Synthesis and evaluation of an RNA editing substrate bearing 2′-deoxy-2′-mercaptoadenosine

Article abstract of DOI:10.1080/15257770902736459

The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in double stranded structure found in various RNA substrates, including mRNAs. Here we describe the synthesis of a phosphoramidite of 2′-deoxy-2′-mercaptoadenosine a

Full text of DOI:10.1080/15257770902736459

Nucleosides, Nucleotides and Nucleic Acids, 28:78–88, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770902736459
SYNTHESIS AND EVALUATION OF AN RNA EDITING SUBSTRATE
BEARING 2ꢀ-DEOXY-2ꢀ-MERCAPTOADENOSINE
Prasanna Jayalath,1 Subhash Pokharel,1,2 Eduardo Ve´liz,1 and Peter A. Beal1
1Department of Chemistry, University of California, Davis, California, USA
2Department of Chemistry, University of Utah, Salt Lake City, Utah, USA
The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine
2
in double stranded structure found in various RNA substrates, including mRNAs. Here we describe
the synthesis of a phosphoramidite of 2 ꢁ-deoxy-2 ꢁ-mercaptoadenosine and its incorporation into an
ADAR substrate. Surprisingly, no deamination product was observed with this substrate indicating
replacing the 2 ꢁ-OH with a 2 ꢁ-SH at the editing site is highly inhibitory. Modeling of nucleotide
binding into the active site suggests the side chain of T375 of human ADAR2 to be in proximity of
the 2 ꢁ-substituent. Mutation of this residue to cysteine caused a greater that 100-fold reduction in
deamination rate with the 2 ꢁ-OH substrate.
Keywords ADARs; RNA substrates; nucleotide binding
INTRODUCTION
The RNA editing adenosine deaminase enzymes (ADARs) convert
adenosines to inosines in various RNAs, including mRNAs. When this
reaction takes place within a coding sequence, the meaning of codons
can be altered.[1–3] Thus, ADARs play a pivotal role in the basic process
of information transfer that takes place during protein expression. A role
for ADARs in nervous system function is implied by the observation that
deletion of ADAR genes leads to significant behavioral defects in model
organisms.[4–6] Interestingly, in addition to the neurological phenotypes
observed, both ADAR1 and ADAR2 knockout mice die prematurely.[4,7]
Received 4 September 2008; accepted 3 December 2008.
P. A. B. acknowledges the National Institutes of Health for financial support in the form of grant
GM061115.
Eduardo Ve´liz is currently affiliated with the Math Science and Technology Division, Nova
Southeastern University, Fort Lauderdale, Florida, USA.
Address correspondence to Peter A. Beal, Department of Chemistry, University of California, Davis,
CA, 95616, USA. E-mail: beal@chem.ucdavis.edu
78

2 ꢁ-Deoxy-2 ꢁ-mercaptoadenosine in an RNA Editing Substrate
79
FIGURE 1 A) A model for nucleotide recognition in the human ADAR2 active site.[8] B) Structural
changes made for this study: 2ꢁ OH → 2ꢁ SH, T375 →T375C.
Thus, the precise role of ADAR activity in adult mammals remains to be
fully defined.
Structural and biochemical studies have shed light on how ADARs
recognize substrates and catalyze adenosine deamination in RNA.[8–14] In
general, members of the ADAR family contain multiple RNA-binding motifs
that bind double-stranded RNA substrates in addition to a zinc-containing
catalytic domain where deamination occurs.[8,15,16] Although no structures
of ADAR/RNA complexes have been reported, the structure of the human
ADAR2 deaminase domain alone has been solved by x-ray crystallography.[8]
Furthermore, modeling of AMP into the zinc-containing active site suggests
residues that may be involved in substrate recognition (Figure 1).[8] In
addition to the residues that bind zinc and the catalytic water molecule
(H394, C451, C516, and E396), R455 and T375 are present in the ADAR2
active site. The nucleotide binding model suggests R455 is positioned near
the purine N7 and T375 is near the 2ꢁ-hydroxyl group of the reactive
nucleotide (Figure 1). Studies in our laboratory with 7-substituted-8-aza-
7-deazaadenosine derivatives in RNA and residue 455 mutants of human
ADAR2 support this proposed model (Maydanovych, Jayalath, Pokharel,
Wang, Tantillo, and Beal, unpublished results). Furthermore, we hypoth-
esized that the close approach of the 2ꢁ substituent and the side chain
of the 375 residue in human ADAR2 might be used to create a covalent
linkage between the enzyme and substrate. For instance, the combination
ꢁ
of an A2 -SH-containing RNA and the T375C mutant of the enzyme might
lead to the formation of a disulfide bond between enzyme and substrate.
Such a stabilization of the enzyme/substrate complex would be useful in its
biophysical characterization. Making and testing these modifications inde-
pendently would also contribute to our understanding of structure/activity
relationships in the ADAR/RNA complex. Here we report the synthesis
of a phosphoramidite of 2ꢁ-deoxy-2ꢁ-mercaptoadenosine and its use in the

80
P. Jayalath et al.
ꢁ
generation and evaluation of an A2 -SH–containing ADAR substrate. We also
analyzed the effect of the T375C mutation on ADAR2 activity.
RESULTS AND DISCUSSION
Synthesis of the phosphoramidite of 2ꢁ-deoxy-2ꢁ-mercaptoadenosine
proceeded from the disiloxyl-protected arabinofuranosyladenine deriva-
tive 1 in analogy to published reports for related nucleosides (Scheme
1).[17,18] The 2ꢁ-triflate was prepared and used without purification in
an SN2 displacement with the sodium salt of tritylmercaptan generating
S-trityl compound 2 in 78% yield.[19,20] Removal of the disiloxyl group
with TBAF in THF provided intermediate 3. Proton NMR analysis of 3
revealed a 1ꢁH-2ꢁH J = 9.6 Hz, consistent with ribose configuration at the
2ꢁ position as expected for the SN2 displacement.[18] Protection of the ex-
ocyclic amino group as the dimethylacetamidine and the 5ꢁ-hydroxyl as the
DMT ether proceeded with good yields. Phosphitylation with 2-cyanoethyl-
(N,N -diisopropylamino)chlorophosphite gave the fully protected phospho-
SCHEME 1

2 ꢁ-Deoxy-2 ꢁ-mercaptoadenosine in an RNA Editing Substrate
81
FIGURE 2 Oligonucleotides used in this study. Abbreviations: A2 STr = 2ꢁ-deoxy-2ꢁ-mercaptoadenosine
ꢁ
protected as S-trityl; A2 SH = 2ꢁ-deoxy-2ꢁ-mercaptoadenosine.
ꢁ
ramidite 6 in 74% yield. This phosphoramidite was used in the solid phase
synthesis of oligoribonucleotides corresponding in sequence to that found
in the pre-mRNA encoding the B subunit of the glutamate receptor near
a known editing site (the R/G site).[21,22] Successful incorporation of the
modified nucleotide into RNA was confirmed by electrospray ionization
mass spectrometry (see Experimental section for details). Figure 2 shows
the different sequences of the oligonucleotides prepared for this study.
Oligonucleotide 9 was prepared for analysis of covalent complex formation
using gel mobility shift assays. Oligonucleotide 12 was generated for analysis
of the effect of 2ꢁ-SH modification on the adenosine deamination rate,
which is more readily assayed when the targeted nucleotide is at the 5ꢁ
end.[22]
Trityl was chosen as the protecting group for the 2ꢁ-mercaptan because
of its ability to survive the conditions of RNA synthesis and deprotection
of the common nucleotides and its lability in the presence of AgNO3, a
reagent known to be compatible with RNA.[17] Once the A2 -STr-containing
ꢁ
oligonucleotide was purified, removal of the S-trityl group was achieved
using AgNO3 essentially as described by Hamm and Piccirilli for cytidine-2ꢁ-
S-trityl-containing RNAs.[17] We confirmed these conditions were effective
at protecting group removal by monitoring the mobility of 32P-labeled
oligonucleotides in a polyacrylamide gel (Figure 3). After treatment with
ꢁ
FIGURE 3 Gel mobility assay to monitor the AgNO3-promoted S-trityl deprotection of an A2 STr
-
containing RNA (see Experimental section for conditions).

82
P. Jayalath et al.
FIGURE 4 Duplex RNA substrate with varying nucleotide X at a known editing site (GluR-B pre-mRNA
ꢁ
R/G site) prepared via hybridization of oligonucleotides 10 + 13 (X = A) or 12 + 13 (X = A2 -SH).
AgNO3 followed by DTT, the rate of migration of the oligonucleotide
through the gel increased consistent with the resulting decrease in molecu-
lar weight. This is apparent when comparing the mobility of oligonucleotide
8 (no AgNO3, -STr 2ꢁ substituent) to oligonucleotide 9 (+ AgNO3, -SH 2ꢁ
substituent) (Figure 3).
We evaluated the effect of replacing the editing site 2ꢁ-hydroxyl group
with a 2ꢁ-mercaptan using duplex structure designed to mimic the R/G
editing site of GluR B pre-mRNA formed by hybridization of either oligonu-
cleotides 10 or 12 with 13 (Figure 2 and Figure 4). Similar duplexes have
been shown previously to support editing by human ADAR2 in vitro. Indeed,
the rate of deamination for the 2ꢁ-OH containing substrate (10 + 13)
is similar to previous reports with the edited base at the duplex 5ꢁ end
(Table 1).[21,22] However, no product was observed with the 2ꢁ-SH substrate
under these conditions (12 + 13), even after prolonged incubation with
the enzyme. Previously, we had shown that although 2ꢁ-deoxyadenosine
and 2ꢁ-deoxy-2ꢁ-fluoroadenosine in RNA were deaminated by ADAR2 with
moderately reduced rates, 2ꢁ-O-methyl modification inhibited deamination
by more than two orders of magnitude.[23] Together these results indicate
that ADAR2 is highly sensitive to modifications at the 2ꢁ-position that are
more sterically demanding than the 2ꢁ-hydroxyl group, an effect that likely
arises from a clash with the T375 residue.
Our interpretation of the low reactivity of the 2ꢁ-SH substrate suggested
the ADAR2 reaction might also be sensitive to mutation at position 375.
Therefore, we evaluated the effect of the T375C mutation on the ADAR2
deamination rate measured in vitro. Indeed, a > 100-fold reduced deamina-
tion rate was observed with this mutant compared to wild type ADAR2 using
the 2ꢁ-OH substrate (10 + 13) (Table 1). It is interesting to note that the
loop of ADAR2 containing T375 is not found in the evolutionarily related
cytidine deaminases and it has been suggested that the adenosine selectivity
TABLE 1 Rate constants for the deamination of duplex substratesa
X
ADAR2
ADAR2 T375C
A
0.08 0.002 min−1
NDb
6.8 0.2 × 10−4 min−1
A-2ꢁSH
ND
aReactions were carried out with 250 nM enzyme, 25 nM RNA substrate.
bND = no product detected.

2 ꢁ-Deoxy-2 ꢁ-mercaptoadenosine in an RNA Editing Substrate
83
of the ADARs arises from the positioning of this loop to prevent cytidine in
RNA from entering the ADAR active site.[8] Thus, this part of the enzyme
appears to have evolved to sense the shape of its cognate substrate. As might
be expected from the results described above, no stable covalent complex
could be observed using electrophoretic mobility shift assays when duplexes
containing the 2ꢁ-SH substrate oligonucleotide 9 were incubated with the
ADAR2 T375C mutant under a variety of conditions.
In summary, we prepared a new phopshoramidite for incorporation of
2ꢁ-deoxy-2ꢁ-mercaptoadenosine into RNA. A duplex RNA mimic of a known
editing site containing 2ꢁ-deoxy-2ꢁ-mercaptoadenosine was evaluated as a
substrate for human ADAR2 in vitro with no deamination product detected.
This result, along with our previous observation that 2ꢁ-O-methlyladenosine
is an extremely poor ADAR2 substrate, suggests the ADAR reaction is highly
sensitive to 2ꢁ modifications more sterically demanding than a hydroxyl
group. Furthermore, we show here the ADAR2 reaction is also sensitive
to cysteine mutation at position 375, a residue likely in proximity to the
2ꢁ-substituent in the enzyme/substrate complex.
EXPERIMENTAL
General Synthetic Procedures
Glassware for all reactions was oven dried at 125◦C overnight and
cooled in a desiccator prior to use. Reactions were carried out under an
atmosphere of dry nitrogen when anhydrous conditions were necessary.
All reagents were purchased from commercial sources (Sigman/Aldrich,
St. Louis, MO, USA or Fischer Scientific, Pittsburgh, PA, USA) and were
used without further purification unless noted otherwise. Liquid reagents
are introduced by oven-dried microsyringes. Tetrahydrofuran was passed
through a column of activated aluminum.[24] Thin layer chromatography
(TLC) was performed with Merck (Whitehouse Station, NJ, USA) silica gel
60 F254 precoated TLC plates, eluting with the solvents indicated. Short
and long wave visualization was performed with a Mineralight multiband
ultraviolet lamp at 254 and 365 nm, respectively. Flash column chromatog-
raphy was performed on Mallinckrodt Baker silica gel 150 (60–200 mesh)
or using an ISCO CombiFlash Companion with Teledyne RediSep Rx
1
columns. H, 13C, and 31P nuclear magnetic resonance spectra of pure
compounds were acquired at 600, 125, and 121 MHz, respectively. Chemical
shifts reported in parts per million (ppm) in the reference to a solvent
peak. The abbreviations such as s, t, m, bs, dd, d stand for singlet, triplet,
multiplet, broad singlet, doublet of doublets and doublet. High-resolution
mass spectra were obtained at Department of Chemistry, University of Utah
or at University of California, Davis mass spectroscopy facility.

84
P. Jayalath et al.
3ꢁ,5ꢁ-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-2ꢁ-deoxy-2ꢁ-
(triphenylmethylthio)adenosine (2). Trifluoromethanesulfonyl chloride
(0.232 mL, 2.17 mmol) was added to a cold (0◦C) stirred solution of 9-[3ꢁ,5ꢁ-
O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-D-arabinofuranosyl]adenine
1 (1.01 g, 1.98 mmol) and DMAP (0.29 g, 2.37 mmol) in anhydrous CH2Cl2
(15 mL). The mixture was stirred at 0◦C for 3 hours and partitioned
between ice-cold AcOH/H2O (1:99) and CH2Cl2 (2 × 100 mL). The
combined organic phases were washed with ice-cold 5% (w/v) aqueous
NaHCO3 (100 mL), brine (150 mL), and dried over anhydrous Na2SO4.
The solvents were evaporated and the residue was dried under reduced
pressure for 6 hours. The above residue was dissolved in DMF (10 mL) and
added dropwise during 30 minutes to a stirred solution of triphenylmethyl
thiol (2.73 g, 9.9 mol) and NaH (0.2 g, 7.92 mmol) in DMF (15 mL) at
0◦C. The reaction mixture was stirred at 0◦C for 3 hours and allowed to
reach room temperature overnight. The reaction mixture was poured into
ice-cold water (150 mL) and extracted into hexane/ethyl acetate (3:7). The
combined organic layers were washed with brine and dried over anhydrous
Na2SO4 and evaporated under reduced pressure. Silica gel chromatography
(3% MeOH in CH2Cl2) of the residue gave compound (2) as a white solid
(1.87 g, 78%). 1H NMR (CDCl3 600 MHz): δ (ppm) 8.07 (s, 1H), 7.41–7.39
(m, 6H), 7.11–7.05 (m, 9H), 5.68 (bs, 2H), 5.50 (d, J = 3.0 Hz, 1H), 5.22
(t, J = 7.2 Hz, 1H), 4.14 (dd, J = 2.4, 7.2 Hz, 1H), 3.97–3.94 (m, 1H), 3.90
(d, J = 3.0, 12.6 Hz, 1H), 3.81 (d, J = 5.4, 12.6 Hz, 1H). 13C NMR (CDCl3
125 MHz): δ (ppm) 155.5, 152.7, 148.9, 144.7, 140.8, 129.6, 128.0, 126.9,
120.2, 90.3, 84.2, 71.8, 67.1, 61.9, 51.5, 17.6, 17.5, 17.49, 17.40, 17.39, 17.36,
17.3, 13.5, 13.3, 13.2, 12.9. ESIHRMS cald for C42H57N5O4SSi2 [M+H]+
768.3435, obsd 768.3457
2ꢁ-Deoxy-2ꢁ-triphenylmethylthioadenosine (3). Compound 2 (0.73 g,
0.95 mmol) was dissolved in anhydrous THF (25 mL) and treated with 1M
n-Bu4NF (1.5 mL, 1.5 mmol) in THF. The mixture was stirred at 0◦C for
3 hours under an argon atmosphere and concentrated. The crude product
was purified by column chromatography using silica gel and CH2Cl2/MeOH
(20:1) to give compound 3 as white solid (0.44 g, 90%). 1H NMR (CDCl3 600
MHz): δ (ppm) 7.95 (s, 1H), 7.89 (s, 1H), 7.12–7.11 (m, 6H), 7.01–6.97 (m,
9H), 5.71 (d, J = 9.6 Hz, 1H), 4.04 (s, 1H), 3.77 (dd, J = 4.2, 10.2 Hz, 1H),
3.68 (dd, J = 1.8, 13.2 Hz, 1H), 3.3 (d, J = 12.6 Hz, 1H). 13C NMR (CDCl3
125 MHz): δ (ppm) 156.1, 152.2, 148.3, 143.9, 141.0, 128.9, 128.1, 127.1,
90.3, 88.4, 88.3, 72.0, 68.0, 63.2, 53.3, 53.2. ESIHRMS cald for C29H27N5O3S
[M+H]+ 526.1913, obsd 526.1912.
N-(Dimethylacetamidine)-2ꢁ-deoxy-2ꢁ-triphenylmethylthioadenosine (4).
To a stirred solution of 3 (0.26 g, 0.49 mmol) in 7 mL of CH2Cl2/MeOH
(4:3) was added N,N -dimethylacetamide dimethyl acetal (0.24 mL, 1.45
mmol) and stirring was continued overnight at room temperature. The
solvents were evaporated under reduced pressure and the resulting residue

2 ꢁ-Deoxy-2 ꢁ-mercaptoadenosine in an RNA Editing Substrate
85
was purified on silica gel column chromatography (CH2Cl2/MeOH (30:1)
1
to give compound 4 (0.24 g) in 84% yield. H NMR (CDCl3 600 MHz): δ
(ppm) 8.33 (s, 1H), 7.98 (s, 1H), 7.18–7.17 (m, 6H), 7.09–7.02 (m, 9H),
6.50 (dd, J = 1.2, 12.0 Hz, 1H), 5.76 (d, J = 10.2 Hz, 1H), 4.10 (s, 1H), 4.03
(d, J = 4.2, 10.2 Hz, 1H), 3.79 (d, J = 13.2 Hz, 1H), 3.40 (t, J = 12.6 Hz,
1H), 3.29 (bs, 3H), 3.16 (bs, 3H), 2.74 (d, J = 4.2 Hz, 1H). 13C NMR (CDCl3
125 MHz): δ (ppm) 162.2, 161.0, 152.5, 149.9, 143.9, 141.9, 129.05, 129.0,
128.3, 128.0, 127.2, 90.2, 88.1, 71.9, 67.9, 63.6, 53.6, 46.4, 38.8, 38.7, 17.8,
11.7. ESIHRMS calcd for C33H35N6O3S [M+H]+ 595.2491, obsd 595.2484.
N-(Dimethylacetamidine)-5ꢁ-O-(4,4ꢁ-dimethoxytriphenylmethyl)-2ꢁ-
deoxy-2ꢁ-triphenylmethylthioadenosine (5). A suspension of 4 (155 mg, 0.26
mmol), and N,N -diisopropylethylamine (50 µL, 313 mmol) in anhydrous
pyridine (1 mL) is treated with 4,4ꢁ-dimethoxytriphenylmethyl chloride
(112 mg, 313 mmol). After stirring at room temperature overnight the
reaction mixture was diluted with EtOAc (25 mL) and washed with
saturated aqueous NaHCO3. The organic layer was removed and dried
over anhydrous Na2SO4 and evaporated under reduced pressure. Silica gel
chromatography (EtOAc) of the residue gave compound (5) as a white solid
(168 mg, 72%). 1H NMR (CD2Cl3 600 MHz): δ (ppm) 8.36 (s, 1H), 8.10 (s,
1H), 7.27–7.09 (m, 24H), 6.70–6.68 (m, 4H), 6.11 (d, J = 9.6 Hz, 1H), 4.07
(dd, J = 4.8, 9.6 Hz, 1H), 4.02 (t, J = 4.2 Hz, 1H), 4.17–4.13 (m, 1H), 3.77
(s, 3H), 3.76 (s, 3H), 3.27 (bs, 3H), 3.22 (dd, J = 4.2, 10.8 Hz, 1H), 3.19
(bs, 3H), 3.03 (dd, J = 3.0, 10.2 Hz, 1H), 2.58 (d, J = 4.8 Hz, 1H), 2.23
(s, 3H). 13C NMR (CD2Cl2 125 MHz): δ (ppm) 158.8, 153.1, 151.8, 144.7,
144.1, 140.8, 135.99, 135.98, 130.2, 130.19, 129.2, 128.5, 128.4, 127.9, 127.3,
127.0, 126.7, 113.2, 86.6, 86.5, 85.4, 70.9, 67.5, 63.9, 55.5, 54.8, 45.0, 17.5.
ESIHRMS calcd for C33H35N6O3S [M+H]+ 897.3798, obsd 897.3816.
N-(Dimethylacetamidine)-5ꢁ-O-(4,4ꢁ-dimethoxytriphenylmethyl)-
3ꢁ-O-[(2-cyanoethoxy)(N,N-diisopropylamino)phosphino]-2ꢁ-deoxy-2ꢁ-
triphenylmethylthioadenosine (6). A solution of 5 (92 mg, 0.1 mmol),
DMAP (13 mg, 0.11 mmol) and N,N -diisopropylethylamine (39 µL,
0.23 mmol) in anhydrous THF (0.8 mL) was treated dropwise with
2-cyanoethyl-(N,N -diisopropylamino)chlorophosphite (53 µL, 0.23 mmol).
After stirring at room temperature for 4 hours the reaction mixture was
diluted with EtOAc (25 mL) and washed with 5% (w/v) aqueous NaHCO3.
The organic layer was removed and dried over anhydrous Na2SO4
and evaporated under reduced pressure. Silica gel chromatography
(CH2Cl2/TEA; 99:1) of the residue gave compound (6) as a white foam (82
mg, 74%). 31P NMR (CD2Cl2, 121 MHz); δ (ppm) 153.10, 150.55. ESIHRMS
calcd for C63H69N8O6PS [M+H]+ 1097.3187, obsd. 1097.3418.
Synthesis, purification and mass spectrometric analysis of RNA. RNA
oligonucleotides were synthesized on a ABI 394 synthesizer (DNA/Peptide
Core Facility, University of Utah, Salt Lake City) using 5ꢁ-DMT protected
β-cyanoethyl phosphoramidites on a 1 µmol scale with coupling times of

86
P. Jayalath et al.
25 minutes for more efficient coupling. The RNA oligonucleotides after
polyacrylamide gel electrophoresis purification were analyzed by mass spec-
trometry at the Mass Spectrometry and Proteomics Core Facility, University
of Utah. Oligonucleotides were analyzed in an acetonitrile solution at pH
= 11 by electrospray ionization on a Quattro-II mass spectrometer (Micro-
mass, Inc., Beverly, MA, USA). The multiply-charged molecular ions were
deconvoluted into a molecular mass spectrum using MaxEnt (Micromass,
Inc.) software. The Quattro-II instrument was operated using Masslynx
software version 3.4 (Micromass, Inc.). ESI-MS analysis results: 2ꢁ-deoxy-2ꢁ-
triphenylmethylthioadenosine 26 mer RNA oligonucleotide 8 calcd. (M-
H)− 9003.7, obsd 9003.2; 2ꢁ-deoxy-2ꢁ-triphenylmethylthioadenosine 22 mer
RNA oligonucleotide 11 calcd. (M-H)− 7427.7; obsd 7427.5.
General Biochemical Procedures. Distilled, deionized water was used for
all aqueous reactions and dilutions. Biochemical reagents were purchased
from Sigma/Aldrich unless otherwise noted. Common enzymes were pur-
chased from Roche (Nutley, NJ, USA), Promega, or New England Biolab
(Ipswich, MA, USA). ADAR2 overexpression and purification were carried
out as previously described.[25] 5ꢁ-End labeling of RNA oligonucleotides
was accomplished using T4 polynucleotide kinase and [γ -32P]ATP, 6000
Ci/mmol (Perkin-Elmer Life Sciences, Waltham, MA, USA) as previously
described.[22]
Removal of S-trityl group to generate oligonucleotides 9 and 12. To
remove the S-trityl protecting group from the RNA oligonucleotide, 750
pmol of RNA dissolved in 7.3 µL of 100 mM TEAA, pH = 6.5 was treated
with 1 µL of 1M aqueous silver nitrate solution. This reaction was mixed
at room temperature by shaking in orbital shaker for 45 minutes. Dithio-
threitol (2 µL of 1M aqueous solution) was added to the reaction and
further mixed by shaking an additional five minutes. TEAA, pH = 6.5 (50
µL of 100 mM) was added, mixed and centrifuged to remove silver/DTT
precipitate. The supernatant containing the deprotected oligonucleotide
was transferred to a new Eppendorf tube. The precipitate was washed with
50 µL of 100 mM TEAA, pH = 6.5 buffer. The supernatants containing
the oligonucleotide were combined. Prior to end labeling, the buffer was
exchanged for 50 mM BisTris, pH = 7.0, 100 mM NaCl, 10 mM MgCl2 with
100 µM TCEP using a Microcon-3 concentrator. For monitoring the depro-
tection reaction, end labeled RNAs 7, 8 and 9 were resolved on a 19% PAGE
gel and visualized using storage phosphor autoradiography (Figure 3).
Preparation of duplex RNA ADAR substrate and deaminase assay. 5ꢁ-
End 32P-labeled duplex RNA substrates were prepared as previously de-
ꢁ
scribed with a minor modification as follows. End labeled A2 -SH-containing
RNA oligonucleotide (12) was mixed with a two-fold excess of unlabeled
12 and 10-fold excess of unlabeled complement strand (13) in pres-
ence of 5 mM DTT. ADAR activity assays were carried out as previously
described.[22]

2 ꢁ-Deoxy-2 ꢁ-mercaptoadenosine in an RNA Editing Substrate
REFERENCES
87
1. Burns, C.M.; Chu, H.; Rueter, S.M.; Hutchinson, L.K.; Canton, H.; Sanders-Bush, E.; Emeson, R.B.
Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 1997, 387, 303–
308.
2. Grosjean, H.; Benne, R. Modification and Editing of RNA. ASM Press, Washington, DC, 1998.
3. Higuchi, M.; Single, F.N.; Koehler, M.; Sommer, B.; Sprengel, R.; Seeburg, P.H. RNA editing of AMPA
receptor subunit GluR-B: A base-paired intron-exon structure determines position and efficiency.
Cell 1993, 75, 1361–1370.
4. Higuchi, M.; Maas, S.; Single, F.N.; Hartner, J.; Rozov, A.; Burnashev, N.; Feldmeyer, D.; Sprengel,
R.; Seeburg, P.H. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the
RNA-editing enzyme ADAR2. Nature 2000, 406, 78–81.
5. Palladino, M.J.; Keegan, L.P.; O’Connell, M.A.; Reenan, R.A. A-to-I Pre-mRNA Editing in Drosophila
is primarily involved in adult nervous system function and integrity. Cell 2000, 102(4), 437–
449.
6. Tonkin, L.A.; Saccomanno, L.; Morse, D.P.; Brodigan, T.; Krause, M.; Bass, B.L. RNA editing
by ADARs is important for normal behavior in Caenorhabditis elegans. EMBO J. 2002, 21(22),
6025–6035.
7. Wang, Q.; Khillan, J.; Gadue, P.; Nishikura, K. Requirement of the RNA editing deaminase ADAR1
gene for embryonic erythropoiesis. Science 2000, 290(5497), 1765–1768.
8. MacBeth, M.R.; Schubert, H.L.; VanDemark, A.P.; Lingam, A.T.; Hill, C.P.; Bass, B.L. Inositol
Hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 2005,
309(5740), 1534–1539.
9. MacBeth, M.R.; Lingam, A.T.; Bass, B.L. Evidence for auto-inhibition by the N terminus of hADAR2
and activation by dsRNA binding. RNA 2004, 10(10), 1563–1571.
10. Cho, D.-S.C.; Yang, W.; Lee, J.T.; Shiekhattar, R.; Murray, J.M.; Nishikura, K. Requirement of
dimerization for RNA editing activity of adenosine deaminases acting on RNA. J. Biol. Chem. 2003,
278(19), 17093–17102.
11. Stefl, R.; Xu, M.; Skrisovska, L.; Emeson, R.B.; Allain, F.H.-T. Structure and specific RNA binding of
ADAR2 double-stranded RNA binding motifs p345. Structure 2006, 14, 345–355.
12. Dawson, T.R.; Sansam, C.L.; Emeson, R.B. Structure and sequence determinants required for the
RNA editing of ADAR2 substrates. J. Biol. Chem. 2004, 279, 4941–4951.
13. Maydanovych, O.; Easterwood, L.M.; Cui, T.; Veliz, E.A.; Pokharel, S.; Beal, P.A. Probing adenosine-
to-inosine editing reactions using RNA-containing nucleoside analogs. Methods Enzymol. 2007, 424,
369–386.
14. Stephens, O.M.; Haudenschild, B.L.; Beal, P.A. The binding selectivity of ADAR2ꢁs dsRBMs con-
tributes to RNA-editing site selectivity. Chem. Biol. 2004, 11(9), 1239–1250.
15. Bass, B.L.; Weintraub, H. A developmentally regulated activity that unwinds RNA duplexes p607.,
Cell 1987, 48(4), 607–613.
16. Doyle, M.; Jantsch, M.F. Distinct in vivo roles for double-stranded RNA-binding domains of the
Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting. J. Cell Biol. 2003, 161(2), 309–
319.
17. Hamm, M.L.; Piccirilli, J.A. Incorporation of 2ꢁ-deoxy-2ꢁ-mercaptocytidine into oligonucleotides via
phosphoramidite chemistry. J. Org. Chem. 1997, 62, 3415–3420.
18. Marriott, J.H.; Mottahedeh, M.; Reese, C.B. Synthesis of 2ꢁ-thioadenosine. Carbohydr. Res. 1991, 216,
257–269.
19. Robbins, M.J.; Hawrelak, S.D.; Hernandez, A.E.; Wnuk, S.F. Nucleic acid related compounds.
71. Efficient general synthesis of purine (amino, azido, and triflate)-sugar nucleoside. Nucleosides
Nucleotides 1992, 11(2–4), 821–834.
20. Gruen, M.; Becker, C.; Beste, A. Synthesis of 2ꢁ-Iodo- and 2ꢁ-bromo-ATP and GTP analogues as
potential phasing tools for x-ray crystallography. Nucleosides Nucleotides 1999, 18(1), 137–151.
21. Easterwood, L.M.; Veliz, E.A.; Beal, P.A. Demethylation of 6-O-methylinosine by an RNA-editing
adenosine deaminase. J. Am. Chem. Soc. 2000, 122, 11537–11538.
22. Stephens, O.M.; Yi-Brunozzi, H.Y.; Beal, P.A. Analysis of the RNA-editing reaction of ADAR2 with
structural and fluorescent analogs of the GluR-B R/G editing site. Biochemistry 2000, 39, (40),
12243–12251.

88
P. Jayalath et al.
23. Yi-Brunozzi, H.Y.; Easterwood, L.M.; Kamilar, G.M.; Beal, P.A. Synthetic substrate analogs for the
RNA-editing adenosine deaminase ADAR-2. Nucleic Acids Res. 1999, 27(14), 2912–2917.
24. Pangborn, A.B.; Giardello, M.A.; Grubbs, R.H.; Rosen, R.K.; Timmers, F.J. Safe and convenient
procedure for solvent purification. Organometallics 1996, 15(5), 1518–1520.
25. MacBeth, M.R.; Bass, B.L. Large-scale overexpression and purification of ADARs from saccha-
romyces cerevisiae for biophysical and biochemical studies. Methods Enzymol. 2007, 424, 319–331.