Novel RNA base air with higher specificity using single selenium atom

Article abstract of DOI:10.1093/nar/gks010

Specificity of nucleobase pairing provides essential foundation for genetic information storage, replication, transcription and translation in all living organisms. However, the wobble base pairs, where U in RNA (or T in DNA) pairs with G instead of A, might compromise the high specificity of the base pairing. The U/G wobble pairing is ubiquitous in RNA, especially in non-coding RNA. In order to increase U/A pairing specificity, we have hypothesized to discriminate against U/G wobble pair by tailoring the steric and electronic effects at the 2-exo position of uridine and replacing the 2-exo oxygen with a selenium atom. We report here the first synthesis of the 2-Se-U-RNAs as well as the 2-Se-uridine (^SeU) phosphoramidite. Our biophysical and structural studies of the ^SeU-RNAs indicate that this single atom replacement can indeed create a novel U/A base pair with higher specificity than the natural one. We reveal that the ^SeU/A pair maintains a structure virtually identical to the native U/A base pair, while discriminating against U/G wobble pair. This oxygen replacement with selenium offers a unique chemical strategy to enhance the base pairing specificity at the atomic level.

Full text of DOI:10.1093/nar/gks010

Published online 9 February 2012
Nucleic Acids Research, 2012, Vol. 40, No. 11 5171–5179
doi:10.1093/nar/gks010
Novel RNA base pair with higher specificity using
single selenium atom
Huiyan Sun, Jia Sheng, Abdalla E. A. Hassan, Sibo Jiang, Jianhua Gan and
Zhen Huang*
Department of Chemistry, Georgia State University, Atlanta, Georgia, 30303, USA
Received October 23, 2011; Revised December 8, 2011; Accepted December 27, 2011
U/A pair (4,5). U/G wobble pair offers unique structural
and thermodynamic features (3–5). On the one hand, the
U/G pairing increases structure and function diversities of
RNA (6). But on the other hand, it may jeopardize the
pairing specificity and can cause potential mutations in
RNA transcription and protein translation. Codon–anti-
codon mismatch or misreading is observed with an error
frequency at 10ꢀ5 or higher, which may affect the accuracy
of synthesized proteins (7–9). For instance, the first
position of the codon–anticodon interaction with wobble
mismatch (U/G) was discovered in Escherichia coli (error
frequency = 0.1%) with 100-fold higher than the normal
error level (9). In this mis-incorporation of serine (codon:
AGC) (9), glycine codon (GGC) in mRNA is recognized
by Ser-charged tRNA (anticodon: GCU) instead of
Gly-charged tRNA (anticodon: GCC). Similarly, the
second position of the codon–anticodon interaction with
wobble mismatch (U/G) was also observed, where Lys
(codon: AAA) is mis-incorporated instead of normal
incorporation of Arg (codon: AGA), with much higher
error frequency (5–12%) (10). To avoid the negative
impact of the wobble pairing on the level of protein syn-
thesis, the genetic codes with degeneracy are used to deal
with the consequence of the wobble pairing. Thus, wobble
pairing is often observed at the third codon position
through the codon degeneracy to limit errors. However,
the codons forming the Watson–Crick pairs with tRNA
anticodons are still preferred (11,12). Study shows that the
third codon position with a Watson–Crick base pair can
reduce the frequency of amino acid mis-incorporation by
nearly 10-fold, and it is much more accurate than that
with a wobble pair for the same amino acid (13).
Nevertheless, the 3-nt genetic codes that accommodate
the wobble pairing are used as the most ideal counter-
measure at the level of protein synthesis in living organ-
isms (14). Clearly, on the basis of the chemical principle,
this degeneracy strategy properly guarantees the transla-
tion accuracy at the protein level by tolerating wobble
pairs and silent mutations at the RNA and DNA levels.
Since the 2-exo-oxygen of uridine plays a significant role
in U/G wobble pair, we hypothesized that tailoring the
ABSTRACT
Specificity of nucleobase pairing provides essential
foundation for genetic information storage, replica-
tion, transcription and translation in all living organ-
isms. However, the wobble base pairs, where U in
RNA (or T in DNA) pairs with G instead of A, might
compromise the high specificity of the base pairing.
The U/G wobble pairing is ubiquitous in RNA, espe-
cially in non-coding RNA. In order to increase U/A
pairing specificity, we have hypothesized to discrim-
inate against U/G wobble pair by tailoring the steric
and electronic effects at the 2-exo position of
uridine and replacing the 2-exo oxygen with a
selenium atom. We report here the first synthesis
of the 2-Se-U-RNAs as well as the 2-Se-uridine
(
SeU) phosphoramidite. Our biophysical and struc-
tural studies of the SeU-RNAs indicate that this
single atom replacement can indeed create a novel
U/A base pair with higher specificity than the natural
one. We reveal that the SeU/A pair maintains a struc-
ture virtually identical to the native U/A base pair,
while discriminating against U/G wobble pair. This
oxygen replacement with selenium offers a unique
chemical strategy to enhance the base pairing
specificity at the atomic level.
INTRODUCTION
DNA and RNA are crucial genetic information carriers
(1,2). The base pairs of DNAs (T/A and C/G) and RNAs
(U/A and C/G) need to be highly specific and accurate for
the purpose of the precise genetic information storage,
replication, transcription and translation. However, the
wobble base pairs, where U in RNA (or T in DNA)
pairs with G instead of A, may compromise the high spe-
cificity of the base pairing. In RNA, especially non-coding
RNA, U/G wobble pair (Figure 1) is ubiquitous (3) and
sometimes it has the similar stability as the Watson–Crick
*To whom correspondence should be addressed. Tel:/Fax: +1 404 413 5535; Email: huang@gsu.edu
ß The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

5172 Nucleic Acids Research, 2012, Vol. 40, No. 11
evaporated under reduced pressure. The residue was
purified by flash column chromatography (10%
methanol in dichloromethane) and pure compound 7
1
was obtained in 95% yield. H NMR (CDCl3) d: 7.87
(d, J = 7.7 Hz, 1H, H-6), 7.44–7.20 (m, 9H, Ar), 6.85
(m, 4H, Ar), 6.11 (br, 1H, OH), 5.88 (d, J = 6.0 Hz, 1H,
H-10), 5.54 (d, J = 7.7 Hz, 1H, H-5), 4.63 (m, 1H, H-40),
4.44 (m, 1H, H-30), 4.24 (d, J = 2.3 Hz, 1H, H-20), 3.75 (d,
J = 3.1 Hz, 6H, OCH3), 3.42 (m, 2H, H-50), 3.40–3.30 (br,
1H, OH), 2.55 (s, 3H, SCH3); 13C NMR (CDCl3) d:
169.19 (C-4), 164.36 (C-2), 158.88 (Ar), 144.49 (Ar),
140.13 (C-6), 135.37 (Ar), 135.22 (Ar), 130.41 (Ar),
130.28 (Ar), 128.32 (Ar), 128.28 (Ar), 127.29 (Ar),
113.54 (Ar), 108.92 (C-5), 91.95 (C-10), 87.35 (C-Ar3),
84.82 (C-40), 75.24 (C-20), 71.63 (C-30), 63.40 (C-50),
55.40 (OCH3), 15.39 (SCH3); High resolution mass
spectra (HRMS) electrospray ionization-time of fight
Figure 1. Native and Se-modified U/A pairs and U/G wobble pairs.
steric and electronic effects at this site may discrimin-
ate against the wobble pair, enabling the modified U/A
base pair with higher specificity. Interestingly, selenium
has been discovered in natural tRNAs in the 2-Se-
uridine form, i.e. 5-methylaminomethyl-2-selenouridine
(mnm5se2U), in the wobble position on the anticodon
loop (15,16). The function of such selenium modification
is not completely clear yet, though it was proposed that
such Se derivatization on tRNAs probably improves the
accuracy and efficiency of protein translation (17).
Similarly, the corresponding sulfur modification has
been observed on natural tRNAs (18). Sulfur was chem-
ically introduced to the 2-position of uridine (19,20). The
S-modified U/G pair is slightly less stable than the native
U/G pair (5), while the SU/A is more stable over the native
U/A pair. Thus, we hypothesized that the 2-oxygen
replacement with selenium (SeU, Figure 1) can destabilize
and discriminate against the U/G wobble pair, because the
(ESI-TOF)
Chemical formula: C31H33N2O7S.
[M+H+]= 577.2003
(calc.
577.2008),
1-(50-O-4,40-dimethoxytrityl-ꢀ-D-ribofuranosyl)-2-
selenouridine 8. A solution of NaSeH was generated by
addition of absolute ethanol (50 ml) to selenium (6.2 g,
78 mmol) and sodium borohydride (NaBH4, 4.43 g,
0.117 mol) at 0ꢁC. The reaction was completed in 2 h
and a clear solution was formed. The ethanolic solution
was added to compound 7 (4.5 g, 7.80 mmol) and the
mixture was stirred for 8 h under argon. The reaction
mixture was then concentrated under reduced pressure
and ethyl acetate (50 ml) was added to the residue. The
organic layer was washed with water several times
(5 ꢂ 30 ml), and then dried over anhydrous magnesium
sulfate. Purification was performed by flash column chro-
matography (4% methanol in dichloromethane) and the
˚
atomic size of selenium (1.16 A) is larger than that of
˚
˚
sulfur (1.02 A) and oxygen (0.73 A). Moreover, selenium
has the least ability to form a hydrogen bond among O, S
and Se, which weakens the hydrogen bond originally
formed by the 2-oxygen of the wobble pair. Thus, it is
expected that this 2-Se-replacement can largely destabilize
U/G pair by generating a steric hindrance against the pair
and significantly weakening the hydrogen bond.
Furthermore, it is expected that the 2-Se-substitution
does not significantly affect the hydrogen bonds within
the U/A pair, since the 2-oxygen is not directly involved
in the U/A base pairing. Therefore, we decided to incorp-
orate selenium into the 2-position of uridine in RNA, in
order to atom-specifically increase the U/A pair specificity
and disrupt the U/G wobble.
1
light yellow compound (8) was obtained (85% yield). H
NMR (CDCl3) d: 10.95 (s, 1H, NH), 8.24 (d, J = 8.2 Hz,
1H, H-6), 7.44–7.19 (m, 9H, Ar), 6.84 (m, 4H, Ar), 6.48 (s,
1H, H-10), 5.66 (d, J = 8.1 Hz, 1H, H-5), 4.48 (m, 2H,
H-40,H-30), 4.22 (m, 1H, H-20), 3.89 (s, 1H, OH), 3.79 (s,
6H, OCH3), 3.58 (dd, J = 23.6, 9.2 Hz, 2H, H-50), 2.97 (br,
1H, OH); 13C NMR (CDCl3) d: 175.74 (C-2), 159.21
(C-4), 158.98 (Ar), 158.94 (Ar), 144.45 (Ar), 140.82
(C-6), 135.38 (Ar), 135.18 (Ar), 130.35 (Ar), 130.27 (Ar),
128.30 (Ar), 128.28 (Ar), 127.45 (Ar), 113.58 (Ar), 108.37
(C-5), 96.86 (C-10), 87.38 (C-Ar3), 84.41 (C-40), 76.33
(C-20), 69.19 (C-30), 61.20 (C-50), 55.48 (OCH3). HRMS
MATERIALS AND METHODS
(ESI-TOF)
Chemical formula: C30H29N2O7Se; UV (MeOH):
max = 311 nm (in methanol).
[M-H+]ꢀ = 609.1136
(calc.
609.1140),
Synthesis of 2-Se-uridine phosphoramidite
1-(50-O-4,40-dimethoxytrityl-ꢀ-D-ribofuranosyl)-2-
ꢁ
methylthiouridine 7. Five grams of dry compound 6
(its synthesis: Scheme S1 in Supplementary Data) was
dissolved in dry N, N-dimethylformamide (DMF),
followed by addition of iodomethane (5.5 ml, 89 mmol).
1,8-Diazabicyclo[5.4.0]undec-7-ene (2 ml, 13.3 mmol) was
then added to the reaction mixture at 0ꢁC. The reaction
was monitored by thin layer chromatography (TLC)
plate (12% methanol in dichloromethane, Rf = 0.4) and
completed in 4 h. Ethyl acetate (50 ml) was poured into the
mixture and DMF was removed by washing the organic
layer with saturated sodium chloride solution. The organic
phase was dried over anhydrous magnesium sulfate and
1-(20-O-tert-butyldimethylsilyl-50-O-4,40-dimethoxytrityl-ꢀ-
D-ribofuranosyl)-2-selenouridine 9a and 1-(30-O-tert-
butyldimethylsilyl-50-O-4,40-dimethoxytrityl-ꢀ-D-ribofu-
ranosyl)-2-selenouridine 9b. 50-DMTr-2-selenouridine 8
(0.5 g, 0.82 mmol) was dissolved in dry DMF, then tert-
butyldimethylsilyl chloride (TBDMSCl, 0.15 g, 0.98 mmol)
and imidazole (0.11 g, 1.64 mmol) were added into the
solution under nitrogen gas. The reaction was monitored
by TLC plate (15% ethyl acetate in dichloromethane,
Rf = 0.8). The mixture was stirred overnight at room tem-
perature and then directly poured into ethyl acetate

Nucleic Acids Research, 2012, Vol. 40, No. 11 5173
O
N
O
N
O
NH
N
NH
S
a
S
CH3
b
N
Se
DMTrO
DMTrO
DMTrO
O
O
O
OH OH
6
OH OH
7
OR3 OR2
8: R2 = H, R3 = H
9: 9a (R2 = TBDMS, R3 = H);
9b (R2 = H, R3 = TBDMS)
c
O
O
d
N
N
Oligonucleotide-5'
O
CN
CN
P
SeU
N
Se
N
Se
f
e
O
DMTrO
DMTrO
-O
O
O
O
O
O
O
OH
OR2
OTBDMS
OR3
P
P
-O Oligonucleotide-3'
N
OCH2CH2CN
10: 10a (R2 = TBDMS, R3 = H);
10b (R2 = H, R3 = TBDMS)
12
11
Scheme 1. Synthesis of SeU-phosphoramidite 11 and RNAs (12). Reagents and conditions: (a) CH3I, DBU, DMF; (b) Se, NaBH4, EtOH;
(c) TBDMS-Cl, imidazole, DMF; (d) ICH2CH2CN, (i-Pr)2NEt, CH2Cl2; (e) (i-Pr2N)2P(Cl)OCH2CH2CN, (i-Pr)2NEt, CH2Cl2; (f) solid-phase
synthesis.
(20 ml) and washed with water (2 ꢂ 20 ml). The organic
layer was dried by anhydrous magnesium sulfate and
evaporated under reduced pressure. Two compounds,
9a and 9b, were obtained. The two regional isomers
(ratio 1:1) were purified together by flash column chroma-
tography (10% ethyl acetate in dichloromethane) and
were not further separated. Since, it was both challenging
and unnecessary to separate each isomer, we decided to
move to the next step of synthesis without separation of
these two isomers. HR-MS (ESI-TOF, 9a and 9b)
[M-H+]ꢀ = 723.1990 (calc. 723.2005). Chemical formula:
C36H43N2O7SeSi.
(d, J = 7.7 Hz, 1H, H-5), 5.60 (d, J = 6.5 Hz, 1H, H-10),
4.61–4.49 (m, 1H, H-40), 4.31 (m, 2H, H-30, H-20), 3.80
(s, 6H, OCH3), 3.54–3.34 (m, 4H,H-50, SeCH2CH2CN),
3.01 (m, 2H, SeCH2CH2CN), 2.91 (s, 1H, OH), 0.94 (s,
9H, SiCMe3), 0.09 (d, 6H, SiMe2); 13C NMR (CDCl3) d:
167.70 (C-4), 159.04 (C-2), 158.51 (Ar), 144.17 (Ar),
139.19 (C-6), 134.91 (Ar), 134.74 (Ar), 130.26 (Ar),
130.17 (Ar), 128.30 (Ar), 128.11 (Ar), 127.58 (Ar),
118.78 (CN), 113.58 (Ar), 110.61 (C-5), 93.13 (C-10),
87.82 (C-Ar3), 85.39 (C-40), 77.27 (C-20), 72.40 (C-30),
63.82 (C-50), 55.44 (OCH3), 25.84 (SiCMe3), 24.06
(SeCH2CH2CN), 18.89 (SeCH2CH2CN), 18.14 (SiCMe3),
ꢀ4.56 (SiCH3), ꢀ4.91 (SiCH3). HRMS (ESI-TOF)
[M+H+]+= 778.2464 (calc. 778.2427). Chemical formula:
C39H48N3O7SeSi. 10b: 1H NMR (CDCl3) d: 8.09 (d,
J = 7.7 Hz, 1H, H-6), 7.32 (m, 9H, Ar), 6.88 (m, 4H,
Ar), 5.75 (d, J = 7.7 Hz, 1H, H-5), 5.65 (d, J = 3.7 Hz,
1H, H-10), 4.46 (m, 1H, H-20), 4.21 (dd, J = 9.3, 5.1 Hz,
1H, H-40), 4.18–4.08 (m, 1H, H-30), 3.83 (s, 6H, OCH3),
3.70 (m, 1H, H-50), 3.55 (m, 1H, H-50), 3.41 (m, 2H,
SeCH2CH2CN), 3.22 (d, J = 5.5 Hz, 1H, OH), 3.08 (m,
2H, SeCH2CH2CN), 0.90 (s, 9H, SiCMe3), 0.11 (d, 6H,
SiMe2). 13C NMR (CDCl3) d: 167.90 (C-4), 159.05 (C-2),
157.82 (Ar), 143.99 (Ar), 138.96 (C-6), 135.02 (Ar), 134.89
(Ar), 130.35 (Ar), 130.34 (Ar), 128.37 (Ar), 128.29 (Ar),
127.58 (Ar), 118.95 (CN), 113.56 (Ar), 113.53 (Ar), 110.49
(C-5), 93.84 (C-10), 87.57 (C-Ar3), 84.72 (C-40), 76.17
(C-20), 71.05 (C-30), 61.71 (C-50), 55.48 (OCH3), 25.82
(SiCMe3), 24.12 (SeCH2CH2CN), 18.92 (SeCH2CH2CN),
18.17 (SiCMe3), ꢀ4.59 (SiCH3), ꢀ4.60 (SiCH3). HRMS
(ESI-TOF) [M+H+]+= 778.2401 (calc. 778.2427).
Chemical formula: C39H48N3O7SeSi.
1-(20-O-tert-butyldimethylsilyl-50-O-4,40-dimethoxytrityl-ꢀ-
D-ribofuranosyl)-2-cyanoethylselanyluridine 10a and 1-(30-
O-tert-butyldimethylsilyl-50-O-4,40-dimethoxytrityl-ꢀ-D-
ribofuranosyl)-2-cyanoethylselanyluridine
10b. The
mixture (0.52 g, 0.72 mmol) of 9a and 9b was dissolved
in dried dichloromethane at 0ꢁC. Iodopropionitrile
(0.78 g, 4.31 mmol) was added to the solution, followed by
addition of diisopropylethylamine (0.37 ml, 2.15 mmol).
The reaction was monitored by TLC plates (30% ethyl
acetate in dichloromethane). After 4-h reaction, the
solvent was removed under reduced pressure and the
residue was partitioned between ethyl acetate (20 ml) and
water (20 ml). The organic phase was dried over
anhydrous magnesium sulfate and evaporated into
dryness. Two crude products were obtained: 1-(20-O-tert-
butyldimethylsilyl-50-O-4,40-dimethoxytrityl-b-D-ribofur-
anosyl)-2-cyanoethylselanyluridine 10a (Rf = 0.35) and 1-
(30-O-tert-butyldimethylsilyl-50-O-4,40-dimethoxytrityl-b-
D-ribofuranosyl)-2-cyanoethylselanyluridine
10b
(Rf = 0.30). These two compounds can be separated by
flash column chromatography (15% ethyl acetate in
dichloromethane). 10a was obtained in 0.228 g (41%
yield) and 10b was obtained in 0.235 g (42% yield). 10a:
1H NMR (CDCl3) d: 7.96 (d, J = 7.7 Hz, 1H, H-6),
7.53–7.11 (m, 9H, Ar), 6.85 (m, 4H, Ar), 5.71
1-[20-O-tert-butyldimethylsilyl-30-O-(2-cyanoethyl-N,N-
diisopropylamino)
thoxytrityl-ꢀ-D-ribofuranosyl)]-2-cyanoethylselanyluridine
11. Diisopropylethylamine (15.5 mg, 0.12 mmol)
and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
phosphoramidite-50-O-(4,40-dime-

5174 Nucleic Acids Research, 2012, Vol. 40, No. 11
(26 mg, 0.11 mmol) were added to a solution of 10a
(100 mg, 0.10 mmol) in dry dichloromethane (5 ml)
at room temperature under nitrogen gas. The mixture
was monitored by TLC (15% ethyl acetate in
dichloromethane). When the reaction was completed in
4 h, rapid Al2O3 column chromatography (dichloro-
methane as the eluent) was performed to remove the
organic salts. The solvent was then evaporated under
reduced pressure and the residue was dissolved in 0.5 ml
dichloromethane and precipitated in dry hexane under
vigorous stirring. The precipitate was collected by filtra-
tion, dried under reduced pressure and directly used for
solid-phase synthesis. 1H NMR (CDCl3) d: 7.95
(d, J = 7.7 Hz, 1H, H-6), 7.30 (m, 9H, Ar), 6.84 (m,4H,
Ar), 5.82 (d, J = 7.6 Hz, 1H, H-10), 5.63 (d, J = 7.7 Hz,
1H, H-5), 4.68 – 4.43 (m, 1H, H-40), 4.43 – 4.29 (m,
1H,), 4.24 (s, 1H), 3.98 (dd, J = 15.6, 8.4 Hz, 2H), 3.80
(s, 6H, OCH3), 3.63 (dd, J = 19.8, 7.3 Hz, 4 H), 3.42
(dd, J = 19.3, 8.7 Hz, 5H), 3.01 (s, 2H), 2.70
(d, J = 5.8 Hz, 2H), 1.20 (d, J = 6.7 Hz, 18H), 1.08
(d, J = 6.5 Hz, 6H), 0.92 (s, 13H), 0.14 – 0.02 (m, 9H);
13C NMR (CDCl3) d: 167.83 (C-4), 159.04 (C-2), 158.71
(Ar), 144.16 (Ar), 139.02 (Ar), 134.96 (Ar), 134.74 (Ar),
130.21 (Ar), 130.14 (Ar), 128.38 (Ar), 128.04 (Ar), 127.58
(Ar), 118.86 (SeCH2CH2CN), 117.75 (OCH2CH2CN),
113.67 (Ar), 110.69 (C-5), 92.50 (C-10), 87.92 (C-Ar3),
85.43 (C-40), 77.15 (C-20), 72.81 (C-30), 63.72 (C-50),
59.39 (OCH2CH2CN), 55.48 (OCH3), 43.22-43.10
(NCMe2), 29.90 (OCH2CH2CN), 26.12-25.97 (NCMe2),
Native RNAs were purchased from Integrated DNA
Technologies. The concentrations of the native,
Se-modified RNAs were adjusted to 1.0 mM in water.
The Se-RNA samples were characterized by matrix
assisted laser desorption/ionization-time of fight mass
spectrometry (MALDI-TOF MS) (Table 1) and HPLC
(Figure 2).
pH titration curve of 2-selenouridine
2-Selenouridine was prepared through detritylation of
1-(50-O-4,40-dimethoxytrityl-b-D-ribofuranosyl)-2-selenour
idine (8) by acid treatment. The 2-selenouridine solutions
Table 1. MALDI-TOF MS of 2-Se-U RNAs
Entry
Oligonucleotide
molecular formula
Measured (calc.) m/z
1
2
3
50-rGUAUASeUAC-30
C76H94N29O53P7Se
[M+H]+= 2558.7 (2558.5)
[M+H]+= 3740.3 (3740.2)
[M+H]+= 3859.4 (3859.3)
50-rAUCACCSeUCCUUA-30
C111H141N38O82P11Se
50-rAAUGCSeUGCACUG-30
C114H142N45O81P11Se
24.85
(SiCMe3),
24.02
(SeCH2CH2CN),
18.94
(SeCH2CH2CN), 18.37 (SiCMe3), ꢀ4.35 (SiCH3), ꢀ4.56
(SiCH3); 31P NMR (CDCl3) d: 148.81, 152.30. HRMS
(ESI-TOF) [M+H+]+= 978.3528 (calc. 978.3505).
Chemical formula: C48H65N5O8PSeSi.
Solid-phase synthesis of the 2-Se-functionalized RNAs
ABI3400 DNA/RNA Synthesizer was used for all the
RNA oligonucleotides synthesis (1.0 mmol scale). All the
non-modified nucleoside phosphoramidite reagents used
were ultra-mild (Glen Research). RNA oligonucleotides
were synthesized in DMTr-on form, cleaved from the
beads and deprotected by the treatment of 0.05 M
K2CO3 methanol solution for 10 h at room temperature.
After evaporating the solution to dryness, the 20-TBDMS
deprotection was performed in TBAF (0.5 ml, 1 M) for
14 h at room temperature. Then the RNAs were treated
with 1 M Tris–HCl buffer (0.5 ml, pH 7.5) for 5 min,
followed by concentrating to 0.5 ml and desalting using
G-25 Sephadex column. The 50-DMTr deprotection was
then performed using Glen-Pek RNA column, followed
by desalting using Sep-Pak Vas column.
HPLC analysis and purification
The RNA oligonucleotides were analyzed and purified by
reversed-phase high performance liquid chromatography
(RP-HPLC), flow rate 6 ml/min [buffer A: 20 mM
triethylammonium acetate (TEAAc, pH 7.1) in water;
buffer B: 20 mM TEAAc (pH 7.1) in 50% acetonitrile].
The HPLC analysis was performed with a linear
gradient from buffer A to 100% buffer B in 20 min.
Figure 2. HPLC analysis of 2-Se-U modified RNA 12-mer
(50-rAUCACCSeUCCUUA-30). (A) The crude DMTr-on Se-RNA,
retention time was 12.2 min. (B) The pure DMTr-off Se-RNA with
same gradient and buffer, retention time was 7.1 min. Samples were
eluted with a linear gradient from buffer A (20 mM triethylammonium
acetate, pH 7.1) to 70% buffer
B (50% acetonitrile, 20 mM
triethylammonium acetate, pH 7.1) in 10 min, to 100% buffer B in
12 min and continuous 100% buffer B to 20 min.

Nucleic Acids Research, 2012, Vol. 40, No. 11 5175
were adjusted to desired pH values in the buffer of 50 mM
Na2HPO4 at room temperature. The UV–Vis spectra were
recorded every 0.1 pH unit between pH 6–8 and every 0.2–
0.5 pH unit between pH 4–6 and pH 8–10. The pH of each
solution was measured before and after its UV–Vis
spectrum collection and the error was within 0.02 pH
unit. The titration data was plotted and shown in Figure 3.
melting experiment was 0.5ꢁC per min. The melting tem-
perature data and curves of the matched and mismatched
duplexes are presented in Table 2 and Figure 4.
Crystallization
The purified RNA oligonucleotide (50-GUAUA-SeU-AC-
30, 1 mM) was heated to 80ꢁC for 2 min, and cooled down
slowly to room temperature. The Nucleic Acid Mini
Screen Kit (Hampton Research) was applied to screen
the crystallization conditions at different temperatures
(10, 20 and 25ꢁC) using the hanging-drop method by
vapor diffusion.
Thermodenaturization of duplex RNAs
The UV-melting temperature studies were carried out by
Cary 300 UV–Vis Spectrophotometer and a temperature
control module. The samples (2 mM RNA duplexes) were
dissolved in buffer of 150 mM NaCl, 2 mM MgCl2 and
10 mM Na2HPO4–NaH2PO4 (pH 6.8). The samples were
heated to 80ꢁC and cooled to room temperature slowly
and then kept in 4ꢁC overnight. The heating rate of
Data collection
Perfluoropolyether was used as a cryoprotectant during
the crystal mounting, and data collection was taken
under the liquid nitrogen stream at ꢀ174ꢁC. The
Se-RNA crystal data were collected at beam line X12B
and X12C in NSLS of Brookhaven National
Laboratory. A number of crystals were screened to
Figure 3. Plot of wavelength (nm) versus pH for 2-selenouridine
nucleoside. The fitted titration curve yields the pKa value (7.29 0.02).
Table 2. Melting temperatures (Tm) of the native, S- and Se-modified
RNA duplexes
Entry
Sequences
Base pairs
Tm (ꢁC)
1
2
3
4
5
I: 50-rAUCACCUCCUUA-30
I+30-rUAGUGGAGGAAU-50
I+30-rUAGUGGGGGAAU-50
I+30-rUAGUGGCGGAAU-50
I+30-rUAGUGGUGGAAU-50
II: 50-rAUCACCSeUCCUUA-30
II+30-rUAGUGGAGGAAU-50
II+30-rUAGUGGGGGAAU-50
II+30-rUAGUGGCGGAAU-50
II+30-rUAGUGGUGGAAU-50
III: 50-rAAUGCUGCACUG-30
III+30-rUUACGACGUGAC-50
III+30-rUUACGGCGUGAC-50
III+30-rUUACGCCGUGAC-50
III+30-rUUACGUCGUGAC-50
U/A
U/G
U/C
U/U
62.8
62.5
50.6
48.8
6
7
8
9
10
11
12
13
14
15
SeU/A
SeU/G
SeU/C
SeU/U
65.8
58.5
50.3
57.3
U/A
U/G
U/C
U/U
64.1
59.4
52.0
51.5
16
17
18
19
20
IV: 50-rAAUGCSeUGCACUG-30
IV+30-rUUACGACGUGAC-50
IV+30-rUUACGGCGUGAC-50
IV+30-rUUACGCCGUGAC-50
IV+30-rUUACGUCGUGAC-50
SeU/A
SeU/G
SeU/C
SeU/U
66.5
55.5
51.9
58.0
Figure 4. Normalized UV-melting curves of RNA duplexes. (A) Native
RNA (50-rAAUGCUGCACUG-30) paired with matched and
mismatched strands. (B) Se-RNA (50-rAAUGCSeUGCACUG-30) with
matched and mismatched strands.
Bold and underlined sequences indicate the pairing and mis-pairing sites.

5176 Nucleic Acids Research, 2012, Vol. 40, No. 11
Table 3. Data collection and refinement statistics of SeU-RNA
to the 2-position of thymidine in DNA (29). This success-
ful strategy has encouraged us to introduce the selenium
functionality to the 2-position of uridine in RNA. Herein,
we report the first synthesis of the 2-selenouridine deriva-
tives and RNAs. The synthesis (Scheme S1 in
Supplementary Data) started from the glycosidation (30)
of the acylated ribofuranose (1) with silylated 2-thiouracil
(3), followed by benzoyl deprotection and trityl protection
of the 50-hyroxyl group to offer 6 (31). After methylation
of 6 to activate the 2-thio-functionality (29), NaSeH was
used to displace the 2-S-functionality and offer the
2-Se-uridine 8 in 85% yield. Following the protections
of the 20-hydroxyl group and the 2-Se-functionality
with ICH2CH2CN, the Se-phosphoramidite 11
was synthesized by phosphitylation of 10 a (29,32,33).
The SeU-phosphoramidite was finally incorporated
into RNAs by solid-phase synthesis. The synthesized
SeU-RNAs (12) were deprotected, purified and con-
firmed by HPLC and MS (Table 1 and Figure 2).
The characterization of the Se-nucleosides and
Se-nucleotides is presented in Supplementary Figures
S1–S23.
Structure (PDB ID)
GUAUA-SeU-AC (3S49)
Data collection
Space group
Cell dimensions: a, b, c (A),
R32
47.095, 47.095, 424.655,
90, 90, 120
˚
a, b, g (ꢁC)
90, 90, 120
Resolution range (A) (last shell)
Unique reflections
Completeness (%)
Rmerge (%)
˚
50.0–2.28 (2.37–2.28)
9870 (959)
99.8 (99.4)
7.7 (23.5)
21.0 (3.9)
18.8 (10.7)
I/s(I)
Redundancy
Refinement
Resolution range (A)
Rwork(%)
Rfree (%)
˚
30.0–2.3
21.4
26.9
Number of reflections
Number of atoms
Nucleic acid (double)
Heavy atoms and ion
Water
8206
1162
7 Se
71
RMS deviations
˚
Bond length (A)
Bond angle
0.007
1.169
Characterization of the 2-Se-functionalized RNA
identify the one with strong anomalous scattering at the
K-edge absorption of selenium. The distance of the
detector to the crystals was set to 150 mm. The wavelength
After cleavage from solid support and deprotection, the
crude DMTr-on RNAs were purified by RP-HPLC and
lyophilized to dryness. As shown in Figure 2A, the
coupling yield is ꢃ90%. The 50-DMTr deprotection of
the oligonucleotides was then performed using Glen-Pek
RNA column. The HPLC analysis of the DMTr-off RNA
was shown in Figure 2B. All the pure seleno-RNA oligo-
nucleotides were characterized by MALDI-TOF
MS (Table 1).
˚
of 0.9795 A was chosen for selenium SAD phasing. The
crystals were exposed for 10 or 15 s/image with 1ꢁ oscilla-
tion, and a total of 180 images were taken for each data
set. All the data were processed using HKL2000 and
DENZO/SCALEPACK (21).
Structure determination and refinement
The structure of the Se-RNA [50-GUAUA-SeU-AC-30]2
was solved by molecular replacement with both CNS
(22) and Phaser (23). The refinement protocol includes
simulated annealing, positional refinement, restrained
B-factor refinement, and bulk solvent correction.
The stereo-chemical topology and geometrical restrain
parameters of DNA/RNA (24) have been applied.
The topologies and parameters for modified uridine with
2-selenium (US) were constructed and applied.
After several cycles of refinement, a number of highly
ordered waters were added. Finally, the occupancies of
selenium were adjusted. Cross-validation (25) with a
5–10% test set was monitored during the refinement.
The sA-weighted maps (26) of the (2 mjFojꢀDjFcj) and
the difference (mjFojꢀDjFcj) density maps were
computed and used throughout the model building
(Table 3).
Thermodenaturization study of 2-Se-uridine RNAs
containing match and mismatch base pairs
UV-melting temperatures (Tm) of the native and
Se-modified duplexes with match and mismatch sequences
are shown in Table 2, Figures 4 and 5. Tm of the Se-RNA
duplex containing the SeU/A Watson–Crick pair was
3.0ꢁC higher for one duplex (or 2.4ꢁC higher for the
other duplex) than those of the corresponding duplexes
containing native U/A pair (Table 2). Comparing with
Se
native U/G, the U/G pair is ꢃ4ꢁC less stable than the
native U/G pair, suggesting that SeU discourages the SeU/
G pair formation. While the SeU/C mis-pair is slightly less
stable than the native U/C mis-pair, the SeU/U mis-pair is
more stable than the native U/U mis-pair. The higher
stability may be attributed to the higher acidity of the
imino group (3-NH) of SeU [pKa = 7.29 0.02,
Figure 3, compared to that of the native uridine
(pKa = 9.18 0.02) (34)], which may promote U/U inter-
action via hydrogen bond. In addition, considering a
RESULTS AND DISCUSSION
˚
selenium atom is 0.43 A larger in atomic radius than an
Synthesis of 2-Se-uridine phosphoramidite
oxygen atom, the 2-Se atom may strengthen the stacking
interaction between SeU and its 30-nucleobase
(Supplementary Figure S24).When directly comparing
the Watson–Crick base pairs (U/A and SeU/A) with
their own corresponding mis-pairs, it is clear that SeU/A
pair has the balanced discrimination against all mis-pairs,
Though selenium was incorporated into uridine four
decades ago (27,28), RNA containing 2-Se-uridine (SeU)
has not been synthesized because of the synthetic chal-
lenge. Recently, our laboratory has successfully developed
a novel strategy to incorporate the selenium functionality

Nucleic Acids Research, 2012, Vol. 40, No. 11 5177
Figure 5. Differencesa of melting temperatures (Tm) of the native and Se-modified U/A pairs and their corresponding mis-pairs. Native (white bar)
refers to the Tm difference between the native U/A pair and the other mis-pairs (U/G, U/C and U/U); Se-Modified (gray bar) refers to the Tm
difference between the SeU/A pair and the other modified mis-pairs (SeU/G, SeU/C and SeU/U).
with the Tm differences of SeU/G (7.3ꢁC for one duplex or
11ꢁC for the other in Table 2, and Figure 5), SeU/C
(15.5ꢁC or 14.6ꢁC), and SeU/U (8.5ꢁC for both sequences).
While maintaining fine discrimination against U/C pair
(Tm difference: 12.2 or 12.1ꢁC) and U/U pair
(Tm difference: 14 or 12.6ꢁC), the native U/A pair
poorly discriminates against U/G wobble pair (the Tm dif-
ferences: 0.3 or 4.7ꢁC in Figure 5 and Table 2). Therefore,
in general, SeU/A has higher base pair fidelity than the
native U/A pair.
Since the Tm differences between the native U/A pair
and U/G wobble pair were relatively small (0.3 and 4.7ꢁC
in Figure 5). The small Tm differences indicate possible
changes between U/A and U/G pairs without a significant
decrease in duplex stability. This is consistent with the
ubiquitous presence of U/G wobble pair in RNAs,
which diversifies the structure and function of RNAs,
especially non-coding RNAs. Such small thermostability
difference between native U/A pair and U/G wobble pair
has been previously observed in the literature (4,5). On the
contrary, the Tm differences between the SeU/A and SeU/G
pairs were significant, such as 7.3ꢁC (versus 0.3ꢁC in the
native) and 11ꢁC (versus 4.7ꢁC in the native) in Figure 5.
The single selenium atom replacement directly decreases
the thermal stability of the U/G wobble pair by 4.0 and
3.9ꢁC in RNA duplexes (Table 2). This experimental ob-
servation reveals that the U/G wobble pair can be greatly
discriminated by incorporating a selenium atom to the
2-position of uridine. The strong discrimination against
U/G pair is mainly attributed to the selenium disruption
of the hydrogen bond formed by the 2-oxygen (Figure 1)
and to the steric effect of the bulky selenium atom at the
2-position. Our results indicate that the 2-Se-modification
on uridine significantly increases the high specificity of the
U/A base pair.
of SeU/A pairing. Similar to the native, the Se-RNA
crystal is also in rhombohedral space group R32. The
˚
Se-RNA structure, determined at 2.3 A resolution, is
˚
virtually identical to the native one (35) (at 2.2 A reso-
lution, Figure 6). Interestingly, the Se-RNA crystals
grew much faster than the native ones. In six days, the
Se-RNA formed diffraction-quality crystals in decent
sizes (approximately 0.05 ꢂ 0.05 mm), while the
corresponding native did not crystallize in 3–4 weeks
under the same conditions. Moreover, the Se-RNA
crystals could form in broader buffer conditions (12 out
of 24 conditions in Hampton buffers) than the
corresponding native (2 out of 24 conditions). This
observation of faster crystal growth of the Se-RNA is
consistent with the Se-facilitated duplex stability. As
shown in Figure 6A, there are seven self-complementary
RNA molecules in a unit cell, and the overall shape of the
duplexes is almost linear (ꢃ8ꢁ inclination to the screw
axes). Although this assembling pattern results in the
discontinued backbones and grooves, the duplexes stack
on top of each other in a head-to-tail fashion, and a
peudo-fiber is formed. The data collection and structure
refinement statistics are summarized in Table 3.
Since 2-exo-oxygen of uridine is not involved in the
hydrogen bond interactions of U/A pairing, it’s expected
that the U/A pair will accommodate the larger selenium
atom at this position (Figures
1 and 6C). The
Se-modification also leads to the acidity increase of the
3-imino group (NH) in the 2-Se-uridine, which strengthens
the hydrogen bond between N3 of U6 and N1 of A11.
Indeed, after the selenium modification, the U/A
hydrogen bond length between N3 of U6 and N1 of
˚
A11 is shortened from the native distance (3 A) to the
˚
Se-modified distance (2.81 A). Moreover, after the
Se-modification (Figure 6D), the U/A hydrogen bond
length between O4 of U6 and N6 of A11 decreases by
Crystallization and data collection of Se-RNA
Consistently, our crystal structure study of the SeU-RNA
[50-rGUAUA-SeU-AC-30] supports the biophysical results
˚
0.47 A from the native distance (3.39 A) to the
˚
˚
Se-modified distance (2.92 A). The shortened H-bond
lengths indicate stronger H-bonds, which may explain

5178 Nucleic Acids Research, 2012, Vol. 40, No. 11
A
B
C
D
3.39 / 2.92Å
3.00 Å
2.81 Å
Se
Se
Figure 6. Global and local structures of the U-containing RNA r[50-GUAUA( U)AC-30]2 with a resolution of 2.3 A. (A) The overall structure of
˚
Se
duplex. (B) The superimpose comparison of one U-RNA duplex (red; PDB ID: 3S49) with its native counterpart [50-r(GUAUAUA)-dC-30]2 (cyan;
PDB ID: 246D) with a RMSD value 0.55. The two red balls represent the selenium atoms. (C) The experimental electron density of SeU6/A11 base
pair with s = 1.0. (D) The superimpose comparison of the local base pair SeU6/A11 (red) and the native U6/A11 (cyan). The numbers indicate the
distance between the corresponding atoms.
the increase of duplex stability after the Se-modification.
On the contrary, the distance between Se2 of U6 and C2 of
A11 in the Se-modified duplex is slightly increased. This
distance increase is likely due to a steric effect. This steric
clash at the position 2 of the Se-uridine can be a driving
force to increase SeU/A pair specificity. Consistent with
our biophysical study, our structure study has
indicated that the selenium bulkiness at the uridine
2-postion discourages the U/G wobble pairing.
Moreover, due to the electronic effect of a selenium
atom, the inability of a Se atom to form a stable
hydrogen bond is another main factor responsible for
the discrimination against U/G wobble pair.
U/A pair, thereby providing a unique chemical strategy to
further enhance base pair fidelity. Furthermore, the
2-Se-modification will provide a useful tool in X-ray
crystal structure studies of RNAs and their protein
complexes. The atom-specific mutagenesis with selenium
opens a new research avenue for investigating base-pair
recognition, fidelity and RNA modification. This novel
base pair (SeU/A) with higher specificity likely enables bet-
ter preservation of genetic information at the RNA level.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Figures 1–24 and Supplementary Methods.
CONCLUSIONS
In summary, we have first synthesized the SeU-RNAs as
well as the SeU-phosphoramidite. Our biophysical and
structural studies on the SeU-RNAs indicate that the
native and Se-modified structures are virtually identical.
Moreover, the 2-Se-modification can largely discriminate
against the U/G wobble pair without significant impact on
ACKNOWLEDGEMENTS
We thank Dr Alex Soares, the mail-in program and PXrr
in Brookhaven National Laboratory (BNL). The National
Synchrotron Light Source (NSLS) at BNL is funded by
NIH’s National Center for Research Resources and the

Nucleic Acids Research, 2012, Vol. 40, No. 11 5179
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