A transition state analogue for an RNA-editing reaction

Article abstract of DOI:10.1021/ja0472073

Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-a

Full text of DOI:10.1021/ja0472073

Published on Web 08/19/2004
A Transition State Analogue for an RNA-Editing Reaction
Brittany L. Haudenschild,† Olena Maydanovych,† Eduardo A. Ve´liz,†
Mark R. Macbeth,‡ Brenda L. Bass,‡ and Peter A. Beal*,†
Contribution from the Department of Chemistry, UniVersity of Utah, 315 South 1400 East,
Salt Lake City, Utah 84112, and Howard Hughes Medical Institute, Department of Biochemistry,
UniVersity of Utah, 50 North Medical DriVe, Salt Lake City, Utah 84132
Received May 12, 2004; E-mail: beal@chem.utah.edu
Abstract: Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at
the corresponding position. Because inosine is decoded as guanosine during translation, this modification
can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double
bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction
catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use
in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate
receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight
binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-
affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2’s
two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue
into the sequence/structural context of a known editing site. These results advance our understanding of
substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR‚
RNA complexes.
Introduction
an ADAR homologue in Drosophila melanogaster leads to a
morphologically normal fly with dramatic behavioral defects,
RNA editing refers to a wide variety of modification reactions
that change the sequence of an RNA molecule from that encoded
by the gene sequence.1 Deamination at C6 of adenosine (A) in
RNA catalyzed by the ADAR family of enzymes generates
inosine (I) at the corresponding nucleotide position.2 Because
inosine is decoded as guanosine during translation, this RNA
modification can lead to codon changes and the introduction of
amino acids into a gene product not encoded in the gene.3,4
Inosines also appear in the untranslated regions of numerous
mRNAs,5,6 and these may regulate whether a double-stranded
RNA enters the RNA interference pathway.7 Interestingly,
several targets of the A to I modification reaction are mRNAs
encoding receptors for neurotransmitters, and editing appears
to be necessary for a properly functioning central nervous system
in metazoa.3,4,8-12 For instance, deletion of the gene encoding
such as tremors, uncoordinated locomotion, and an inability to
fly or jump.11 Likewise, C. elegans containing deletion muta-
tions in each ADAR gene show defects in behaviors such as
chemotaxis.12 Thus, it appears the protein structural diversity
that arises through editing of mRNA by adenosine deamination
is used in the nervous system to produce complex behavior.
The study of the effect changes in levels of RNA editing have
on human behavior has only recently been initiated and appears
to link abnormal editing to psychiatric disorders.13-15 During
our ongoing studies to define the mechanism of the ADAR
reaction and discover inhibitors of this process, we have
endeavored to develop a mimic of the reaction transition
state.16-20 Such a mimic would aid in the structural character-
(10) Wang, Q.; Khillan, J.; Gadue, P.; Nishikura, K. Science 2000, 290, 1765-
1768.
† Department of Chemistry.
‡ Department of Biochemistry.
(11) Palladino, M. J.; Keegan, L. P.; O’Connell, M. A.; Reenan, R. A. Cell
2000, 102, 437-449.
(1) Grosjean, H.; Benne, R. Modification and Editing of RNA; ASM Press:
Washington, DC, 1998.
(12) Tonkin, L. A.; Saccomanno, L.; Morse, D. P.; Brodigan, T.; Krause, M.;
Bass, B. L. EMBO J. 2002, 21, 6025-6035.
(2) Bass, B. L.; Nishikura, K.; Keller, W.; Seeburg, P. H.; Emeson, R. B.;
O’Connell, M. A.; Samuel, C. E.; Herbert, A. RNA 1997, 3, 947-949.
(3) Burns, C. M.; Chu, H.; Rueter, S. M.; Hutchinson, L. K.; Canton, H.;
Sanders-Bush, E.; Emeson, R. B. Nature 1997, 387, 303-308.
(4) Higuchi, M.; Single, F. N.; Kohler, M.; Sommer, B.; Sprengel, R.; Seeburg,
P. H. Cell 1993, 75, 1361-1370.
(13) Niswender, C. M.; Herrick-Davis, K.; Dilley, G. E.; Meltzer, H. Y.;
Overholser, J. C.; Stockmeier, C. A.; Emeson, R. B.; Sanders-Bush, E.
Neuropsychopharmacology 2001, 24, 478-491.
(14) Iwamoto, K.; Kato, T. Neurosci. Lett. 2003, 346, 169-172.
(15) Gurevich, I.; Tamir, H.; Arango, V.; Dwork, A. J.; Mann, J. J.; Schmauss,
C. Neuron 2002, 34, 349-356.
(5) Morse, D. P.; Bass, B. L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6048-
6053.
(16) Yi-Brunozzi, H.-Y.; Easterwood, L. M.; Kamilar, G. M.; Beal, P. A. Nucleic
Acids Res. 1999, 27, 2912-2917.
(6) Morse, D. P.; Aruscavage, P. J.; Bass, B. L. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 7906-7911.
(17) Stephens, O. M.; Yi-Brunozzi, H.-Y.; Beal, P. A. Biochemistry 2000, 39,
12243-12251.
(7) Tonkin, L. A.; Bass, B. L. Science 2003, 302, 1725.
(8) Hoopengardener, B.; Bhalla, T.; Staber, C.; Reenan, R. Science 2003, 301,
832-836.
(18) Yi-Brunozzi, H.-Y.; Stephens, O. M.; Beal, P. A. J. Biol. Chem. 2001,
276, 37827-37833.
(9) Higuchi, M.; Maas, S.; Single, F. N.; Hartner, J.; Rozov, A.; Burnashev,
N.; Feldmeyer, D.; Spengle, R.; Seeburg, P. H. Nature 2000, 406, 78-81.
(19) Easterwood, L. M.; Veliz, E. A.; Beal, P. A. J. Am. Chem. Soc. 2000, 122,
11537-11538.
9
10.1021/ja0472073 CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 11213-11219
11213

A R T I C L E S
Haudenschild et al.
Figure 1. Proposed mechanism for the ADAR-catalyzed RNA-editing reaction. Hydration of adenosine forms a tetrahedral intermediate, and loss of ammonia
from this structure generates the inosine-containing product.
ization of the protein-RNA complex and could lead to new
approaches for inhibiting editing activity in vivo.
The development of an effective transition state analogue that
binds an ADAR with high affinity requires knowledge of the
RNA substrate recognition properties of the enzyme and the
deamination reaction mechanism. ADARs recognize their
substrates, at least in part, via an RNA-binding domain
containing multiple copies of the double-stranded RNA binding
Figure 2. Hydration of 8-azanebularine in RNA generates a mimic of the
proposed transition state for the ADAR RNA-editing reaction.
motif (dsRBM).21 Consistent with the involvement of dsRBMs,
duplex RNA secondary structure in the substrate is a requirement
for the ADAR-catalyzed reaction.22 ADARs also have conserved
Aza substitution at the 8-position of the purine ring system
sequences similar to the consensus sequence that makes up the
increases its susceptibility to covalent hydration.32 Thus, the
active site of cytidine deaminases (CDAs).23,24 CDA uses a zinc-
increased rate of deamination for 8-azaadenosine-containing
activated water molecule to carry out deamination of its
RNAs suggests that hydration is rate limiting for the ADAR2
nucleoside substrate via attack at C4 of the pyrimidine and loss
reaction with these substrates. Consistent with the large 8-aza
of ammonia.25 Using the comparison to CDAs, amino acids
effect observed for the ADAR2 reaction, 8-azanebularine, an
identified as possible active site residues for the ADARs,
analogue with a hydrogen in place of the C6 amino group,
including putative zinc-binding ligands and a residue involved
inhibits the reaction at high concentrations (IC50 ) 15 mM).20
8-Azanebularine inhibition can be explained if one considers
in proton transfers (E396 in ADAR2), have been altered by site-
directed mutagenesis with a corresponding loss of editing
that, once bound in the ADAR2 active site, the nucleoside can
activity.26,27 It is believed that once an ADAR recognizes its
be hydrated to form a good mimic of the transition state for the
duplex RNA substrate, it flips the reactive nucleotide into an
hydrolytic deamination of adenosine (Figure 2). However,
active site similar to that found in CDAs.17,28 In analogy to the
because this nucleoside lacks the duplex RNA structure found
mechanism of CDA, a metal-bound hydroxide ion then attacks
in RNA-editing substrates, the interaction with the enzyme is
the purine ring to form a tetrahedral intermediate (Figure 1).
weak. For the generation of a high-affinity ligand for the
This intermediate collapses, ejecting ammonia and the inosine-
enzyme, it would likely be necessary to incorporate 8-azanebu-
containing product. Nucleoside analogues that mimic the
larine into an RNA structure recognized by ADAR2.
transition state leading to the tetrahedral intermediate, including
Here, we report the synthesis of a phosphoramidite of
compounds that are susceptible to covalent hydration of the
8-azanebularine and its use in the preparation of an inter-
pyrimidine ring, are excellent inhibitors of CDAs.29,30 This
molecular duplex RNA similar to a hairpin structure found at a
includes 5-fluorozebularine, which has been shown by X-ray
known editing site for ADAR2. The binding properties of the
modified RNAs were analyzed in gel mobility shift experiments
crystallography to bind CDA as the covalent hydrate.31
8-Azaadenosine substitution in various RNA substrates ac-
using deletion and point mutants of ADAR2. These studies
celerates the rate of deamination at these sites by ADAR2.20
indicate that a tight binding ligand for an ADAR can be
generated by incorporation of 8-azanebularine into a duplex
(20) Veliz, E. A.; Easterwood, L. M.; Beal, P. A. J. Am. Chem. Soc. 2003, 125,
10867-10876.
RNA. The observed high-affinity binding is dependent on a
functional ADAR2 active site, the presence of dsRBM II, and
correct placement of the nucleoside analogue into the sequence/
structural context of a known editing site. These results have
implications for future structural studies of ADAR‚RNA
complexes and in the development of inhibitors of adenosine
deamination RNA editing.
(21) Doyle, M.; Jantsch, M. F. J. Struct. Biol. 2003, 140, 147-153.
(22) Bass, B. L.; Weintraub, H. Cell 1987, 48, 607-613.
(23) Kim, U.; Wang, Y.; Sanford, T.; Zeng, Y.; Nishikura, K. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 11457-11461.
(24) Melcher, T.; Maas, S.; Herb, A.; Sprengel, R.; Seeburg, P. H.; Higuchi,
M. Nature 1996, 379, 460-464.
(25) Carter, C. W. Biochemie 1995, 77, 92-98.
(26) Lai, F.; Drakas, R.; Nishikura, K. J. Biol. Chem. 1995, 270, 17098-17105.
(27) Cho, D.-S.; Yang, W.; Lee, J. T.; Shiekhattar, R.; Murray, J. M.; Nishikura,
K. J. Biol. Chem. 2003, 278, 17093-17102.
(28) Hough, R. F.; Bass, B. L. RNA 1997, 3, 356-370.
(29) Liu, P. S.; Marquez, V. E.; Kelley, J.; Driscoll, J. S. J. Org. Chem. 1980,
45, 5225-5227.
Experimental Section
General Synthetic Procedures. All reagents were purchased from
Sigma/Aldrich or Fischer Scientific and were used without further
(30) Jeong, L. S.; McCormack, J. J.; Cooney, D. A.; Hao, Z.; Marquez, V. E.
J. Med. Chem. 1998, 41, 2572-2578.
(31) Betts, L.; Xiang, S.; Short, S. A.; Wolfenden, R.; Carter, C. W. J. Mol.
Biol. 1994, 235, 635-656.
(32) Erion, M. D.; Reddy, M. R. J. Am. Chem. Soc. 1998, 120, 3295-3304.
9
11214 J. AM. CHEM. SOC. VOL. 126, NO. 36, 2004

RNA-Editing Transition State Analogue
A R T I C L E S
purification unless noted otherwise. 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. Liquid reagents were introduced
by oven-dried microsyringes. Tetrahydrofuran was distilled from sodium
metal and benzophenone; acetonitrile was distilled from CaH2. Thin-
layer chromatography (TLC) was performed with Merck silica gel 60
F254 precoated TLC plates, eluting with the solvents indicated. Short-
and long-wave visualization was performed with a Mineralight multi-
band ultraviolet lamp at 254 and 365 nm, respectively. Flash column
chromatography was performed on Mallinckrodt Baker silica gel 150
10% CH3OH/CHCl3) to give a light-yellow soft solid (54 mg, 81%).
Spectroscopic data agreed with reported values.34
5′-O-(4,4′-Dimethoxytrityl)-8-azanebularine (5). 8-Azanebularine
ribonucleoside (360 mg, 1.42 mmol) was dissolved in freshly distilled
THF (15 mL). Anhydrous pyridine (690 µL, 8.53 mmol), 4,4′-
dimethoxytrityl chloride (530 mg, 1.56 mmol), and AgNO3 (265.7 mg,
1.56 mmol) were added sequentially to this solution. The reaction
mixture was stirred at room temperature overnight. The mixture was
diluted with EtOAc (25 mL), filtered, and washed with saturated
aqueous NaHCO3 (1 × 40 mL). The organic layer was dried (Na2-
SO4), filtered, and concentrated under reduced pressure. The crude
product was purified by flash column chromatography (DCM/MeOH/
1
(60-200 mesh). H, 13C, and 31P nuclear magnetic resonance spectra
1
of pure compounds were performed at 300, 75, and 121 MHz,
respectively. Chemical shifts are reported in parts per million in
reference to the solvent peak. Chemical shifts for phosphorus NMR
are reported in parts per million using 85% phosphoric acid as an
external standard. The abbreviations s, d, dd, t, and m represent singlet,
doublet, doublet of doublets, triplet, and multiplet, in that order.
Reversed-phase HPLC using a C-18 Vydac column, 218TP510, 5 µm
particle size, 1 × 2.5 cm was performed with a Waters 600E system
controller. UV peaks were monitored on a Waters 490 programmable
multiwavelength detector at 260 nm. All HRFABMS spectra were
obtained on a Finnigan MAT 95. All MALDI analyses were performed
at the University of Utah Mass Spectrometry and Proteomics Core
Facility on a Voyager-DE STR MALDI mass spectrometer.
TEA 98:1:1) to give a light-orange foam (511 mg, 65%). H NMR
(CD2Cl2, 300 MHz): δ (ppm) 9.54 (s, 1H), 9.13 (s, 1H), 7.37-7.34
(m, 2H), 7.26-7.23 (m, 4H), 7.19-7.16 (m, 3H), 6.73 (dd, J ) 9, 3
Hz, 4H), 6.59 (d, J ) 6 Hz, 1H), 5.18-5.16 (m, 1H), 4.82-4.78 (m,
1H), 4.35-4.30 (m, 1H), 3.74 (s, 6H), 3.40 (dd, J ) 9, 3 Hz, 1H),
3.31-3.26 (m, 2H). 13C NMR (CD2Cl2, 75 MHz): δ (ppm) 159.0,
156.9, 152.3, 149.6, 145.3, 136.5, 136.3, 136.2, 130.5, 128.5, 128.2,
127.2, 113.5, 90.3, 86.7, 84.7, 74.6, 72.1, 64.1, 55.7.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)-8-aza-
nebularine (6). 5′-O-(4,4′-Dimethoxytrityl)-8-azanebularine (460 mg,
0.828 mmol) was dissolved in freshly distilled THF (10 mL). Triethy-
lamine (219 µL, 1.57 mmol) was added to the solution followed by
the addition of TBDMSCl (137 mg, 0.911 mmol). After 5 min, AgNO3
(155 mg, 0.911 mmol) was added to the mixture. After being stirred
for 8 h at room temperature, the reaction mixture was diluted with
EtOAc (30 mL), filtered, and washed with saturated aqueous NaHCO3
(1 × 35 mL). The organic layer was dried (Na2SO4), filtered, and
concentrated under reduced pressure. The crude product was purified
by flash column chromatography on silica gel (EtOAc/hexanes 1:8) to
2′,3′,5′-Tri-O-acetyl-8-azaadenosine (1) was synthesized according
to a literature procedure. Spectroscopic data agreed with reported
values.33
2′,3′,5′-Tri-O-acetyl-6-bromo-8-azanebularine (2). 2′,3′,5′-Tri-O-
acetyl-8-azaadenosine (470 mg, 1.19 mmol) was dissolved in freshly
distilled acetonitrile (40 mL). To this solution were added tBuONO
(1.43 mL, 11.9 mmol) and TMS-Br (315 µL, 2.38 mmol). The reaction
mixture was allowed to stir at 0 °C for 1 h. The mixture was diluted
with EtOAc (15 mL) and washed with water (1 × 15 mL) and brine (1
× 15 mL). The organic layer was dried (Na2SO4), filtered, and
concentrated under reduced pressure. The crude product was purified
by flash column chromatography (CHCl3 followed by CHCl3/CH3OH
99:1) to give a white foam (328 mg, 60%). 1H NMR (CDCl3, 300
MHz): δ (ppm) 8.88 (s, 1H), 6.62 (d, J ) 3 Hz, 1H), 6.17-6.14 (m,
1H), 5.80 (t, J ) 6 Hz, 1H), 4.53-4.41 (m, 1H), 4.23-4.18 (m, 2H),
2.13 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H). 13C NMR (CDCl3, 75 MHz):
δ (ppm) 170.3, 169.4, 169.1, 155.7, 148.6, 146.3, 136.7, 87.8, 81.0,
73.0, 70.6, 62.5, 20.5, 20.3, 20.2. HRFABMS: calcd for C15H17BrN5O7
(M + H)+ 458.0312, obsd 458.0306.
2′,3′,5′-Tri-O-acetyl-8-azanebularine (3). 2′,3′,5′-Tri-O-acetyl-6-
bromo-8-azanebularine (200 mg, 0.436 mmol) was dissolved in
methanol (7 mL). 10% Pd/C (12 mg) and anhydrous NaOAc (72 mg,
0.873 mmol) were added to the solution. The flask was evacuated and
refilled with hydrogen gas. The mixture was shaken in a Parr apparatus
for 8 h under 2.5 atm of pressure. The mixture was filtered through
Celite, and the residue was washed with methanol. The filtrate was
concentrated under reduced pressure. The residue was redissolved in
EtOAc (4 mL) and washed with brine (1 × 3 mL). The organic layer
was dried (Na2SO4), filtered, and concentrated under reduced pressure.
The crude product was purified by flash column chromatography (CH2-
Cl2) to give a light-yellow syrup (133 mg, 80%). Spectroscopic data
agreed with reported values.34
1
give a white foam (234 mg, 42%). H NMR (CD2Cl2, 300 MHz): δ
(ppm) 9.61 (s, 1H), 9.17 (s, 1H), 7.49-7.46 (m, 2H), 7.36 (dd, J ) 9,
3 Hz, 4H), 7.27-7.20 (m, 3H), 6.82-6.77 (m, 4H), 6.57 (d, J ) 6 Hz,
1H), 5.41-5.38 (m, 1H), 4.56-4.51 (m, 1H), 4.37-4.30 (m, 1H), 3.77
(s, 6H), 3.51-3.46 (m, 1H), 3.31-3.28 (m, 2H), 2.79 (d, J ) 6 Hz,
1H), 0.86 (s, 9H), 0.05 (s, 3H), -0.13 (s, 3H). 13C NMR (CD2Cl2, 75
MHz): δ (ppm) 159.1, 157.1, 152.4, 150.0, 145.5, 136.7, 136.5, 136.3,
130.6, 128.6, 128.3, 127.2, 113.5, 90.2, 86.8, 85.4, 75.4, 72.4, 64.2,
55.7, 25.9, 18.3, -4.71, -4.76. HRFABMS: calcd for C36H44N5O6Si
(M + H)+ 670.3062, obsd 670.3024.
5′-O-(4,4′-Dimethoxytrityl)-3′-O-[(2-cyanoethoxy)(N,N-diiso-
propylamino)phosphino]-2′-O-(tert-butyldimethylsilyl)-8-azanebul-
arine (7). 5′-O-(4,4′-Dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)-
8-azanebularine (230 mg, 0.343 mmol) was dissolved in freshly distilled
THF (2.0 mL). N,N-Diisopropylethylamine (359 µL, 2.06 mmol)
followed by 2-cyanoethyl-(N,N-diisopropylamino)chlorophosphite (153
µL, 0.687 mmol) was added to the solution. The solution was allowed
to stir at room temperature. After 8 h, the reaction mixture was diluted
with EtOAc (45 mL), filtered, and washed with 5% (w/v) aqueous
NaHCO3 (2 × 25 mL). The organic layer was dried (Na2SO4), filtered,
and concentrated under reduced pressure. The crude product was
purified by flash column chromatography on silica gel (EtOAc/hexanes/
TEA 14:85:1) to give a light-straw foam (232 mg, 78%). 31P NMR
(121 MHz, CH2Cl2, 85% H3PO4 as external standard): δ (ppm) 152.9,
151.0. HRFABMS: calcd for C45H70N7O7PSi (M + H)+ 870.4140, obsd
870.4136.
Deprotection and Purification of 8-Azanebularine-Xontaining
RNA Oligonucleotides. The solid-phase synthesis of RNA oligonucle-
otides using 2′-O-TBDMS-protected â-cyanoethyl phosphoramidites
was carried out as previously described.20 For deprotection, the
controlled pore glass containing fully protected oligonucleotide was
transferred to 1.5 mL of a saturated solution of NH3 in methanol and
incubated at room temperature for 24 h. Solution was removed by pipet
and lyophilized to give a slightly yellow solid. The TBDMS depro-
tection was allowed to proceed at room temperature for 24 h by
8-Azanebularine Ribonucleoside (4). 2′,3′,5′-Tri-O-acetyl-8-aza-
nebularine (100 mg, 0.264 mmol) was dissolved in methanol saturated
with NH3 (1.16 mL), and the mixture was allowed to stand at 4 °C
overnight. The mixture was concentrated under reduced pressure and
purified by flash column chromatography (preadsorbed on silica gel,
(33) Seela, F.; Munster, I.; Lochner, U.; Rosemeyer, H. HelV. Chim. Acta 1998,
81, 1139-1155.
(34) Nair, V.; Chamberlain, S. D. Synthesis 1984, 401-403.
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A R T I C L E S
Haudenschild et al.
Scheme 1 a
Figure 3. Putative hydrolysis product of 8-azanebularine in RNA depro-
tected with TBAF/THF.
incubation of the RNA oligonucleotide with 1 mL of Et3N‚3HF. For
trinucleotides prepared to test the sensitivity to solid-phase synthesis
and deprotection conditions, the mixture was subjected to Waters
reversed-phased C-18 cartridge and the oligonucleotide was eluted with
a mixture of acetonitrile/water. After lyophilization of the oligonucle-
otide-containing fraction, the resulting solid was purified by reversed-
phase HPLC using mobile phase A (0.1 M triethylammonium acetate
(TEAA) buffer, pH ) 7) and mobile phase B (a ratio of 80:20
acetonitrile:0.1 M TEAA buffer) over 48 min with a flow rate 1.5 mL/
min and a solvent gradient 0-75% mobile phase B. Solvent was
a (a) tBuONO, TMSBr, CH3CN, 0 °C, 60%; (b) H2, 10% Pd/C, NaOAc,
CH3OH, 80%; (c) (i) NH3/CH3OH, 4 °C, 81%; (ii) DMTCl, pyridine,
AgNO3, THF, 65%; (iii) TBDMSCl, AgNO3, TEA, THF, 42%; (iv)
ClP(OCH2CH2CN)(N(iPr)2), DIEA, THF, 78%.
removed by lyophilization. MALDI analysis: calcd for trimer 5′-A8aza
-
NG-3′ (M + H)+, 928.19; obsd 928.30. For oligonucleotides longer
than trimers, triethylamine trihydrofluoride was removed by dialysis,
and the oligonucleotides were purified by gel electrophoreses as
previously described.20 Deprotection conditions leading to modification/
fragmentation of 8-azanebularine were as follows. Deprotection and
purification of the trinucleotide was carried out as described above
except the TBDMS deprotection was accomplished in the presence of
600 µL of 1.0 M TBAF/THF(wet) at room temperature for 48 h.
MALDI analysis: calcd for 5′-AXG-3′, where X is a 5-amino-1,2,3-
triazole-4-carbaldehyde ribosyl residue (see Figure 3) (M - H)-,
917.17; obsd 917.14.
azaN at position 24) or ∼10 pM (8-azaN at R/G site) 5′-32P end-labeled
RNA duplex, incubated at 30 °C for 10 min and treated as above.
Variation in the incubation period from 10 to 30 min did not effect the
measured dissociation constant. The data were analyzed by performing
volume integrations of the regions corresponding to free RNA, protein-
RNA complex, and background sites using ImageQuant software. The
data were fit to the equation: fraction bound ) A*[protein]/([protein]
+ KD), where KD is the fitted dissociation constant and A is the fitted
maximum fraction RNA bound at that dissociation constant, using the
least-squares method of KaleidaGraph.
General Biochemical Procedures. Distilled, deionized water was
used for all aqueous reactions and dilutions. Biochemical reagents were
obtained from Sigma/Aldrich unless otherwise noted. Common enzymes
were purchased from Roche, Promega, or New England Biolabs. [γ-32P]-
ATP (6000 Ci/mmol) was obtained from Perkin-Elmer Life Sciences.
Storage phosphor autoradiography was performed utilizing imaging
plates from Eastman Kodak Co. and a Molecular Dynamics Typhoon
9400. Full-length human ADAR2 (hADAR2a-LV(H)6), R-D (MS-
(H)10ENLYFQG-hADAR2a216-711), and ADAR2300-711 (MS(H)10-
ENLYFQG-hADAR2a300-711) were overexpressed in Saccharomyces
cereVisiae and purified as previously described.35 After purification,
R-D was treated with TEV protease to remove the (H)10 tag, which
had a minimal effect on the measured dissociation constant.
Preparation of Duplex Structures. Oligonucleotides were 5′-end-
labeled using [γ-32P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase,
purified on a 19% denaturing polyacrylamide gel, visualized by storage
phosphor autoradiography, excised, and extracted by the crush and soak
method. The labeled oligonucleotide was hybridized to its unlabeled
complement strand by heating to 95 °C for 5 min in TE buffer (10
mM Tris-HCl, pH 7.5, 0.1 mM EDTA) with 200 mM NaCl followed
by slow cooling.
Gel Mobility Shift Assay. 5′-32P end-labeled RNA duplex (∼60
pM) was added to 300 nM R-D, R-D (E396A), or ADAR2300-711 in 15
mM Tris-HCl, pH 7.5, 3% glycerol, 0.5 mM DTT, 60 mM KCl, 1.5
mM EDTA, 0.003% NP-40, 160 units/mL RNasin, 0.1 mg/mL BSA,
and 1.0 µg/mL yeast tRNAPhe, and the reactions were incubated at 30
°C for 30 min. Samples were loaded onto a running 6% nondenaturing
polyacrylamide gel (79:1 acrylamide:bisacrylamide) and electrophoresed
in 0.5 X TBE buffer at 4 °C for 45 min. Storage phosphorimaging
plates (Kodak) were pressed flat against the dried electrophoresis gels
and exposed in the dark. For the determination of dissociation constants,
varying concentrations of ADAR proteins were added to ∼30 pM (8-
Results
To accomplish the goal of incorporating 8-azanebularine into
RNA, we synthesized the corresponding phoshoramidite (Scheme
1). Our synthesis begins with the known 2′,3′,5′-tri-O-acetyl-
8-azaadenosine (1), which underwent diazotization/bromo-
dediazoniation using a variant of the procedure recently reported
by Francom and Robins to give the 6-bromonucleoside 2
(Scheme 1) in good yield.36 This compound was reductively
dehalogenated using H2 and Pd/C to give acetyl protected
8-azanebularine 3. Although other synthetic routes to 8-aza-
nebularine have been previously reported with similar overall
yields,20,34 the generation of 6-bromonucleoside 2 in our scheme
offers the advantage of ready access to other C6 derivatives of
the 8-azapurine ring system by simple substitution reactions.37
Elaboration of 3 to the 5′-O-DMT, 3′-O-TBDMS â-cyanoethyl-
di-iso-propyl phosphoramidite 7 was carried out using standard
reaction conditions (see Experimental Section).
8-Azanebularine survives conditions of automated RNA
synthesis and deprotection using NH3/MeOH followed by Et3N‚
3HF as indicated by mass spectrometric analysis of trinucle-
otides containing the analogue (see Experimental Section).
However, when TBAF/THF was used for removal of silyl
protecting groups, apparent modification at the nucleoside
analogue occurred. The mass of the modified oligonucleotide
product is consistent with a degradation pathway previously
observed for 8-azapurines treated for extended periods with
(36) Francom, P.; Robins, M. J. J. Org. Chem. 2003, 68, 666-669.
(37) Veliz, E. A.; Beal, P. A. J. Org. Chem. 2001, 66, 8592-8598.
(35) Macbeth, M. R.; Lingam, A. T.; Bass, B. L. RNA 2004, in press.
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11216 J. AM. CHEM. SOC. VOL. 126, NO. 36, 2004

RNA-Editing Transition State Analogue
A R T I C L E S
Figure 4. (A) Sequence of an RNA-editing substrate from the R/G site in
GluR-B pre-mRNA, where X ) adenosine (A) or 8-azanebularine (8-azaN)
(R/G 27 mer).17 (B) Domain map for ADAR2 indicating approximate
locations of dsRBM I, dsRBM II, and various amino acid positions.
Figure 6. Quantitative gel mobility shift data for the binding of R-D to
R/G 27 mer RNA shown in Figure 4 with 8-azanebularine at either the
R/G site (blue) or the A24 site (red).
The calculated equilibrium constant for hydration of the
8-azapurine ring favors the dehydrated, fully aromatic system,
and the hydrate has not been detected in solution.32 Thus, if
hydration of the 8-azapurine ring system is required for stable
complex formation as we have suggested, it is most likely
facilitated by the enzyme. Therefore, one would predict that
stable complex formation with 8-azanebularine-containing RNA
would require a functional ADAR2 active site. We tested this
hypothesis by analyzing the binding of the catalytically inactive
E396A mutant of R-D. The analogous residue in E. coli CDA
(E104) is critically important in forming the key tetrahedral
intermediate for the CDA reaction.25 It is believed to both
deprotonate the zinc-bound water and protonate the pyrimidine
ring during its formation. Significantly, the production of a
stable, high-affinity complex with 8-azanebularine-containing
RNA is inhibited by the E396A mutation in R-D (Figure 5,
lane 5). Indeed, the extent of binding observed is similar to the
small amount seen with R-D and the RNA duplex with
adenosine at the editing site (compare Figure 5, lanes 2 and 5)
and likely arises from duplex RNA binding by dsRBM II. We
also assessed the ability of a mutant of ADAR2 lacking both
dsRBMs (ADAR2300-711) to bind the duplex with 8-azane-
bularine at the R/G editing site. Although this protein bears all
of the residues conserved with CDAs and a demonstrated ability
to catalyze deamination of adenosine in RNA, micromolar
concentrations are required to bind the R/G 27 mer containing
the aza analogue at the editing site with no binding observed at
300 nM (Figure 5, lane 6 and data not shown). Together these
results indicate that both a functional active site and dsRBM II
are required for the high-affinity binding to the nucleoside
analogue-containing RNA. Interestingly, the dissociation con-
stant for the analogue-containing RNA binding to full length
ADAR2 is similar to that observed with R-D (see below),
suggesting that dsRBM I of ADAR2 contributes little to specific
binding to analogue-containing RNA.
Figure 5. Gel mobility shift analysis of the binding of 8-azanebularine-
containing RNA to ADAR2 mutants. Lane 1, unmodified GluR-B R/G
duplex; lane 2, unmodified GluR-B R/G duplex + 300 nM R-D; lane 3,
GluR-B R/G duplex with 8-azanebularine at the R/G site; lane 4, GluR-B
R/G duplex with 8-azanebularine at the R/G site + 300 nM R-D; lane 5,
GluR-B R/G duplex with 8-azanebularine at the R/G site + 300 nM R-D
(E396A); lane 6, GluR-B R/G duplex with 8-azanebularine at the R/G site
+ 300 nM ADAR2300-711
.
dilute aqueous acid or base involving covalent hydration
followed by ring opening and fragmentation to give a 5-amino-
1,2,3-triazole-4-carbaldehyde residue (Figure 3).38 Replacement
of TBAF/THF with Et3N‚3HF eliminated this unwanted side
reaction. We used phosphoramidite 7 and the appropriate
deprotection conditions to prepare mimics of the glutamate
receptor B-subunit (GluR-B) pre-mRNA near the R/G editing
site (Figure 4A).17
We chose initially to analyze the effect 8-azanebularine has
on the ADAR2 RNA binding affinity using a deletion mutant
of the enzyme lacking dsRBM I (ADAR2216-711) (Figure 4B).
We refer to this protein as R-D because it contains one dsRBM
and the Deaminase domain. R-D has been shown to retain
efficient RNA-editing activity at the GluR-B R/G site.35 By
deleting dsRBM I from ADAR2, we anticipated the effects on
RNA binding arising from interactions with the deaminase
domain would be maximized. Importantly, a stable, slow moving
complex is observed in gel mobility shift experiments upon
addition of 300 nM R-D to the RNA duplex containing
8-azanebularine at the R/G editing site. Little binding is observed
under these conditions when adenosine is at the editing site
(Figure 5). Under the conditions of these experiments, adenosine
at the R/G site is efficiently converted to inosine, so these
observations indicate the protein-RNA complex is significantly
less stable with inosine at the editing site.35 However, a more
stable complex is formed with the 8-azanebularine at the reactive
site in the duplex (Figure 5, lane 4).
To determine to what extent the tight binding was dependent
on the sequence context of the natural editing site, we used a
quantitative gel mobility shift assay to measure dissociation
constants for binding of R-D to the RNA duplex with 8-aza-
nebularine at the R/G editing site and to the duplex modified at
a site that is not normally edited by ADAR2 (adenosine at
position 24) (Figure 6).18 The RNA with the analogue at the
editing site was bound with a KD ) 2.0 ( 1.6 nM, whereas
modification with the analogue at position 24 caused the RNA
to be bound with a KD ) 271 ( 101 nM (Table 1). The latter
(38) Albert, A.; Lin, C. J. J. Chem. Soc., Perkins Trans. 1 1977, 1819-1822.
9
J. AM. CHEM. SOC. VOL. 126, NO. 36, 2004 11217

A R T I C L E S
Haudenschild et al.
Table 1. Dissociation Constants (KD) for Complexes with ADAR2
nistic similarities, such as the ability to displace alternate leaving
groups from C6 and the 8-aza effect on the deamination rate.20
A crystal structure of ADA bound to nebularine (purine
ribonucleoside) has been solved.42 Importantly, nebularine binds
ADA as the covalent hydrate, a structure that is remarkably
similar to the proposed hydrolytic deamination transition state.
These studies suggested to us that 8-azanebularine, which is
predicted to undergo covalent hydration more readily than does
nebularine,32 might bind tightly in the ADAR active site as a
covalent hydrate. Indeed, high concentrations of 8-azanebularine
inhibit ADAR2, whereas similar concentrations of nebularine
do not.20 Here, we show that 8-azanebularine can be incorpo-
rated into a known RNA ligand of ADAR2 and this leads to
tighter binding to that RNA. This is consistent with the inhibition
results with the free nucleoside and the observation that
8-azaadenosine in RNA is deaminated at a higher rate than is
adenosine; that is, the 8-azapurine ring system is well-suited
for interaction with the ADAR2 active site. However, the affinity
achieved by the 8-azanebularine-containing RNA for R-D (KD
) 2 nM) is still several orders of magnitude lower than that
observed for the transition state analogue coformycin binding
to ADA (KI ≈ 10 pM).43 This may be due to the requirement
for formation of the unfavored hydrate of 8-azanebularine for
ADAR binding, whereas coformycin, with its stable tetrahedral
center at the reactive carbon, does not require this step for ADA
binding. Furthermore, these observations suggest room for
improvement in the development of new high-affinity ligands
for the ADARs.
Mutants and 8-Azanebularine-Modified RNA Duplexesa
site of 8-azanebularine modification
protein
R/G site (KD, nM)
A24 site (KD, nM)
ADAR2full
R-D
R-D (E396A)
3.2 ( 1.6
2.0 ( 1.6
308 ( 39
30 ( 6.7
271 ( 101
NDb
a Dissociation constants were measured using quantitative gel mobility
shift assays and are reported as the average ( standard deviation (see
Experimental Section). b Not determined.
dissociation constant is similar in magnitude to that observed
for unmodified RNA (KD ≈ 1 µM) (data not shown) and for
R-D (E396A) binding to the RNA duplex with 8-azanebularine
at the R/G editing site (KD ) 308 ( 39 nM). Thus, over a 100-
fold affinity difference is realized by positioning the analogue
in the sequence/structural context of the GluR-B R/G editing
site as compared to a site not edited in the same RNA structure.
Furthermore, this increase in affinity requires the functional
active site. Analysis of the binding of full length ADAR2 to
the two RNAs again shows higher affinity binding to editing
site-modified RNA, albeit with a smaller difference in the
measured dissociation constants (∼10-fold) (KD ) 3.2 ( 1.6
nM for the R/G site; KD ) 30 ( 6.7 nM for position 24) (Table
1). The difference is due to increased binding to position 24-
modified RNA as R/G site-modified RNA binds the two proteins
with the same affinity. The higher affinity measured for full
length ADAR2 binding to position 24-modified RNA is a result
of increased nonspecific binding arising from the presence of
dsRBM I, because we have measured a similar affinity (KD )
22 ( 4 nM) for the unmodified duplex binding to full length
ADAR2.17
The data reported here support the hypothesis that the tight
binding observed is in the form of a complex with 8-azane-
bularine at the R/G site bound into the ADAR2 active site. First,
this binding requires substitution at a site on the duplex that is
deaminated by ADAR2 (the R/G editing site). The presence of
an adenosine within duplex RNA does not, by itself, indicate
whether it will be deaminated by ADAR2. Its position on the
duplex and the local sequence context play important roles in
determining an adenosine’s susceptibility to ADAR-catalyzed
deamination.44,45 The R/G site adenosine is deaminated by
ADAR2 to ∼80% yield with a kobs ≈ 1 min-1 in the duplex
substrate shown in Figure 4, whereas no ADAR2-catalyzed
deamination has been observed at the adenosine 24 position.17,18
Also, we have shown previously that the fluorescence of
2-aminopurine positioned at the R/G editing site in this duplex
is enhanced by ADAR2, consistent with enzyme-induced base
flipping at that site.18 No such change in fluorescence is observed
when 2-aminopurine is placed at position 24.18 Here, we
demonstrate that the increase in binding affinity arising from
incorporation of 8-azanebularine into the RNA ligand is also
dependent on its positioning on the duplex. Incorrect positioning
prevents ADAR2 from productive interaction with the analogue.
This may be because the A24 site is not in the right position
relative to preferred binding registers of ADAR2’s dsRBMs or
the flanking sequence may prevent the catalytic domain from
maximum productive binding with adjacent nucleotides. Both
Discussion
There are currently no reports of high-resolution structural
data for ADAR proteins or ADAR-RNA complexes. Such
structural information would be extremely valuable in under-
standing protein-RNA recognition for these enzymes and for
further definition of the deamination reaction mechanism.
Crystallography, NMR spectroscopy, and even simple foot-
printing studies of dsRBM-containing RNA-binding proteins,
like the ADARs, are complicated by the lack of sequence
specificity for the dsRBMs.39,40 This leads to multiple protein-
RNA complexes of similar affinities in solution. One approach
to address this problem for the ADARs is to form a high-affinity
complex involving a tight-binding deamination substrate ana-
logue or transition state analogue in the RNA. The results
presented here indicate that 8-azanebularine-containing RNA
will be useful in these efforts, and relevant studies are currently
underway. Furthermore, 8-azanebularine-containing oligonucle-
otides are likely to be selective inhibitors of ADARs and could
be used to control ADAR-catalyzed deamination reactions.
8-Azanebularine is a known inhibitor of the nucleoside-
modifying enzyme adenosine deaminase (ADA).41 ADA is
structurally unrelated to ADARs, but does share some mecha-
(42) Wilson, D. K.; Rudolph, F. B.; Quiocho, F. A. Science 1991, 252, 1278-
(39) Ryter, J. M.; Schultz, S. C. EMBO J. 1998, 17, 7505-7513.
(40) Ramos, A.; Grunert, S.; Adams, J.; Micklem, D. R.; Proctor, M. R.; Freund,
S.; Bycroft, M.; St. Johnston, D.; Varani, G. EMBO J. 2000, 19, 997-
1009.
(41) Shewach, D. S.; Krawczyk, S. H.; Acevedo, O. L.; Townsend, L. B.
Biochem. Pharm. 1992, 44, 1697-1700.
1284.
(43) Agarwal, R. P.; Spector, T.; Parks, R. E. Biochem. Pharmacol. 1977, 26,
359-367.
(44) Polson, A. G.; Bass, B. L. EMBO J. 1994, 13, 5701-5711.
(45) Dawson, T. R.; Sansam, C. L.; Emeson, R. B. J. Biol. Chem. 2004, 279,
4941-4951.
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11218 J. AM. CHEM. SOC. VOL. 126, NO. 36, 2004

RNA-Editing Transition State Analogue
A R T I C L E S
of these factors could be contributing, and additional studies
will be necessary to determine their relative importance.
there simply may not be enough recognition surface for dsRBM
I, dsRBM II, and the catalytic domain to bind this RNA at the
same time. Alternatively, for dsRBM I and the catalytic domain
to bind concurrently, a conformational change in the protein
that disrupts favorable protein-protein interactions may be
necessary.35 This could be compensated for by binding interac-
tions between dsRBM I and RNA. For binding by the protein
without dsRBM I (R-D), no conformational change would be
necessary, but no additional affinity from dsRBM I‚RNA
binding would be possible either. The two processes would be
energetically equivalent. The difference in affinity observed for
the two proteins for the A24 site-modified RNA can simply be
attributed to nonspecific duplex RNA binding arising from the
dsRBMs, where full length ADAR2 binds more tightly because
it carries two dsRBMs.
In summary, we have shown that 8-azanebularine can be
incorporated into an RNA duplex substrate of ADAR2 via the
phosphoramidite, creating a tight binding ligand for the enzyme.
The observed high-affinity binding is dependent on a functional
ADAR2 active site, the presence of dsRBM II, and correct
placement of the nucleoside analogue into the sequence/
structural context of a known editing site. ADAR2’s dsRBM I
contributes little to the observed affinity for analogue-containing
RNA. These results have implications in our understanding of
substrate recognition during ADAR-catalyzed RNA editing, for
future structural studies of ADAR‚RNA complexes, and in the
development of inhibitors of adenosine deamination RNA
editing.
The high-affinity binding to 8-azanebularine-containing RNA
also requires a functional ADAR2 active site. Mutation of the
critical conserved glutamate residue in the ADAR active site
has been shown to inhibit enzyme activity, but has a minimal
effect on RNA binding to unmodified RNA.26 However, we
show here that this mutation dramatically reduces the affinity
with which R-D binds the RNA duplex modified at the R/G
site with 8-azanebularine (Figure 5, Table 1). This is most likely
because tight binding requires formation of the covalent hydrate,
as observed in the nebularine-ADA crystal structure, and
hydration requires the catalytic residues of the enzyme.42
Importantly, formation of the covalent hydrate of 5-fluoroze-
bularine in the active site of E. coli CDA is prevented by the
E104A mutation in that enzyme as well.46
High-affinity binding to RNA modified at the R/G site also
requires ADAR2’s dsRBM II. Comparing the affinities of R-D,
which lacks dsRBM I, and ADAR2300-711, which lacks both
dsRBMs, for R/G site-modified RNA clearly indicates a
significant role for dsRBM II (at least a 1000-fold difference
in affinity). Thus, in the complex leading to deamination at the
R/G site, dsRBM II contacts the duplex and contributes to
overall affinity. The presence of extensive duplex structure 3′
to the editing site undoubtedly positions the enzyme on the
substrate with dsRBM II bound in this location. Furthermore,
preferred binding registers on the RNA for dsRBM II may fine-
tune the binding and positioning of the catalytic domain.
Acknowledgment. P.A.B. acknowledges support from the
NIH (GM61115) and a Camille Dreyfus Teacher Scholar Award.
We would also like to acknowledge the National Science
Foundation (CHE-9002690) and the University of Utah for funds
necessary to purchase the Finnigan MAT 95 mass spectrometer.
Interestingly, tight binding to 8-azanebularine-containing
RNA does not require dsRBM I. Full length ADAR2 and R-D
bind RNA modified at the R/G site with nearly identical
dissociation constants (Table 1). The presence of dsRBM I does
contribute to nonspecific binding considering the 10-fold
difference in binding of these two proteins to RNA modified at
position 24 (Table 1). How could dsRBM I contribute to
nonspecific binding but not to specific binding to RNA
containing the aza-analogue at the R/G site? It may be that
dsRBM I and the catalytic domain of ADAR2 do not bind the
RNA simultaneously. For the short RNA duplexes studied here,
Supporting Information Available: NMR spectra for com-
pounds 2, 5-7. MALDI mass spectra for trinucleotides contain-
ing 8-azanebularine after different deprotections conditions.
Autoradiograms and plots of fraction RNA bound versus protein
concentration for quantitative gel mobility shift assays. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(46) Carlow, D. C.; Short, S. A.; Wolfenden, R. Biochemistry 1996, 35, 948-
954.
JA0472073
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