Synthesis, oxidation behavior, crystallization and structure of 2′-methylseleno guanosine containing RNAs

Article abstract of DOI:10.1021/ja0621400

We have recently introduced a basic concept for the combined chemical and enzymatic preparation of site-specifically modified 2′-methylseleno RNAs which represent useful derivatives for phasing of X-ray crystallographic data during their three-dimensional structure determination. Here, we introduce the first synthesis of an appropriate guanosine phosphoramidite, which complements the thus far established set of 2′-methylseleno-modified uridine, cytidine, and adenosine building blocks for solid-phase synthesis. The novel building block was readily incorporated into RNA. Importantly, it was the 2′-methylselenoguanosine-labeled RNA that allowed us to reveal the reversible oxidation/reduction behavior of the Se moiety and thus it represents a valuable contribution to the understanding of the action of threo-1,4-dimercapto-2,3-butanediol (DTT) required during solid-phase synthesis, deprotection, and crystallization of selenium-containing RNA. In addition, we investigated 2′-methylseleno RNA with respect to crystallization properties. Our studies revealed that the Se modification significantly increases the range of conditions leading to crystal growth. Moreover, we determined the crystal structures of model RNA helices and showed that the Se modification can affect crystal packing interactions, thus potentially expanding the possibilities for obtaining the best crystal form.

Full text of DOI:10.1021/ja0621400

Published on Web 07/12/2006
Synthesis, Oxidation Behavior, Crystallization and Structure
of 2′-Methylseleno Guanosine Containing RNAs
Holger Moroder,† Christoph Kreutz,† Kathrin Lang,† Alexander Serganov,*,‡ and
Ronald Micura*,†
Contribution from the Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI),
Leopold-Franzens UniVersity, 6020 Innsbruck, Austria, and Memorial Sloan-Kettering Cancer
Center, 1275 York AVe, Box 557, New York, New York
Received March 29, 2006; E-mail: serganoa@mskcc.org; ronald.micura@uibk.ac.at
Abstract: We have recently introduced a basic concept for the combined chemical and enzymatic
preparation of site-specifically modified 2′-methylseleno RNAs which represent useful derivatives for phasing
of X-ray crystallographic data during their three-dimensional structure determination. Here, we introduce
the first synthesis of an appropriate guanosine phosphoramidite, which complements the thus far established
set of 2′-methylseleno-modified uridine, cytidine, and adenosine building blocks for solid-phase synthesis.
The novel building block was readily incorporated into RNA. Importantly, it was the 2′-methylseleno-
guanosine-labeled RNA that allowed us to reveal the reversible oxidation/reduction behavior of the Se
moiety and thus it represents a valuable contribution to the understanding of the action of threo-1,4-
dimercapto-2,3-butanediol (DTT) required during solid-phase synthesis, deprotection, and crystallization
of selenium-containing RNA. In addition, we investigated 2′-methylseleno RNA with respect to crystallization
properties. Our studies revealed that the Se modification significantly increases the range of conditions
leading to crystal growth. Moreover, we determined the crystal structures of model RNA helices and showed
that the Se modification can affect crystal packing interactions, thus potentially expanding the possibilities
for obtaining the best crystal form.
Introduction
In nucleic acid crystallography, selenium-labeled DNA and
RNA oligonucleotides have become recognized recently to
represent useful derivatives for convenient phasing of X-ray
crystallographic data. Pioneering work by Egli, Huang, and co-
workers led to the successful MAD phasing of a short Z-form
DNA duplex via phosphoroselenoate backbone modifications
and of an A-form DNA duplex via 2′-methylseleno uridine
modifications.1,2 Then, our laboratory introduced advanced
procedures for the preparation of 2′-methylseleno-modified
RNA, site-specifically labeled at single uridines, cytidines, and
adenosines (Figure 1).3,4 Thereby, the application of threo-1,4-
dimercapto-2,3-butanediol (DTT) during all crucial steps of
RNA preparation, including the solid-phase synthesis cycle, was
a major breakthrough for the high performance of the Se
approach. This led to the preparation of high-purity RNAs with
up to a hundred nucleotides containing site-specifically incor-
porated, multiple Se labels, exemplified by the adenine ribo-
switch aptamer domain.4,10 Successful applications of the Se-
derivatized RNAs developed in the course of this project have
Figure 1. Se-modified RNA for X-ray crystallography. (a) The synthesis
of a 2′-methylseleno-modified guanosine building block appropriate for RNA
solid-phase synthesis is presented in this study. (b) 2′-Methylseleno
nucleoside containing RNA is readily accessible by chemical synthesis and
enzymatic ligation3,4 and represents a valuable derivative in X-ray structure
determination using advanced techniques for phase determination, such as
MAD (multiwavelength anomalous dispersion), SAD (single-wavelength
anomalous diffraction), or SIRAS (single isomorphous replacement with
anomalous scattering).5-9 (c) Crystallization screening and structure com-
parison of native vs Se-modified model RNA helices is addressed in the
present study to shed light on the influence of Se modification on crystal
packing interactions.
been reported recently and refer to the X-ray structure of the
group I intron with both exons,11 and to the recent structure
† Leopold-Franzens University.
‡ Memorial Sloan-Kettering Cancer Center.
(5) Hendrickson, W. A.; Ogata, C. M. Methods Enzymol. 1997, 276, 494-
523.
(1) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang, Z. J.
Am. Chem. Soc. 2002, 124, 24-25.
(6) Podjarny, A.; Schneider, T. R.; Cachau, R. E.; Joachimiak A. Methods
Enzymol. 2003, 374, 321-341.
(2) Wilds, C. J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M. J. Am. Chem.
Soc. 2002, 124, 14910-14916.
(7) Hendrickson, W. A. Trends Biochem. Sci. 2000, 25, 637-643.
(8) Doublie´, S. Methods Enzymol. 1997, 276, 523-530.
(9) Strub, M. P.; Hoh, F.; Sanchez, J. F.; Strub, J. M.; Bock, A.; Aumelas, A.;
Dumas, C. Structure 2003, 11, 1359-1367.
(3) Ho¨bartner, C.; Micura, R. J. Am. Chem. Soc. 2004, 126, 1141-1149.
(4) Ho¨bartner, C.; Rieder, R.; Kreutz, C.; Puffer, B.; Lang, K.; Polonskaia,
A.; Serganov, A.; Micura, R. J. Am. Chem. Soc. 2005, 127, 12035-12045.
9
10.1021/ja0621400 CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 9909-9918
9909

A R T I C L E S
Moroder et al.
determination of the Diels-Alder ribozyme solved by the SAD
technique via the corresponding 2′-methylseleno pyrimidine
derivatives synthesized in our laboratory.4,12
arabinose 2′-hydroxyl group while retaining the guanine N2
monoacetylated, representing the proper protection for the final
nucleoside phosphoramidite at an early stage of the synthesis.
Hydrolysis product 5 was then reacted with trifluoromethane-
sulfonyl chloride (Tf-Cl) to furnish nucleoside 6, which was
isolated and subsequently substituted (SN2) by sodium meth-
ylselenide, producing key diastereoisomer 7 in high yield.
Deprotection of the TIPDS moiety and simultaneous release of
the O6-(4-nitrophenyl)ethyl group proceeded straightforwardly
using tetrabutylammonium fluoride (TBAF). Derivative 8 was
transformed into the dimethoxytritylated compound 9, which
was phosphitylated to achieve the actual 2′-methylseleno
guanosine phosphoramidite building block 10. We point out that
initial attempts to apply O6-(N,N-diphenylcarbamoyl)16 instead
of O6-(4-nitrophenyl)ethyl protection failed because this group
was unstable and partly substituted during treatment with sodium
methylselenide. Starting with 9-[â-D-arabinofuranosyl]guanine
1, our route provides phosphoramidite 10 in a 6% overall yield
in nine steps with eight chromatographic purifications; in total,
0.8 g of 10 was obtained in the course of this study (Scheme
1).
Chemical Synthesis and Deprotection of Se-Guanosine-
Containing RNA. The preparation of RNA with 2′-methylse-
leno-modified guanosines relies on the 2′-O-TOM-methodology
for strand assembly,17,18 and on a previously established protocol
for the synthesis of RNA with site-specific 2′-methylseleno
uridine, 2′-methylseleno cytidine, and 2′-methylseleno adenosine
modifications.4 Therein, the solid-phase synthesis cycle is
substantially changed from standard RNA synthesis by the
insertion of a step treating the oligonucleotide chain on the solid
support with threo-1,4-dimercapto-2,3-butanediol (DTT). The
repeated exposure of the growing chain to DTT is an absolute
requirement for the reliable synthesis of RNAs (>25 nt)
containing multiple Se labels (>2).4
Along these lines, the novel phosphoramidite 10 was suc-
cessfully incorporated into oligoribonucleotides with coupling
yields higher than 98%, monitored via the UV-trityl assay.
Cleavage from the solid support and deprotection of the 2′-
methylseleno-guanosine-modified RNAs were also performed
in the presence of DTT, added in millimolar amounts to the
deprotection solutions of CH3NH2 in ethanol/H2O and of
tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF).
After deprotection, DTT was removed by size exclusion
chromatography on a Sephadex G25 column. RNAs were then
purified by anion-exchange chromatography under strong de-
naturating conditions (6 M urea, 80 °C; Figure 2). The molecular
weights of the purified RNAs were confirmed by liquid
chromatography (LC) electrospray-ionization (ESI) mass spec-
trometry (MS). Exemplarily, four RNAs containing single 2′-
methylseleno guanosine labels (11a-14a) were prepared with
the sequences listed in Table 1. Sequences 12 and 13 represented
self-complementary 12 nt and 16 nt RNAs, each containing two
isolated G•A mispairs. The structures of the corresponding
unmodified duplexes were previously solved by Leonard et al.
in 1994 and by Sundaralingam et al. in 1999 and are therefore
considered ideal for verifying the guanosine Se approach.19,20
The replacement of natural 2′-OH by 2′-methylseleno groups
does not significantly interfere with the global fold of RNA if
the labels are properly positioned. Provided that the 2′-
methylseleno moieties reside in the covariant, double helical
regions of the target fold, and provided that the 5′- and 3′-ends
of the RNA comprise the same functional groups (phosphates,
triphosphates, cyclophosphates, or hydroxyls) as used to crystal-
lize the native RNA, the crystallization behavior and structure
of the selenium-modified RNAs compare well with their
nonlabeled counterparts.1,4,13 Nevertheless, sugar moieties of
RNA often participate in the formation of intermolecular
contacts in the crystal lattice, providing valuable hydrogen bonds
and van der Waals interactions.14 In this sense, it is an absolute
requirement to make all four standard nucleosides available as
2′-methylseleno building blocks for RNA solid-phase synthesis
in order to not encounter any limitation in adequate positioning
of the Se labels. Here, we present the first synthesis of a 2′-
methylseleno guanosine phosphoramidite appropriate for RNA
solid-phase synthesis and its incorporation into oligoribonucle-
otides. This phosphoramidite completes the set of four standard
RNA nucleoside building blocks with 2′-methylseleno groups.
In addition, we report on the reversible redox behavior of Se-
containing RNA and on the influence of Se modifications on
the crystallization and structure of model RNA helices.
Results and Discussion
Synthesis of a 2′-Methylseleno Guanosine Phosphorami-
dite. Our route began with the simultaneous protection of the
3′- and 5′-hydroxyl groups of commercially available 9-[â-D-
arabinofuranosyl]guanine 1 using 1,3-dichloro-1,1,3,3-tetraiso-
propyldisiloxane (TIPDSiCl2). Then, treatment of 3′,5′-protected
2 with acetic anhydride yielded a mixture of N2,2′-O-diacetylated
and N2,N2,2′-O-triacetylated nucleosides, 3a and 3b. Whereas
3a was readily separated from 3b by column chromatography,
3b resisted isolation in pure form and could only be obtained
together with its diacetylated counterpart 3a. A complete
separation and individual characterization of di- and triacetylated
compounds, however, was achieved after protection of the
guanine lactam moiety with a O6-(4-nitrophenyl)ethyl group
introduced under Mitsunobu conditions,15 resulting in 4a and
4b. Protection of O6 was mandatory since this functional group
is known to be reactive toward trifluoromethanesulfonyl chloride
which was required in a later step of the synthesis. For efficient
large-scale synthesis, separation of the di- and triacetylated
nucleoside derivatives 4a and 4b was not required. Starting with
a mixture of them, the basic aqueous conditions of the
subsequent hydrolysis step were optimized to liberate the
(10) Serganov, A.; Yuan, Y. R.; Pikovskaya, O.; Polonskaia, A.; Malinina, L.;
Phan, A. T.; Ho¨bartner, C.; Micura, R.; Breaker, R. R.; Patel, D. J. Chem.,
Biol. 2004, 11, 1729-1741.
(11) Adams, P. L.; Stahley, M. R.; Kosek, A. B.; Wang, J.; Strobel, S. A. Nature
2004, 430, 45-50.
(12) Serganov, A.; Keiper, S.; Malinina, L.; Tereshko, V.; Skripkin, E.;
Ho¨bartner, C.; Polonskaia, A.; Phan, A. T.; Wombacher, R.; Micura, R.;
Dauter, Z.; Jaschke, A.; Patel, D. J. Nat. Struct. Mol. Biol. 2005, 12, 218-
224.
(13) Teplova, M.; Wilds, C. J.; Wawrzak Z.; Tereshko V.; Du Q.; Carrasco,
N.; Huang, Z.; Egli, M. Biochimie 2002, 84, 849-858.
(14) Shah, S. A.; Brunger, A. T. J. Mol. Biol. 1998, 285, 1577-1588.
(15) Ho¨bartner, C.; Kreutz, C.; Flecker, E.; Ottenschla¨ger, E.; Pils, W.;
Grubmayr, K.; Micura R. Monatsh. Chem. 2003, 134, 851-873.
(16) Hirao, I.; Harada, Y.; Kimoto, M.; Mitsui, T.; Fujiwara, T.; Yokoyama, S.
J. Am. Chem. Soc. 2004, 126, 13298-13305.
(17) Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. HelV. Chim. Acta
2001, 84, 3773-3795.
(18) Micura, R. Angew. Chem., Int. Ed. Engl. 2002, 41, 2265-2267.
9
9910 J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006

Selenium-Derivatized RNA for X-ray Crystallography
A R T I C L E S
Scheme 1. Synthesis of 2′-Methylseleno Guanosine Phosphoramidite 10a
a (a) 1.05 equiv of TIPDSiCl2, in DMF/pyridine, room temperature, 16 h, 98%; (b) 10 equiv of acetic anhydride, in DMF/pyridine, 80 °C, 16 h, 64%; (c)
1.3 equiv of NPE-OH, 1.4 equiv of PPh3, 1.3 equiv of DIAD, in dioxane, room temperature, 3 h, 64%; (d) aqueous ammoniumhydroxide, in THF/methanol/
water, 0 °C, 5 min, 57%; (e) 1.5 equiv of trifluoromethanesulfonyl chloride, 1.5 equiv of DMAP, 2.5 equiv of NEt3, in CH2Cl2, 0 °C, 2 h, 69%; (f) 6 equiv
of NaBH4, 2 equiv of CH3SeSeCH3, in THF, 20 min, 87%; (g) 1 M TBAF, in THF, room temperature, 2.5 h, 79%; (h) 1.1 equiv of DMT-Cl, in pyridine,
room temperature, 16 h, 59%; (i) 1.5 equiv of (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite, 10 equiv of CH3CH2N(CH3)2, in CH2Cl2, room temperature,
2 h, 88%; (DIAD diisopropyl azodicarboxylate, DMAP 4-(dimethylamino)-pyridine, DMT dimethoxytrityl, NPE 2-(4-nitrophenyl)ethyl, TBAF tetrabutyl-
ammonium fluoride, TIPDSiCl2 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane).
of 1 min in retention time (Figure 2a). Both peaks were isolated,
and LC-ESI-MS analysis revealed the expected molecular
weight for the slow-migrating RNA and a molecular weight of
16 mass units higher for the fast-migrating RNA. This observa-
tion gave evidence that the 2′-methylseleno group had been
oxidized to the corresponding 2′-methylselenoxide group.21 We
further corroborated this hypothesis by the finding that purified
11a was rapidly transformed into 11b by exposure to a solution
of 2,4,6-trimethylpyridine/water/acetonitrile containing 2 mM
iodine. We were then able to reconvert 11b back into 11a upon
treatment with 10 mM DTT in ethanol/water. It is remarkable
that both oxidation and reduction proceeded without any side
products as detected by HPLC. Moreover, the oxidation reaction
seems to proceed under diastereoselective control due to
observation of a single-product peak. The reversible redox
behavior was also investigated and confirmed for the other Se-
guanosine-containing sequences, 12-14 (Figure 3).
Figure 2. HPLC traces of crude, deprotected 2′-methylseleno guanosine
It has been noted in the literature that uniform oxidation of
Se-methionine-containing protein crystals to the corresponding
modified RNA (anion exchange HPLC: Dionex DNAPac (4 × 250 mm),
80 °C, 1 mL/min, 0-60% B in 45 min; (A) 25 mM Tris-HCl, 6 M urea,
selenoxides and selenones results in a higher anomalous
signal.22-24 Likewise, the consistent transformation of RNA 2′-
methylseleno groups into their corresponding selenoxides could
be beneficial, provided the oxidation was complete, the sele-
noxide moieties were not structurally perturbing, and the RNA
derivatives were stable enough over the period of crystallization
and data collection. Our ongoing work is focused on these issues.
pH 8.0; (B) same as A + 0.5 M NaClO4). (a) Comparison of crude
rACGGSeUC 11, prepared without (resulting in 11a and oxidized product
11b; top, see also Figure 3) and with (resulting in 11a exclusively; bottom)
threo-1,4-dimercapto-2,3-butandiol (DTT) treatment during solid-phase
synthesis and deprotection. (b) and (c) Crude and purified (inset) rCGSe-
CGAAUUAGCG 12a and rGCAGSeAGUUAAAUCUGC 13a prepared with
DTT treatment. Deprotection procedure includes three steps: (1) 150 mM
DTT in EtOH/H2O, 1-3 h, room temperature; (2) CH3NH2 in EtOH/H2O,
150 mM DTT, 4-6 h, room temperature; (3) 1 M TBAF in THF, 150 mM
DTT, 12-16 h, room temperature.
Crystallization of 2′-Methylseleno-Guanosine-Containing
RNAs. Virtually identical structures of the Se-modified and
nonmodified Diels-Alder ribozymes have proven that Se
modification does not affect RNA conformation and does not
necessarily change RNA packing in the crystals.12 However,
sugars often participate in the crystal packing of RNA and, if
placed in contacting regions, 2′-methylseleno modifications may
Sequence 14 represented a GSe derivative of the 22 nt selenium-
derivatized RNA/DNA hybrid that was crystallized in a ternary
complex of the group I intron with both exons.3,11
Redox Behavior of 2′-Methylseleno-Guanosine-Containing
RNAs. When the RNA hexamer 11 was prepared without the
use of DTT, the HPLC profile of the crude deprotected material
showed two dominating peaks, 11a and 11b, with a difference
(21) Chen, T.; Greenberg, M. M. J. Am. Chem. Soc. 1998, 120, 3815-3816.
(22) Moroder, L. J. Peptide Sci. 2005, 11, 187-214.
(23) Sharff, A. J.; Koronakis, E.; Luisi, B.; Koronaki, V. Acta Crystallogr. D
2000, 56, 785-788.
(24) Ali, M. A.; Peisach, E.; Allen, K. N.; Imperiali, B. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 12183-12188.
(19) Leonard, G. A.; McAuley-Hecht, K. E.; Ebel, S.; Lough, D.; Brown, T.;
Hunter, W. N. Structure 1994, 2, 483-494.
(20) Pan, B.; Mitra, S. N.; Sundaralingam, M. Biochemistry 1999, 38, 2826-
2831.
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A R T I C L E S
Moroder et al.
Table 1. RNAs Containing 2′-Methylseleno Guanosines Prepared by Solid Phase Synthesis
isolated yield
OD
molecular weight
length
calcd
foundb
(amu)
no.
sequencea
(nt)
scale (
µ
mol)
260 nm
nmol
(amu)
11a
12a
13a
14a
5-ACGGSeUC-3
5-CGSeCGAAUUAGCG-3
5-GCAGSeAGUUAAAUCUGC-3
5-AAGCCACACAAACC(dA)(dG)(dA)CGSeGCC-3
6
12
16
22
1
27
78
90
19
190
466
486
92
1951.2
3910.3
5182.1
7057.4
1951.4
3911.4
5182.6
7056.1
2
2
1
a GSe, 2′-methylseleno guanosine; dA, 2′-deoxy adenosine; dG, 2′-deoxy guanosine. b LC-ESI MS.
Figure 4. Location of the guanosine residues chosen for the 2′-methylseleno
modifications (circled in red) in the structures of the nonmodified duplex
RNAs, 12-mer19 and 16-mer20 (a). Crystal contact regions between two RNA
molecules (gray and cyan sticks) are apparent in the surface representation
of 12-mer (b) and 16-mer (c) RNAs. Nucleotides shown in cyan in (b) and
(c) are indicated in RNAs (a). 2′-Oxygens for 2′-methylseleno modifications
are shown as red spheres and the minimal distances to neighboring RNA
molecules are indicated.
Figure 3. Redox behavior of rCGSeCGAAUUAGCG 12. (a) Chemical
structures of reduced and oxidized selenium-modified RNA; reaction
conditions (oxidation): 1.5 nmol of RNA, 2 mM iodine solution in H2O/
CH3CN/sym-collidine; reaction conditions (reduction): 1.5 nmol of RNA,
2 mM DTT in 150 µL of H2O. (b) HPLC traces of isolated 12a (selenide)
and 12b (selenoxide) and co-injection (anion exchange; for conditions see
Figure 2). (c) LC-ESI-MS spectra of 12a (top; molecular weight, calcd
3911.25, found 3911.37) and 12b (bottom; molecular weight, calcd 3927.25,
found 3928.05).
1 for the 12-mers and 13 versus 7 for the 16-mers). Importantly,
successful crystallization conditions for the nonmodified and
Se-modified RNAs overlapped only partially (10 for the 12-
mers and 19 conditions for the 16-mers, respectively). A large
number of the Se-modified RNAs was crystallized in new
unique conditions. This means that Se modification can be
viewed as a new sequence variation of RNA targets to find
diffraction-quality crystals. It is not clear, however, if this
increase can be explained by the particular placement of
selenium atoms or by the lower solubility of the 2′-methylseleno-
modified RNAs. Nevertheless, such behavior of Se-modified
RNAs can be paralleled with well-known properties of Se-Met-
modified proteins, which sometimes do not produce crystals in
the established conditions and require adjustment of crystal-
lization conditions or even new crystallization screenings.
Crystal Structure of the 2′-Methylseleno-Modified 16 nt
RNA. Among many solution conditions suitable for crystal-
lization of the Se-modified RNAs, two, based on lithium sulfate
and PEG4000, respectively, yielded large well-diffracting
crystals of the 16 nt RNA 13a (see Experimental Section).
Remarkably, the nonmodified 16-mer produced only micro-
crystals in the same conditions. Both “lithium sulfate” and
“PEG4000” crystals belonged to the R3 space group (Table 2),
originally found in the crystals of the nonmodified RNA.20 The
crystals had similar cell unit parameters with a/b dimensions
6.0% and 8.9% longer than those in the original structure, clearly
interfere with the formation of crystal contacts.14 To investigate
the impact of these modifications on RNA crystallization, we
placed 2′-methylseleno guanosine residues into the 12 nt and
16 nt RNAs in positions (Figure 4a) which are located close to
(12-mers, Figure 4b) or within (16-mers, Figure 4b) the regions
of intermolecular crystal contacts.19,20 Since these RNAs form
double-stranded helices in the crystal lattice that are packed in
a head-to-tail fashion with just a few side-to-side contacts, the
effect of Se modification on crystallization should be maximized
at least in the 16 nt RNA 13a.
Interestingly, both Se-modified RNAs, 12a and 13a, were
not crystallized in the conditions published for the nonmodified
RNAs,19,20 although control nonmodified RNAs readily pro-
duced diffracting crystals. To investigate the ability of the Se-
modified RNAs to crystallize, we performed a search for
crystallization conditions using commercial sparse matrix kits.
Both 12 nt and 16 nt Se-modified RNAs along with their
nonmodified counterparts were screened using 146 unique
conditions at 4 and 20 °C. Remarkably, the Se-modified RNAs
12a and 13a produced crystals with a larger number of solutions
than their nonmodified counterparts did (total 17 versus 15 for
the 12-mers and 33 versus 24 for the 16-mers). However, this
increase was predominantly due to the appearance of thin
needle-shaped crystal plates growing from the common center
(‘RNA flowers’)25 for 12a (7 conditions) and a higher number
of microcrystals for 13a (26 versus 11). A count of large-shaped
crystals was clearly greater for the nonmodified RNAs (3 versus
(25) Hollbrook, S. R.; Hollbrook, E. I.; Walukiewicz, H. E. Cell. Mol. Life Sci.
2001, 58, 234-243.
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Selenium-Derivatized RNA for X-ray Crystallography
A R T I C L E S
Table 2. X-ray Data Collection and Refinement Statistics.a,b
16-mer PEG4000
16-mer PEG4000
16-mer Li2SO4
Data Collection
space group
R3
R3
R3
cell dimensions (hexagonal settings)
a, b, c (Å)
45.06, 45.06, 128.20
90, 90, 120
20-3.0 (3.11-3.0)
45.47, 45.47, 128.91
90, 90, 120
20-2.9 (3.0-2.9)
46.31, 46.31, 128.35
90, 90, 120
20-2.9 (3.0-2.9)
R, â, γ (deg)
resolutiona
peak
inflection
0.979113
10.1 (36.3.9)
16.3 (4.0)
99.9 (100)
11097
remote
wavelength (Å)
0.978901
10.6 (34.4)
14.6 (6.0)
99.9 (100)
11079
0.968634
10.4 (34.8)
16.4 (6.7)
99.8 (100)
11061
0.979096
10.9 (31.6)
32.3 (10.1)
99.9 (100)
23518
0.978948
11.2 (32.0)
12.7 (6.5)
97.2 (99.5)
13676
R
sym (%)a
I /σ(I)a
completeness (%)a
measured reflections
unique reflectionsa
1970 (194)
1975 (193)
1970 (194)
2198 (220)
2227 (221)
Refinement
resolution (Å)
number of reflections
working set
20-2.9 (2.97-2.9)
20-2.9 (2.97-2.9)
2095
2118
1966
2016
test set
99
102
completeness
R
number of atoms
RNA
99.68 (100.0)
23.0/25.5
680
680
52.9
97.2 (99.4)
21.2/23.9
695
680
35.2
work/Rfree (%)a
mean B-factor (Å2)
rmsd from ideality
bond lengths (Å)
bond angles (deg)
estimated coordinate error (Å)b
0.005
1.103
0.47
0.005
1.125
0.32
a Values for the highest resolution shell are in parentheses. b Estimated coordinate error based on maximum likelihood was calculated with REFMAC
(ref 49).
suggesting possible alterations in the crystal packing. The X-ray
Intermolecular Contacts in the Crystal Lattice. Our
analysis of the crystal contacts revealed that the RNA helices
of 13a are packed in the head-to-tail fashion, making a few
side-to-side interactions with two neighboring RNA helices. In
contrast to the nonmodified RNAs (Figure 5c), the central region
of the Se-modified RNAs is not involved in packing interactions
(Figure 5d). In addition, two stacked helices (green and gray,
Figure 5c) of the nonmodified RNA make side-to-side contacts
with the same neighboring helix (blue), while the Se-modified
helices do not form such overlapping interactions (Figure 5d,
Supporting Information Tables S1 and S2). Another rather
noticeable difference between the two types of packing is the
orientation of G4 and G20 (red nucleotides on Figure 5c,d). In
the nonmodified RNA, these nucleotides face the opposite strand
of the neighboring helix, but in the presence of Se modifications
(Figure 5e) they are positioned against the same nucleotides.
Moreover, three Se atoms from neighboring molecules are
located at a close (∼4.0 Å) distance from each other (Figure
5f). The atoms form an equilateral triangle in the center of the
packing interactions, thus placing nine nucleotides from three
RNA molecules into close proximity, and therefore directly and
significantly contributing toward bringing these RNA helices
together. Since these packing interactions are exclusive for the
Se-modified RNAs and are characteristic of the crystals grown
in rather different conditions with either salt or organic polymer
as precipitating agents, we suggest that the 2′-methylseleno
modification is an important driving force in the packing of
RNA helices.
structures determined from the lithium sulfate and PEG4000
crystals were virtually identical (rmsd 0.55 Å) and, therefore,
only the 2.9 Å lithium sulfate structure is described below.
The Se-modified 16 nt RNA 13a was crystallized as a right-
handed A-form duplex with two nonadjacent G•A mispairs
separated by four Watson-Crick base pairs as described for
the nonmodified RNA structure.20 Using all-atom superimposi-
tion, the two structures can be superimposed with a rather large
rmsd of 1.377 Å due to an almost doubled (∼8°) end-to-end
bending in the Se-modified RNA calculated by CURVES
(Figure 5a).26 Characteristic nucleobase interactions of the
original duplex, in particular the G(syn)•A+(anti) mispairs, can
also be modeled in the Se-modified structure (Figure 5b).
Nevertheless, original N6(A)-O6(G) and N1(A)-N7(G) hy-
drogen bond patterns are not preserved due to longer N1(A)-
N7(G) distances (4.38 and 3.53 Å). Therefore, in the Se-
modified RNA both G•A+ mispairs are held by O6(G)-N1(A)
hydrogen bonds (2.52 and 2.71 Å) with the potential formation
of an O6(G6)-N6(A27) hydrogen bond (2.78 Å). It is worth
mentioning that the electron density map for the G•A mispairs
(Figure 5b) is slightly worse than the base pairs closer to the
RNA tips, suggesting some “breathing” within the structure.
Such “breathing” is supported by higher B-factor values in the
middle part of the duplex and it may even cause alternate G•A
pairing in different molecules of the crystal. The formation of
different G•A base pairs at the same sequence position is not
that surprising since the conformation of these mismatches is
sensitive to various factors.27-32
Conclusion
With the synthesis of the guanosine phosphoramidite 10 and
its facile incorporation into RNA by solid-phase synthesis, we
(26) Lavery, R.; Sclenar, H. J. Biomol. Struct. Dyn. 1989, 4, 655-667.
(27) SantaLucia, J.; Turner, D. H. Biochemistry 1993, 32, 12612-12623.
(28) Wu, M.; Turner, D. H. Biochemistry 1996, 35, 9677-9689.
(29) Wu, M.; SantaLucia, J.; Turner, D. H. Biochemistry 1997, 36, 4449-4460.
(30) Heus, A. H.; Wijmenga, S. S.; Hoppe, H.; Hilbers, C. W. J. Mol. Biol.
1997, 271, 147-158.
(31) Gao, X.; Patel, D. J. J. Am. Chem. Soc. 1988, 110, 5178-5182.
(32) Carbonnaux, C.; van der Marel, G. A.; van Boom, J. H.; Guschlbauer, W.;
Fazakerley, G. V. Biochemistry 1991, 30, 5449-5458.
9
J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006 9913

A R T I C L E S
Moroder et al.
Figure 5. Crystal structure of the 2′-methylseleno-modified RNA duplex (rGCAGSeAGUUAAAUCUGC)2 (sequence 13a) and comparison with its nonmodified
counterpart. (a) Superimposition of the 2′-methylseleno-modified (green) and nonmodified (red) RNA structures and their curved helical axes (pink and light
green, respectively). (b) G(syn)•A+(anti) mispair shown with omit Fo - Fc electron density map, contoured at 2σ level. Hydrogen bond is shown by dashed
line. (c) Crystal packing of the nonmodified 16-mer RNA20 in stereo representation. Side-to-side packing contacts are shown for three neighboring helices
(green, blue, and gray) depicted in stick representations. Guanosines chosen for the Se modifications are in red. (d) Crystal packing of the 2′-methylseleno-
modified 16-mer RNA presented in the same way as Figure 5c. Guanosines with the 2′-methylseleno modifications are in red. (e) Omit 2Fo - Fc electron
density map for the region around 2′-methylseleno-modified residue G4, calculated without G4 and contoured at 1σ level. Red sphere and stick show Se
atom and methyl group. (f) Packing contacts between three Se-modified RNAs (green, yellow, and blue sticks and surfaces) involving 2′-methylseleno
groups (red spheres for Se atoms and sticks for methyl groups).
have complemented the set of previously reported 2′-methyl-
seleno-modified uridine, cytidine, and adenosine building bocks
and continue to establish Se-modified RNA as a powerful tool
in RNA X-ray structure analysis. Moreover, our detailed
investigations on the redox behavior of 2′-methylseleno-
guanosine-containing RNA shed light on the requirement to
expose Se-RNA to DTT during synthesis, deprotection, and
crystallization since the presence of DTT reduces selenoxides
and therefore guarantees a uniformly labeled RNA.
For X-ray structure analysis, we consider RNA with covalent
2′-methylseleno groups best applicable for sizes up to about 80
nucleotides. Se-RNA of this dimension can be readily obtained
by solid-phase synthesis in combination with enzymatic ligation
procedures as shown previously.4 For RNAs up to about 35 nt,
the Se approach is in competition with 5-iodo and 5-bromo
pyrimidine derivatization.33-39 We render the Se approach
superior since all four 2′-methylseleno nucleoside phosphor-
amidites are now available, and therefore a great flexibility for
adequate positioning within the RNA target is attained. In
addition, 5-halogen pyrimidine derivatives are highly photore-
active species.40-42 Inherent radiation damage of 5-halogen-
modified nucleic acids during MAD data collection has been
reported as a limitation.37 For medium-size RNA (up to 100
nt), the Se approach competes with heavy metal ion derivati-
zation.43-46 Heavy atom search is a time-consuming process
which requires soaking of the RNA crystals with dozens of
compounds at various concentrations, therefore demanding many
reasonably good crystals. This can be a serious obstacle, as had
been encountered for the Diels-Alder ribozyme where the Se
(37) Ennifar, E.; Carpentier, P.; Ferrer, J. L.; Walter, P.; Dumas, P. Acta
Crystallogr., D 2002, 58, 1262-1268.
(38) Nowakowski, J.; Shim, P. J.; Stout, D.; Joyce, G. F. J. Mol. Biol. 2000,
300, 93-102.
(39) Correll, C. C.; Freeborn, B.; Moore, P. B.; Steitz, T. A. Cell 1997, 91,
705-712.
(40) Gott, J. M.; Wu, H.; Koch, T. H.; Uhlenbeck, O. C. Biochemistry 1991,
30, 6290-6295.
(33) Shui, X.; Peek, M. E.; Lipscomb, L. A.; Gao, Q.; Ogata, C.; Roques, B.
P.; Garbay-Jaureguiberry, C.; Wilkinson, A. P.; Williams, L. D. Curr. Med.
Chem. 2000, 7, 59-71.
(41) Xu, Y.; Sugiyama, H. J. Am. Chem. Soc. 2004, 126, 6274-6279.
(42) Zeng, Y.; Wang, Y. J. Am. Chem. Soc. 2004, 126, 6552-6553.
(43) Golden, B. L. Methods Enzymol. 2000, 317, 124-132.
(44) Ennifar, E.; Walter, P.; Dumas, P. Acta Crystallogr. 2001, D57, 330-332.
(45) Francois, B.; Lescoute-Phillips, A.; Werner, A.; Masquida, B. In Handbook
of RNA Biochemistry Volume 1; Hartmann, R. K., Bindereif, A., Scho¨n,
A., Westhof, E., Eds.; Wiley-VCH: New York, 2005; pp 438-452.
(46) Ke, A.; Doudna, J. A. Methods 2004, 34, 408-414.
(34) Deng, J.; Xiong, Y.; Sundaralingam M. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 13665-13670.
(35) Wing, R.; Drew, H.; Takano, T.; Broka, C.; Tanaka, S.; Itakura, K.;
Dickerson, R. E. Nature 1980, 287, 755-758.
(36) Zhang, L.; Doudna, J. A. Science 2002, 295, 2084-2088.
9
9914 J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006

Selenium-Derivatized RNA for X-ray Crystallography
A R T I C L E S
ESI-MS (m/z): calcd for C22H39N5O6Si2 [M + H]+ 526.76, found
526.10, [M + Na]+ 548.74, found 548.17.
approach finally delivered the key derivative to enable structure
determination.12 Despite the similarity between the native and
Se-modified Diels-Alder ribozymes, we anticipated that 2′-
methylseleno groups may interfere with RNA crystallization if
placed in the regions of the crystal packing interactions. Our
crystallization experiments clearly indicate that even in the worst
case, when modifications are located in the regions of inter-
molecular contacts and perturb established crystallization pro-
tocols, additional screening may identify many more crystalli-
zation conditions. These conditions, however, can produce
crystals with the same space group but different crystal packing
interactions, necessitating the careful analysis of the crystal
isomorphism prior usage of native and selenium data in phase
calculations. Nevertheless, the X-ray structures presented here
demonstrate that even in the presence of a novel set of packing
interactions, possibly driven by 2′-methylseleno modifications,
overall RNA conformation and even fine structural details are
largely preserved. Our crystallization experiments, therefore,
suggest a novel role for 2′-methylseleno modifications, namely,
expanding the search for new crystal forms and potentially even
forcing crystallization of former noncrystallizable RNA mol-
ecules.
N2-Acetyl-9-[2′-O-acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-â-D-arabinofuranosyl]guanine (3a) and N2,N2-Diacetyl-9-
[2′-O-acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-â-D-
arabinofuranosyl]guanine (3b). Compound 2 (7.00 g, 13.3 mmol) was
coevaporated with dry pyridine three times and then suspended in DMF
(80 mL), pyridine (80 mL), and acetic anhydride (80 mL) under an
argon atmosphere. The reaction mixture was stirred for 16 h at 80 °C.
The solvents were removed under vacuum and the residual brown
viscous oil was partitioned between dichloromethane (500 mL) and an
aqueous solution containing 5% citric acid. The organic layer was
separated and the aqueous layer was extracted four times with
dichloromethane (700 mL). The combined organic layers were washed
with saturated aqueous NaCl (600 mL), dried over Na2SO4, and
concentrated under vacuum. The crude product was purified by column
chromatography on SiO2 (CH2Cl2/MeOH, 99/1-92/8 v/v). Yield: 2.91
g of 3a; 5.20 g of 3a + 3b as colorless foam (64%). A mixture of 3a
and 3b can be used in the next reaction step. TLC (CH2Cl2/MeOH,
9/1): Rf ) 0.57 (3b), 0.63 (3a). Characterization data of 3a: 1H NMR
(300 MHz, CDCl3): δ 0.95-1.05 (m, 28H, (iPr)4Si2O); 1.75 (s, 3H,
COCH3); 2.30 (s, 3H, NHCOCH3); 3.87 (m, 1H, H-C(4′)); 4.08 (m,
2H, H1-C(5′) and H2-C(5′)); 4.64 (m, 1H, H-C(3′)); 5.49 (m, 1H,
H-C(2′)); 6.26 (d, J ) 6.4 Hz, 1H, H-C(1′)); 7.97 (s, 1H, H-C(8));
9.58 (s, 1H, H-N(1)); 12.03 (s, 1H, HN-C(2)) ppm. 13C NMR (75
MHz, CDCl3): δ 12.38, 12.85, 13.03, 13.35, 16.70, 16.72, 16.83, 16.89,
17.26, 17.34, 17.42 (iPr)4Si2O); 20.07 (COCH3); 24.29 (NHCOCH3);
60.60 (C(5′)); 71.14 (C(3′)); 76.40 (C(2′)); 80.36 (C(4′)); 80.44 (C(1′));
120.81 (C(ar)); 137.61 (C(8)); 147.52, 148.14, 155.66 (C(ar)); 169.83,
172.11 (2 × COCH3) ppm. UV (MeOH): λmax ) 256 nm. ESI-MS
(m/z): calcd for C26H43N5O8Si2 [M + H]+ 610.82, found 610.10,
[M + Na]+ 632.81, found 632.26.
N2-Acetyl-9-[2′-O-acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-â-D-arabinofuranosyl]-O6-[2-(4-nitrophenyl)ethyl]gua-
nine (4a) and N2,N2-Diacetyl-9-[2′-O-acetyl-3′,5′-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)-â-D-arabinofuranosyl]-O6-[2-(4-
nitrophenyl)ethyl]guanine (4b). Compound 3a (4.65 g, 7.48 mmol),
triphenylphosphine (2.94 g, 11.2 mmol), and 2-(4-nitrophenyl)ethanol
(1.88 g, 11.2 mmol) were coevaporated with dry dioxane and then
suspended in dioxane (150 mL). After 30 min of stirring, diisopropyl
azodicarboxylate (2.36 mL, 12.0 mmol) was added and stirring
continued for 45 min. The solvents were removed under vacuum and
the residue was partitioned between dichloromethane (500 mL) and
saturated sodium bicarbonate solution. The organic layer was separated,
and the aqueous layer was extracted twice with dichloromethane (700
mL). The combined organic layers were dried over Na2SO4 and
evaporated. The crude product was purified by column chromatography
on SiO2 (hexanes/EtOAc, 9/1-5/5 v/v). Yield: 3.63 g of 4a as
yellowish foam (64%). Likewise, a mixture of 3a and 3b was used as
starting material. Separation of 4a and 4b was performed by column
chromatography on SiO2 (hexanes/EtOAc, 9/1-5/5 v/v). TLC (hexanes/
EtOAc, 2/8): Rf ) 0.55 (4a), 0.72 (4b). Characterization data of 4a:
1H NMR (300 MHz, CDCl3): δ 0.98-1.25 (m, 28H, (iPr)4Si2O); 1.71
(s, 3H, COCH3); 2.56 (s, 3H, NHCOCH3); 3.31 (t, J ) 6.8 Hz, 2H,
CH2-C6H4-NO2); 3.90 (m, 1H, H-C(4′)); 4.10 (m, 2H, H1-C(5′)
and H2-C(5′)); 4.69 (m, 1H, H-C(3′)); 4.74 (m, 2H, O6-CH2); 5.56
(m, 1H, H-C(2′)); 6.41 (d, J ) 6.2 Hz, 1H, H-C(1′)); 7.50 (d, J )
8.5 Hz, 2H, 4-nitrophenyl H-C(2)/H-C(6)); 7.84 (s, 1H, HN-C(2));
8.09 (s, 1H, H-C(8)); 8.18 (d, J ) 8.5 Hz, 2H, 4-nitrophenyl H-C(3)/
H-C(5)) ppm. 13C NMR (75 MHz, CDCl3): δ 12.40, 12.97, 13.03,
13.39, 16.73, 16.83, 16.90, 17.26, 17.32, 17.39, 17.42 ((iPr)4Si2O); 20.02
(COCH3); 25.13 (NHCOCH3); 35.05 (CH2-C6H4-NO2); 60.55 (C(5′));
66.90 (O6-CH2); 71.29 (C(3′)); 76.38 (C(2′)); 80.54 (C(4′)); 80.56
(C(1′)); 117.28 (C(ar)); 123.78 (4-nitrophenyl C(2)/C(6)); 129.92 (4-
nitrophenyl C(3)/C(5)); 140.28 (C(8)); 145.48, 146.97, 152.23, 152.78,
Experimental Section
Synthesis of 2′-Methylseleno Guanosine Phosphoramidite (10).
1
General. H, 13C, and 31P NMR spectra were recorded on a Bruker
DRX 300 MHz, or Varian Unity 500 MHz instrument. The chemical
shifts are reported relative to TMS and referenced to the residual proton
signal of the deuterated solvents: CDCl3 (7.26 ppm), d6-DMSO (2.50
ppm) for 1H NMR spectra; CDCl3 (77.0 ppm) or d6-DMSO (39.5 ppm)
for 13C NMR spectra. 31P shifts are relative to external 85% phosphoric
acid. 1H- and 13C-assignments were based on COSY and HSQC
experiments. UV-spectra were recorded on a Varian Cary 100 spec-
trophotometer. Analytical thin-layer chromatography (TLC) was carried
out on silica 60F-254 plates. Flash column chromatography was carried
out on silica gel 60 (230-400 mesh). Packing of silica gel columns
was performed with 1% Et3N added to the corresponding starting eluent.
All reactions were carried out under an Ar atmosphere. 9-[â-D-
Arabinofuranosyl]guanine 1 was obtained from MetkinenOy, Finland.
Chemical reagents and solvents were purchased from commercial
suppliers and used without further purification. Organic solvents for
reactions were dried overnight over freshly activated molecular sieves
(4 Å).
9-[3′,5′-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-â-D-arabino-
furanosyl]guanine (2). 9-[â-D-Arabinofuranosyl]guanine 1 (4.16 g; 14.7
mmol) was coevaporated with dry pyridine three times and then
suspended in DMF (160 mL) and pyridine (14 mL) under an argon
atmosphere. After dropwise addition of 1,3-dichloro-1,1,3,3-tetraiso-
propyldisiloxane (4.86 g, 15.4 mmol), the reaction mixture was stirred
at room temperature for 16 h and subsequently precipitated by being
poured into ice-water that was vigorously stirred (2.6 L). The crude
product was collected on a glass-fritted Bu¨chner funnel and used without
further purification for the next step. For analysis, a small portion of
crude product was crystallized from ethanol. Yield: 7.60 g of 2 as
white powder (98%). TLC (CH2Cl2/MeOH, 85/15): Rf ) 0.65. 1H NMR
(300 MHz, d6-DMSO): δ 1.06 (m, 28H, (iPr)4Si2O); 3.74 (m, 1H,
H-C(4′)); 3.95 (m, 2H, H1-C(5′) and H2-C(5′)); 4.30 (m, 1H,
H-C(3′)); 4.42 (m, 1H, H-C(2′)); 5.78 (d, J ) 5.9 Hz, 1H, HO-
C(2′)); 5.95 (d, J ) 6.4 Hz, 1H, H-C(1′)); 6.43 (s, 2H, NH2); 7.60 (s,
1H, H-C(8)); 10.57 (s, 1H, H-N(1)) ppm. 13C NMR (75 MHz, d6-
DMSO): δ 11.93, 12.26, 12.40, 12.77, 16.73, 16.81, 16.88, 17.09,
17.11, 17.13, 17.26 (iPr)4Si2O); 61.23 (C(5′)); 74.48 (C(2′)); 75.34
(C(3′)); 79.44 (C(4′)); 80.49 (C(1′)); 115.79 (C(ar)); 135.86 (C(8));
151.25, 153.64, 156.67 (C(ar)) ppm. UV (MeOH): λmax ) 252 nm.
160.55 (C(ar)); 169.35, 172.00 (COCH3) ppm. UV (MeOH): λmax
)
9
J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006 9915

A R T I C L E S
Moroder et al.
269 nm. ESI-MS (m/z): calcd for C34H50N6O10Si2 [M + H]+ 759.97,
found 759.26, [M + Na]+ 781.96, found 781.42. Characterization data
of 4b: 1H NMR (300 MHz, CDCl3): δ 0.99-1.17 (m, 28H, (iPr)4Si2O);
1.69 (s, 3H, COCH3); 2.27 (s, 6H, 2 × COCH3); 3.30 (t, J ) 6.9 Hz,
2H, CH2-C6H4-NO2); 3.91 (m, 1H, H-C(4′)); 4.10 (m, 2H, H1-
C(5′) and H2-C(5′)); 4.78 (m, 1H, H-C(3′)); 4.79 (m, 2H, O6-CH2);
5.55 (dd, J ) 6.4, 8.0 Hz, 1H, H-C(2′)); 6.44 (d, J ) 6.4 Hz, 1H,
H-C(1′)); 7.46 (d, J ) 8.7 Hz, 2H, 4-nitrophenyl H-C(2)/H-C(6));
7.84 (s, 1H, HN-C(2)); 8.15 (d, J ) 8.7 Hz, 2H, 2H, 4-nitrophenyl
H-C(3)/H-C(5)); 8.26 (s, 1H, H-C(8)) ppm. 13C NMR (75 MHz,
CDCl3): δ 12.38, 12.97, 12.99, 13.33, 16.72, 16.84, 16.89, 17.26, 17.33,
17.38, 17.42 ((iPr)4Si2O); 19.88 (COCH3); 26.01 (2 × COCH3); 35.02
(CH2-C6H4-NO2); 61.08 (C(5′)); 67.33 (O6-CH2); 72.04 (C(3′)); 76.72
(C(2′)); 80.76 (C(4′)); 81.28 (C(1′)); 120.42 (C(ar)); 123.77 (4-
nitrophenyl C(2)/C(6)); 129.92 (4-nitrophenyl C(3)/C(5)); 142.75 (C(8));
145.27, 147.00, 152.53, 152.87, 161.26 (C(ar)); 169.43, 171.87
(COCH3) ppm. UV (MeOH): λmax ) 260 nm. ESI-MS (m/z): calcd
for C36H52N6O11Si2 [M + H]+ 802.00, found 801.07, [M + Na]+ 823.99,
found 823.31.
17.20, 17.30, 17.37 ((iPr)4Si2O); 25.06 (COCH3); 35.03 (CH2-C6H4-
NO2); 61.00 (C(5′)); 67.02 (O6-CH2); 72.67 (C(3′)); 80.00 (C(1′)); 80.86
(C(4′)); 87.72 (C(2′)); 117.45 (C(ar)); 123.80 (4-nitrophenyl C(2)/C(6));
129.90 (4-nitrophenyl C(3)/C(5)); 139.71 (C(8)); 145.40, 147.00,
152.31, 152.77, 160.70 (C(ar)); 170.59 (COCH3) ppm. UV (MeOH):
λmax ) 268 nm. ESI-MS (m/z): calcd for C33H47F3N6O11SSi2 [M +
H]+ 849.99, found 849.37, [M + Na]+ 871.98, found 871.27.
N2-Acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-O6-[2-
(4-nitrophenyl)ethyl]-2′-deoxy-2′-methylselenoguanosine (7). Sodium
borohydride (64 mg, 1.70 mmol) was placed in a sealed 25 mL two
necked round-bottom flask, dried on high vacuum for 15 min to deplete
oxygen, kept under argon, and suspended in dry THF (2.3 mL).
Dimethyl diselenide (55 µL, 0.57 mmol) was slowly injected into this
suspension, followed by the dropwise addition of anhydrous ethanol;
0.4 mL was required until gas bubbles started to appear in the yellow
mixture. The solution was stirred at room temperature for 1 h and the
almost colorless solution was injected into a solution of 6 (230 mg,
0.27 mmol) in dry THF (2.7 mL). The reaction mixture was stirred at
room temperature for 20 min. Then, aqueous 0.2 M triethylammonium
acetate buffer (5 mL, pH 7) was added and the solution was reduced
to half of its volume by evaporation. Dichloromethane was added and
the organic layer was washed twice with 0.2 M triethylammonium
acetate buffer and, finally, with saturated sodium chloride solution. The
organic layer was dried over Na2SO4 and the solvent was evaporated.
The crude product was purified by column chromatography on SiO2
(hexanes/EtOAc, 85/15-4/6 v/v). Yield: 186 mg of 7 as colorless foam
N2-Acetyl-9-[3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-â-
D-arabinofuranosyl]-O6-[2-(4-nitrophenyl)ethyl]guanine (5). Com-
pound 4a (3.41 g, 4.49 mmol) was dissolved in THF/MeOH (5/4; 130
mL) and stirred at 0 °C, and NaOH (0.5 M, 150 mL) was added. After
7 min acetic acid (0.5 M, 160 mL) was added. The solvents were
evaporated and the residue was dissolved in dichloromethane (400 mL),
washed with water (400 mL), dried over Na2SO4, and evaporated. The
crude product was purified by column chromatography on SiO2 (CH2-
Cl2/MeOH, 99/1-98/2 v/v). Yield: 1.84 g of 5 colorless foam (57%).
Likewise, a mixture of 4a and 4b was used as starting material. TLC
1
(87%). TLC (hexanes/EtOAc, 4/6): Rf ) 0.49. H NMR (300 MHz,
CDCl3): δ 0.97-1.10 (m, 28H, (iPr)4Si2O); 1.93 (s, 3H SeCH3); 2.55
(s, 3H, COCH3); 3.31 (t, J ) 6.8 Hz, 2H, CH2-C6H4-NO2); 3.94 (dd,
J ) 4.4, 4.5 Hz, 1H, H-C(2′)); 4.06 (m, 2H, H1-C(5′) and H2-
C(5′)); 4.16 (m, 1H, H-C(4′)); 4.75 (s, 1H, H-C(3′)); 4.78 (m, 2H,
O6-CH2); 6.21 (d, J ) 4.5 Hz, 1H, H-C(1′)); 7.49 (d, J ) 8.7 Hz, 2H,
4-nitrophenyl H-C(2)/H-C(6)); 7.87 (s, 1H, HN-C(2)); 8.07 (s, 1H,
H-C(8)); 8.16 (d, J ) 8.7 Hz, 2H, 4-nitrophenyl H-C(3)/H-C(5))
ppm. 13C NMR (75 MHz, CDCl3): δ 12.68, 13.03, 13.14, 13.51, 16.85,
16.96, 16.97, 17.11, 17.27, 17.28, 17.35, 17.45 ((iPr)4Si2O); 25.13
(COCH3); 35.03 (CH2-C6H4-NO2); 47.05 (C(2′)); 61.85 (C(5′)); 66.91
(O6-CH2); 71.69 (C(3′)); 84.65 (C(4′)); 89.68 (C(1′)); 118.34 (C(ar));
123.76 (4-nitrophenyl C(2)/C(6)); 129.93 (4-nitrophenyl C(3)/C(5));
139.87 (C(8)); 145.46, 146.97, 152.03, 152.31, 160.60 (C(ar)); 170.57
(COCH3) ppm. UV (MeOH): λmax ) 269 nm. ESI-MS (m/z): calcd
for C33H50N6O8SeSi2 [M + H]+ 794.92, found 794.97, [M + Na]+
816.93, found 817.15.
1
(hexanes/EtOAc, 2/8): Rf ) 0.46. H NMR (300 MHz, CDCl3): δ
1.01-1.14 (m, 28H, (iPr)4Si2O); 2.37 (s, 3H, COCH3); 2.29 (t, J )
6.8 Hz, 2H, CH2-C6H4-NO2); 3.86 (m, 1H, H-C(4′)); 4.05 (m, 2H,
H1-C(5′) and H2-C(5′)); 4.51 (t, J ) 7.3 Hz, 1H, H-C(3′)); 4.30 (s,
1H, HO-C(2′)); 4.64 (m, 1H, H-C(2′)); 4.74 (m, 2H, O6-CH2); 6.14
(d, J ) 5.7 Hz, 1H, H-C(1′)); 7.49 (d, J ) 8.7 Hz, 2H, 4-nitrophenyl
H-C(2)/H-C(6)); 8.07 (s, 1H, HN-C(2)); 8.14 (s, 1H, H-C(8)); 8.16
(d, J ) 8.7 Hz, 2H, 4-nitrophenyl H-C(3)/H-C(5)) ppm. 13C NMR
(75 MHz, CDCl3): δ 12.46, 13.00, 13.08, 13.50, 16.89, 16.95, 17.05,
17.32, 17.39, 17.48 (iPr)4Si2O); 24.95 (COCH3); 35.02 (CH2-C6H4-
NO2); 61.49 (C(5′)); 67.01 (O6-CH2); 74.59 (C(3′)); 76.75 (C(2′)); 81.75
(C(4′)); 83.82 (C(1′)); 117.67 (C(ar)); 123.79 (4-nitrophenyl C(2)/C(6));
129.91 (4-nitrophenyl C(3)/C(5)); 141.43 (C(8)); 145.44, 146.96,
151.56, 152.47, 160.21 (C(ar)), 169.87 (COCH3) ppm. UV (MeOH):
λmax ) 260 nm. ESI-MS (m/z): calcd for C32H48N6O9Si2 [M + H]+
717.93, found 717.11, [M + Na]+ 739.92, found 739.29.
N2-Acetyl-2′-deoxy-2′-methylselenoguanosine (8). Compound 7
(735 mg, 0.726 mmol) was dissolved in THF (25 mL) and treated with
1 M TBAF in THF (6 mL). The solution was stirred at room
temperature for 2.5 h. The solvent was evaporated and the product was
isolated by column chromatography on SiO2 (CH2Cl2/MeOH, 97/3-
93/7 v/v). Yield: 230 mg of 8 as colorless foam (79%). TLC (CH2-
N2-Acetyl-9-{2′-O-[(trifluoromethyl)sulfonyl]-3′,5′-O-(1,1,3,3-tet-
raisopropyldisiloxane-1,3-diyl)-â-D-arabinofuranosyl}-O6-[2-(4-ni-
trophenyl)ethyl]guanine (6). Compound 5 (600 mg, 0.837 mmol) was
coevaporated with dry pyridine and dissolved in CH2Cl2 (100 mL).
DMAP (153 mg, 1.26 mmol) and triethylamine (169 mg, 233 µL, 1.67
mmol) were added and the reaction mixture was cooled to 0 °C. Then,
trifluoromethanesulfonyl chloride (211 mg, 158 µL, 1.26 mmol) was
added and the mixture was stirred for 2 h. The reaction mixture was
diluted with dichloromethane, washed with saturated sodium bicarbon-
ate solution, dried over Na2SO4, and evaporated. The crude product
was purified by column chromatography on SiO2 (hexanes/EtOAc, 9/1-
5/5 v/v). Yield: 490 mg of 6 as colorless foam (69%). TLC (hexanes/
1
Cl2/MeOH, 85/15): Rf ) 0.45. H NMR (300 MHz, d6-DMSO): δ
1.63 (s, 3H SeCH3); 2.18 (s, 3H, COCH3); 3.57 (m, 2H, H1-C(5′)
and H2-C(5′)); 3.97 (m, 1H, H-C(4′)); 4.01 (m, 1H, H-C(2′)); 4.31
(s, 1H, H-C(3′)); 5.02 (t, J ) 5.5 Hz, 1H, HO-C(5′)); 5.82 (d, J )
4.6 Hz, 1H, HO-C(3′)); 6.14 (d, J ) 9.1 Hz, 1H, H-C(1′)); 8.30 (s,
1H, H-C(8)); 11.68 (s, 1H, HN-C(2)); 12.05(s, 1H, H-N(1)) ppm.
13C NMR (75 MHz, d6-DMSO): δ 2.90 (SeCH3); 24.21 (COCH3);
46.82 (C2′); 62.05 (C(5′)); 73.23 (C(3′)); 87.71 (C(4′)); 88.89 (C(1′));
120.46 (C(ar)); 138.24 (C(8)); 148.53, 149.31, 155.19, (C(ar)); 173.91
(COCH3) ppm. UV (MeOH): λmax ) 277 nm. ESI-MS (m/z): calcd
for C13H17N5O5Se [M + H]+ 403.26, found 403.76, [M + Na]+ 425.25,
found 426.04.
N2-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-methylselenogua-
nosine (9). Compound 8 (178 mg, 0.443 mmol) was coevaporated with
dry pyridine and then dissolved in pyridine (2 mL). The solution was
treated with 4,4′-dimethoxytrityl chloride (165 mg, 0.487 mmol) in two
portions over a period of 1 h. Stirring was continued overnight. The
1
EtOAc, 50/50): Rf ) 0.44. H NMR (300 MHz, CDCl3): 1.00-1.16
(m, 28H, (iPr)4Si2O); 2.55 (s, 3H, COCH3); 3.35 (t, J ) 6.8 Hz, 2H,
CH2-C6H4-NO2); 3.94 (m, 1H, H-C(4′)); 4.12 (m, 2H, H1-C(5′)
and H2-C(5′)); 4.83 (t, J ) 6.8 Hz, 2H, O6-CH2); 4.95 (m, 1H,
H-C(3′)); 4.30 (s, 1H, HO-C(2′)); 5.46 (m, 1H, H-C(2′)); 6.42 (d,
J ) 5.9 Hz, 1H, H-C(1′)); 7.49 (d, J ) 8.7 Hz, 2H, 4-nitrophenyl
H-C(2)/H-C(6)); 7.88 (s, 1H, HN-C(2)); 8.05 (s, 1H, H-C(8)); 8.17
(d, J ) 8.7 Hz, 2H, 4-nitrophenyl H-C(3)/H-C(5)) ppm. 13C NMR
(75 MHz, CDCl3): δ 12.64, 12.97, 13.10, 13.23, 16.59, 16.72, 16.80,
9
9916 J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006

Selenium-Derivatized RNA for X-ray Crystallography
A R T I C L E S
solvents were removed under vacuum and the residue was dissolved
in dichloromethane, washed with 5% citric acid, water, and saturated
sodium bicarbonate solution, dried over Na2SO4, and evaporated. The
crude product was purified by column chromatography on SiO2 (CH2-
Cl2/MeOH, 98/2-95/5 v/v). Yield: 184 mg of 9 as colorless foam
solution was completely evaporated, tetrabutylammonium fluoride
trihydrate (TBAF‚3H2O) in THF (1 M, 0.95 mL) was added. The
reaction mixture was slowly shaken for at least 12 h at room temperature
to remove the 2′-O-silyl ethers. The reaction was quenched by the
addition of triethylammonium acetate (TEA) (1 M, pH 7.4, 0.95 mL).
The volume of the solution was reduced to 1 mL and the solution was
loaded on a Amersham HiPrep 26/10 Desalting (2.6 × 10 cm; Sephadex
G25). The crude RNA was eluted with H2O and dried.
Analysis of crude RNA products after deprotection was performed
by anion-exchange chromatography on a Dionex DNAPac100 column
(4 × 250 mm) at 80 °C. Flow rate: 1 mL/min; eluant A: 25 mM
Tris-HCl (pH 8.0), 6 M urea; eluant B: 25 mM Tris-HCl (pH 8.0), 0.5
M NaClO4, 6 M urea; gradient: 0-60% B in A within 45 min; UV
detection at 265 nm. Crude RNA products (trityl-off) were purified on
a semipreparative Dionex DNAPac100 column (9 × 250 mm). Flow
rate: 2 mL/min; gradient: ∆5-10% B in A within 20 min. Fractions
containing RNA were loaded on a C18 SepPak cartridge (Waters/
Millipore), washed with 0.1-0.2 M (Et3NH)HCO3 and H2O, and eluted
with H2O/CH3CN (6/4). RNA fractions were lyophilized.
1
(59%). TLC (CH2Cl2/MeOH, 9/1): Rf ) 0.56. H NMR (300 MHz,
CDCl3): δ 1.76 (s, 3H SeCH3); 1.87 (s, 3H, COCH3); 3.27 (m, 1H,
H1-C(5′)); 3.45 (m, 1H, H2-C(5′)); 3.73, 3.74 (2s, 6H, OCH3); 3.90
(m, 1H, HO-C(3′)); 4.11 (m, 1H, H-C(2′)); 4.29 (m, 1H, H-C(4′));
4.57 (s, 1H, H-C(3′)); 5.98 (d, J ) 9.0 Hz, 1H, H-C(1′)); 7.18-7.47
(m, 13H, trityl-H); 7.87 (s, 1H, H-C(8)); 9.63 (s, 1H, HN-C(2));
12.11(s, 1H, H-N(1)) ppm. 13C NMR (75 MHz, CDCl3): δ 4.05
(SeCH3); 23.78 (COCH3); 49.03 (C(2′)); 55.25 (2 × OCH3); 64.10
(C(5′)); 72.94 (C(3′)); 85.45 (C(4′)); 96.48 (C(ar)); 88.88 (C(1′));
113.24, 121.47, 127.12, 127.95, 128.05, 130.03, 135.54, 135.80 (C(ar));
138.36 (C(8)); 144.83, 147.53, 148.85, 155.82, 158.73 (C(ar)); 172.49
(COCH3) ppm. UV (MeOH): λmax ) 260 nm. FT-ICR-ESI-MS (m/z):
calcd for C34H35N5O7Se [M + H]+ 706.17833, found 706.17730.
N2-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-methylselenogua-
nosine 3′-(2-cyanoethyl diisopropylphosphoramidite) (10). Com-
pound 9 (220 mg, 0.312 mmol) was dissolved in a mixture of
ethyldimethylamine (228 mg, 337 µL, 2.24 mmol) in dry dichlo-
romethane (2.5 mL) under argon. After 15 min at room temperature,
2-cyanoethyl N,N-diisopropylchlorophosphoramidite (111 mg, 0.468
mmol) was slowly added and the solution was stirred at room
temperature for 2 h. The crude product was purified by column
chromatography on SiO2 (CH2Cl2/MeOH, 99.5/0.5-98.5/1.5 v/v).
Yield: 248 mg of 10 as colorless foam (88%). TLC (CH2Cl2/MeOH,
97/3): Rf ) 0.63. 1H NMR (500 MHz, CDCl3): δ 1.00-1.27 (m, 24H,
2 × ((CH3)2CH)2N); 1.37, 1.48 (s, 6H, 2x SeCH3); 1.69, 1.73 (s, 6H,
COCH3); 2.24, 2.28 (2 × m, 2H, CH2CN); 2.62, 2.66 (2 × t, 2H, CH2-
CN); 3.15 (m, 2H, H1-C(5′)); 3.54-3.63 (m, 4H, ((CH3)2CH)2N, 2H,
H2-C(5′), 2H, POCH2); 3.77, 3.78 (2s, 12H, OCH3); 3.91-3.99, 4.10-
4.22 (2m, 2H, POCH2); 4.36, 4.41 (m, 2H, H-C(4′)); 4.45 (m, 2H,
H-C(2′)); 4.71, 4.76 (m, 1H, H-C(3′)); 6.01, 6.06 (2 × d, J ) 9.0
Hz, 9.34 Hz, 2H, H-C(1′)); 6.80, 7.26, 7.40, 7.55 (4 × m, 26H, trityl-
H); 7.80 (s, 2H, H-C(8)); 7.67 (s, 2H, HN-C(2)); 11.93 (s, 2H,
H-N(1)) ppm. 31P NMR (121 MHz, CDCl3): δ 151.3, 150.4 ppm;
UV (MeOH): λmax ) 235 nm. FT-ICR-ESI-MS (m/z): calcd for
C34H35N5O7Se [M + H]+ 906.28639, found 906.28685.
Solid-Phase Synthesis of 2′-Methylseleno Nucleoside Containing
RNAs. 2′-O-TOM standard nucleoside phosphoramidites and the
corresponding CPG supports (1000 Å) were obtained from GlenRe-
search. All oligoribonucleotides containing 2′-methylseleno nucleosides
were synthesized on Pharmacia instrumentation (Gene Assembler Plus
or Special) following modified DNA/RNA standard methods containing
an additional cycle step of treatment with DTT; detritylation (2.0
min): dichloroacetic acid/1,2-dichloroethane (4/96); coupling (3.0
min): phosphoramidites/acetonitrile (0.1 M × 120 µL) were activated
by benzylthiotetrazole/acetonitrile (0.35 M × 360 µL); capping (3 ×
0.4 min): (A) Ac2O/sym-collidine/acetonitrile (20/30/50), (B) 4-(di-
methylamino)pyridine/acetonitrile (0.5 M), A/B ) 1/1; oxidation (1.0
min): I2 (10 mM) in acetonitrile/sym-collidine/H2O (10/1/5); DTT
treatment (2.0 min): DTT (100 mM) in ethanol/ H2O (2/3). A ready-
to-use synthesis method file is documented in ref 4. Solutions of
standard amidites, tetrazole solutions, and acetonitrile were dried over
activated molecular sieves overnight. Solutions of 2′-methylseleno
guanosine phosphoramidites were only dried for 4-6 h over activated
molecular sieves before consumption.
Oxidation of 2′-Methylseleno-Modified RNA. Oxidation was
carried out by treating 2′-methylselenoguanosine-containing oligo-
nucleotides (1.5 nmol) with 2 mM iodine in a solution of H2O/CH3-
CN/sym-collidine (65/10/1, v/v/v; 150 µL) for 5 min. Before analysis
by ion-exchange column chromatography (Figure 3; Dionex DNAPac-
100, conditions as above), the solution was extracted with CHCl3/amyl
alcohol (25/1, v/v; 50 µL) twice to remove excess iodine.
Reduction of 2′-Methylselenoxide-Modified RNA. Reduction of
2′-methylselenoxide containing oligonucleotides was achieved by
treatment with DTT in water (1.5 nmol of RNA, 2 mM DTT in 150
µL of H2O) for 5 min. The reaction mixture was directly analyzed by
ion-exchange column chromatography (Figure 3; Dionex DNAPac-100,
conditions as above).
Mass Spectrometry of 2′-Methylseleno Guanosine Containing
RNAs. All experiments were performed on a Finnigan LCQ Advantage
MAX ion trap instrumentation connected to an Amersham Ettan micro
LC system. RNAs were analyzed in the negative-ion mode with a
potential of -4 kV applied to the spray needle. LC: Sample (250 pmol
of RNA dissolved in 20 µL of 20 mM EDTA solution; average injection
volume: 10-20 µL); column (Amersham µRPC C2/C18; 2.1 × 100
mm) at 21 °C; flow rate: 100 µL/min; eluant A: 8.6 mM TEA, 100
mM 1,1,1,3,3,3-hexafluoro-2-propanol in H2O (pH 8.0); eluant B:
methanol; gradient: 0-100% B in A within 30 min; UV detection at
254 nm.
RNA Crystallization and Data Collection. For crystallization
screening, RNAs (1 mM) were annealed in a buffer containing 50 mM
potassium acetate (pH 6.9) and 5 mM MgCl2 at 90 °C for 2 min and
quickly cooled on ice. Crystals were grown by the hanging-drop vapor
diffusion method using Crystal Screen, Crystal Screen 2, and Natrix
kits (Hampton Research). The RNA and reservoir solutions were mixed
in a 1:1 ratio (total volume 2 µL) and incubated at 4 and 20 °C for
several days. Nonmodified and Se-modified RNAs were grown in
parallel in the same wells. Crystals of dimensions 0.1 mm × 0.1 mm
× 0.05 mm from the wells with Crystal Screen #15 solution (1.0 M
Li2SO4, 0.1 M Na-citrate pH 5.6, 0.5 M (NH4)2SO4) and Crystal Screen
#9 solution (30% PEG4000 (w/v), 0.1 M Na-citrate pH 5.6, 0.2 M
NH4-aceteate) were frozen in liquid nitrogen without cryoprotection.
Native and MAD data were collected at beamline X25 at the
Brookhaven National Synchrotron Light Source (NSLS) from the
crystals cooled at 100 K.
Deprotection and Purification of 2′-Methylseleno Nucleoside
Containing RNAs. Prior to deprotection and cleavage from the solid
support the Se-containing RNAs were treated with DTT in ethanol/
H2O 1/1 (150 mM, 200 µL) for 1-3 h at room temperature. Then,
MeNH2 in EtOH (8 M, 0.60 mL), MeNH2 in H2O (40%, 0.60 mL),
and DTT in EtOH/H2O 1/1 (2 M, 95 µL; final DTT concentration 150
mM) were added and deprotection was continued for 5-6 h. After the
Structure Determination. X-ray data were processed with HKL2000
(HKL Research, VA) (Table 1). The “PEG4000” structure was
determined using MAD selenium data. Heavy atom search and phasing
was performed using SHARP,47 including the solvent flattening
procedure. Anomalous phasing power at peak was 1.08, and figure of
(47) de La Fortelle, E.; Bricogne, G. Methods Enzymol. 1997, 276, 472-494.
9
J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006 9917

A R T I C L E S
Moroder et al.
merit before/after solvent flattening was 0.45/0.87. Electron density
maps were calculated using CCP448 up to 3.0 Å resolution limit. The
RNA model was built manually using TURBO-FRODO (http://
afmb.cnrs-mrs.fr/rubrique113.html) and refined with REFMAC up to
2.9 Å resolution limit using data collected from a similar crystal.49
Alternatively, the structure can be solved by molecular replacement
using MOLREP48,50 and the native 16-mer RNA structure as a search
model. The best MOLREP solution, however, places the RNA model
to the wrong position within an infinite RNA helix. The model has to
be shifted along the helical axis using the anomalous Se signal and the
interruptions of the RNA backbone as beacons. The noncanonical
G6•A27 and A11•G27 pairs were modeled using omit 2Fo - Fc and
Fo - Fc electron density maps calculated either without these purines
or their bases. Due to limited resolution and rather poor quality of the
omit maps, we attempted to build all major glycosidic conformations
of these residues followed by refinement with REFMAC and analysis
of the 2Fo - Fc and Fo - Fc electron density maps. The G(syn)•A+-
(anti) conformation showed the best refinement statistics and best fit
to the maps and was chosen over the second best G(anti)•A+(anti)
conformation after analysis and comparison of the electron density maps
calculated using data collected from a few independently grown crystals.
Note that different molecules in the crystals may contain different
conformations of the G•A pairs. The “Li2SO4” structure was solved
by molecular replacement using MOLREP and the RNA model from
the “PEG4000” structure and refined with REFMAC. Sulfate ions were
added to the model based on analysis of 2Fo - Fc and Fo - Fc electron
density maps. All RNA residues were traced in the maps. Coordinates
have been deposited in the Protein Data Bank (accession code 2H1M).
Acknowledgment. We gratefully acknowledge the Austrian
Science Fund FWF (P17864) and the Tiroler Wissenschaftsfonds
(GZ UNI-404/39) for funding and Kathrin Breuker (Cornell
University and University of Innsbruck) for FT-ICR mass
spectra. We are grateful to Dr. Dinshaw J. Patel for support
(NIH GM073618), and Drs. L. Malinina and V. Kuryavyi for
discussion. We thank M. Becker and the staff of beamline X25
(NSLS) for help in data collection.
Supporting Information Available: 1H and 13C NMR spectra
of compounds 2-9; 1H NMR spectrum of compound 10; tables
of intermolecular distances for the 16-mer RNA duplex (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
(48) Collaborative Computational Project, Number 4. Acta Crystallogr., D 1994,
50, 760-763.
(49) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr., D 1997,
53, 240-245.
(50) Vagin, A.; Teplyakov, A. J. Appl. Crystallogr. 1997, 30, 1022-1025.
JA0621400
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9918 J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006