethoff et al., 2012).
8.4 13C ZZ Exchange NMR Spectroscopy
In gene regulatory riboswitch RNAs, slower dynamics occuring at a few
milliseconds to seconds can be of special importance for their biological
function. This class of noncoding RNAs switches their tertiary and/or sec-
ondary structure in response to the presence or absence of a low-molecular

488
Christoph H. Wunderlich et al.
Table 1 Summary of Exchange Data Obtained from 13C-CPMG RD Experiments of the
A-Site RNA at 288 K
kex/s21
pA/%a
pB/%a
Δδ(13C)/ppmb
Global parameters
1204ꢃ256
98.1ꢃ0.8
1.9ꢃ0.8
U6
2.42ꢃ0.4
0.87ꢃ0.6
U95
apA refers to the ground state and pB to the excited state population.
b13C-chemical shift difference between ground state A and excited state B for the residues exhibiting
nonflat dispersion profiles are given.
weight metabolites (Haller et al., 2011). We identified a bistable terminator
antiterminator sequence partition (Fig. 12A, dashed box; Rieder et al.,
2010). To address the equilibrium refolding kinetics of this naturally
occurring bistable RNA element, a single 6-13C-cytidine substitution
was introduced, and 13C-longitudinal exchange NMR spectroscopy was
1
applied. Integration of the H–13C-correlation peaks in a standard HSQC
spectrum yielded the equilibrium constant favoring fold B (KAB ¼3).
Subsequently, the forward and backward rates of the secondary structure
equilibrium were determined at 33 °C using an approach introduced
by analyzing longitudinal relaxation rates with and without exchange
contribution (Fig. 12B). The forward rate constant kAB amounts to
3.3ꢃ0.1 sꢁ1 and the rate kBA for the folding process from state B to
A to 1.4ꢃ0.3 sꢁ1.
8.5 Chemical Exchange Saturation Transfer
To characterize the nature of riboswitch RNA transitions on timescales
slower than CPMG but faster than ZZ exchange, CEST experiments
are carried out by varying the position of a very weak 13C radiofrequency
(rf ) B1 field and quantified indirectly using the intensities of the major
state (NMR-visible) correlations (Bouvignies & Kay, 2012a,2012b;
Fawzi, Ying, Ghirlando, Torchia, & Clore, 2011; Forse´n & Hoffman,
1963; Longhini et al., 2015; Vallurupalli, Bouvignies, & Kay, 2012;
Zhao, Hansen, & Zhang, 2014). As expected, when the B1 field position
coincides with or is close to the resonance frequency of the 13C resonant
peak in the ground state, the intensity of this ground state peak decreases
because of rf saturation effect (Forse´n & Hoffman, 1963). Significantly,
when the weak B1 field is applied close to the position of the
corresponding peak from the excited (NMR-invisible) state, the

Figure 12 (A) Competing terminator antiterminator RNA folds and the influence of preQ1 binding on the 2° structure equilibrium. (B) 13
longitudinal exchange spectrum at a mixing time of 200 ms and at 33 °C.
C

490
Christoph H. Wunderlich et al.
ground-state peak intensity also decreases due to the transfer of the rf per-
turbation from the excited state to the ground state by chemical exchange.
A plot of the intensity of the ground-state (observed) peak as a function of
the position of the B1 field indicates a pair of dips, with the larger dip at the
resonance frequency of the carbon in the ground state and the smaller dip
at the frequency of the corresponding 13C resonant peak in the excited
state (Fig. 13). Thus, a visual inspection of the plotted data readily reveals
that the NMR-invisible state is shifted into a carbon frequency region
expected for residues in a Watson–Crick base-pair configuration
(Fig. 13). Data were recorded at 16.4 T, and the CEST profiles were fitted
using the Bloch–McConnell equations assuming a two-site exchange pro-
cess (Longhini et al., 2015). Global fitting of CEST profile to a single two-
state model resulted in kex ¼687ꢃ24 sꢁ1 and minor excited population
pES ¼2.3ꢃ0.1%, and a 13C-chemical shift difference between ground state
1
and excited state of 3.3 ppm. With our selective labels, the large JC1 -C2
0
0
(ꢄ45 Hz) couplings do not complicate the measurements and need not
been taken explicitly into account in the data analysis of sugar (C10) CEST
profiles as required in previous studies using uniformly labeled RNA (Zhao
et al., 2014).
9. CONCLUSIONS
Here, we presented a broad and versatile 13C-labeling approach for
RNA using 6-13C-pyrimidine phosphoramidite building blocks or
chemoenzymatic coupling approach, which can be introduced into RNA
by oligonucleotide solid phase or enzymatic synthesis. The 6-13C-
pyrimidine labels are generally applicable to study conformational dynamics
and the refolding kinetics in RNAs comprising up to 30–40 nucleotides
using standard NMR techniques. For larger RNAs, the labeling pro-
tocol can be used in combination with enzymatic ligation strategies or
chemoenzymatic approaches as outlined. In that case, pulse sequences com-
prising a TROSY element are particularly useful. The building blocks are
currently used to site-specifically modify functional nucleic acids, such as
riboswitch aptamer domains or ribozymes, to give fundamental insights into
the functional dynamics of these RNAs. We anticipate that this methodology
of incorporating modified or labeled nucleotides into specifically designated
positions or regions of RNA is very general and will open up new avenues
for not only studying the structure and dynamics of large RNAs but will also
be useful in biotechnological applications such as RNA biosensors.