Unnatural base pairs mediate the site-specific incorporation of an unnatural hydrophobic component into RNA transcripts

Article abstract of DOI:10.1016/j.bmcl.2004.02.072

Site-specific incorporation of a hydrophobic nucleotide analog into RNA, by T7 transcription mediated by unnatural base pairs, was developed. The nucleotide analog, 5-phenylethynyl-3-(β-D-ribofuranosyl)pyridin-2-one 5-triphosphate (denoted by Ph-yTP), was chemically synthesized and then site-specifically incorporated by T7 RNA polymerase into RNA opposite the pairing partner, 2-amino-6-(2-thienyl)purine (denoted by s) in DNA templates. The introduction of Ph-y into a theophylline-binding RNA aptamer, in which a uridine in the internal loop was replaced by Ph-y, raised the thermal stability of the aptamer. Thus, this unnatural nucleotide analog would be useful for stabilizing RNA tertiary structures and complexes between RNA and other molecules.

Full text of DOI:10.1016/j.bmcl.2004.02.072

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2593–2596
Unnatural base pairs mediate the site-specific incorporation of an
unnatural hydrophobic component into RNA transcripts
Masayuki Endo,a Tsuneo Mitsui,b Taeko Okuni,a Michiko Kimoto,a Ichiro Hiraoa,b,* and
Shigeyuki Yokoyamaa,c,d,
*
aProtein Synthesis Technology Team, Protein Research Group, RIKEN Genomic Sciences Center (GSC), 1-7-22 Suehiro-cho,
Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
bResearch Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
cDepartment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
dRIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
Received 27 November 2003; revised 19 February 2004; accepted 19 February 2004
Abstract—Site-specific incorporation of a hydrophobic nucleotide analog into RNA, by T7 transcription mediated by unnatural
base pairs, was developed. The nucleotide analog, 5-phenylethynyl-3-(b-D-ribofuranosyl)pyridin-2-one 5-triphosphate (denoted by
Ph-yTP), was chemically synthesized and then site-specifically incorporated by T7 RNA polymerase into RNA opposite the pairing
partner, 2-amino-6-(2-thienyl)purine (denoted by s) in DNA templates. The introduction of Ph-y into a theophylline-binding RNA
aptamer, in which a uridine in the internal loop was replaced by Ph-y, raised the thermal stability of the aptamer. Thus, this
unnatural nucleotide analog would be useful for stabilizing RNA tertiary structures and complexes between RNA and other
molecules.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Unnatural base pairs that can work with the natural A–
T and G–C base pairs in transcription enable the site-
specific incorporation of extra components into RNA,
generating novel RNAs with increased functionality.1;2
Recently, we developed unnatural base pairs between 2-
amino-6-(N,N-dimethylamino)purine (x)3 and (1H)pyr-
idin-2-one (y), and 2-amino-6-(2-thienyl)purine (s) and
y4 (Fig. 1). The nucleoside 50-triphosphate of y (yTP)
was site-specifically incorporated into RNA opposite s
or x in DNA templates by T7 RNA polymerase. In
particular, the specificity of the s–y pairing was as high
as those of the natural base pairings in transcription.4 In
addition, a modified yTP, a nucleoside 50-triphosphate
of 5-iodo-(1H)pyridine-2-one (I-y), was also specifically
incorporated into RNA at desired positions.5 The I-y
components in RNA molecules have the potential to
function as photoactivated crosslinking residues as well
X
5
X
(a)
H C
(b)
N
H
H
H
H
6
4
3
CH
N
y
y
S
N
3
3
N
N
N
1
R
R
2
N
O
O
x
s
H
H
N
N
R
N
N
N
N
R
H
H
y: (X=H)
I-y: (X=I)
Ph-y: (X=phenylethynyl)
Figure 1. Unnatural x–y and s–y base pairs that function in tran-
scription.
as heavy atoms to facilitate X-ray crystallography of
large RNA molecules and RNA–protein complexes. In
addition to these functions, a series of modified y
derivatives can be chemically synthesized from the
nucleoside of I-y.
Keywords: Unnatural base pair.
* Corresponding authors. Tel.: +81-45-503-9644; fax: +81-45-503-9645
(I.H.); tel.: +81-3-5841-4413; fax: +81-3-5841-8057 (S.Y.); e-mail
addresses: ihirao@postman.riken.jp; hirao@mkomi.rcast.u-tokyo.
ac.jp; yokoyama@biochem.s.u-tokyo.ac.jp
Here, we report the synthesis of a nucleoside 50-tri-
phosphate of a hydrophobic y analog, 5-phenylethynyl-
3-(b-
D
-ribofuranosyl)pyridin-2-one
5-triphosphate
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.072

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M. Endo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2593–2596
(Ph-yTP), and the specificity of the Ph-yTP incorpora-
tion into RNA fragments by T7 RNA polymerase.
Furthermore, Ph-y was introduced into an RNA apt-
amer, in which a uridine residue in the single-stranded
region was replaced by Ph-y, and the thermal stability of
the modified RNA aptamer was examined. The phenyl
and ethynyl residues in the y analog would be expected
to stabilize RNA tertiary structures6–8 and the interac-
tions between RNA and other molecules, especially be-
tween RNA and proteins.9
pH 7.0 was determined by quantitative analysis of the
phosphorus in the compound.14
The efficiency and specificity of the Ph-yTP incorpora-
tion into RNA were assessed using a short DNA tem-
plate (35-mer) containing s at a specific position or a
template (35-mer) consisting of natural bases as a con-
trol, from which 17-mer transcripts were obtained (Fig.
2a). The 35-mer template strands included the promoter
sequence for T7 RNA polymerase, followed by a short
sequence consisting of C and T, with s or A at position
+11, and G at position +16. To facilitate the detection
and quantification of the mis-incorporation of Ph-yTP
opposite A and G, the template strands included only
one A and/or G. These template strands were annealed
with a 21-mer nontemplate strand, and the transcription
was carried out by T7 RNA polymerase with 1 mM
natural NTPs, 10 mM GMP, and 0.1 lCi/ll [a-32P]ATP
in the presence or absence of Ph-yTP (1 mM) at 37 ꢁC
for 3 h. The internally labeled transcripts obtained by T7
transcription in the presence or absence of
Ph-yTP were analyzed by gel electrophoresis (Fig. 2b).
The nucleoside derivatives of Ph-y (3 and 4) were syn-
thesized, as shown in Scheme 1. The introduction of the
phenylethynyl group into the nucleoside of y was carried
out by coupling the ribonucleoside of I-y (2)5 and phen-
ylacetylene with Pd(Ph3P)4, CuI, and triethylamine,10
with a 92% yield.11 The nucleoside 3 was converted to
the nucleoside 50-triphosphate (4)12 according to the
literature.13 The triphosphate was purified by DEAE-
Sephadex column chromatography and C18 reversed-
phase HPLC. The molar absorption coefficient of
Ph-yTP (14,000 at 260 nm and 26,000 at kmax: 286 nm) at
I
NH
NH
NH
NH
O
P
O
P
O
P
O
O
O
O
HO
O
O
HO
HO
HO
O
O
O
O
O
a
b
c,d
O-
O-
O-
OH OH
OH OH
OH OH
OH OH
4 (Ph-yTP)
1
2
3
Scheme 1. (a) I2, KI, Na2CO3, 100 ꢁC, 4 h, (b) phenylacetylene, Pd(Ph3P)4, Cul, Et3N, DMF, rt, 4–6 h, (c) POCl3, 1,8-bis(dimethylamino)naph-
thalene, (CH3O)3PO, 0 ꢁC, 2 h, (d) bis(tri-n-butylammonium)pyrophosphate, tri-n-butylamine, 0 ꢁC, 10 min.
Figure 2. Site-specific incorporation of Ph-y into RNA by T7 RNA polymerase. (a) Schemes of the experiments. (b) Gel-electrophoresis of the
transcripts. Lane 1: transcription using the template (N¼s) in the presence of Ph-yTP, lane 2: transcription using the template (N¼s) in the absence of
Ph-yTP. (c) Two-dimensional TLC for nucleotide-composition analyses of transcripts using the templates (N¼s or A) in the presence of Ph-yTP. The
quantitative data are shown in Table 1.

M. Endo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2593–2596
Table 1. Nucleotide composition analysis of T7 transcriptsa
2595
Entry
Template
Ph-yTP (mM)
Composition of nucleotides incorporated as 50 neighbor of A
Apb
Gp
Cp
Up
Ph-yp
1
2
3
N¼s
1
1
0
1.06c
[1]d
4.01
[4]
0.91
[1]
0.03
[0]
0.98
[1]
N¼A
1.07
[1]
4.03
[4]
0.87
[1]
1.03
[1]
0.004
[0]
N¼A
1.05
[1]
4.06
[4]
0.86
[1]
1.03
[1]
––e
[0]
a Transcription was carried out using a solution (20 lM) containing 2 lM of each template, 1 mM each natural NTP, 2 lCi of [a-32P]ATP, 10 mM
GMP, and 50 units of T7 RNA polymerase at 37 ꢁC for 3 h.
b Composition of nucleotides incorporated as 50 neighbor of p*A, as shown in Figure 2a.
c The values were determined using the following formula: (radioactivity of each nucleotide)/(total radioactivity of all nucleotides) · (total number of
nucleotides at 50 neighbor of A).
d The theoretical number of each nucleotide is shown in brackets.
e Not analyzed.
The full length transcript (17-mer) was generated by the
transcription in the presence of Ph-yTP (Fig. 2b, lane 1)
using the template containing s. Although the tran-
scription in the absence of Ph-yTP also gave products
corresponding to a 17-mer and a 16-mer (Fig. 1b, lane
2), the transcription efficiency was lower than that in the
presence of Ph-yTP. In addition, the 17-mer transcript
consisting of natural ribonucleotides showed slightly
higher mobility than that of the 17-mer transcript con-
taining Ph-y. These results indicate that Ph-yTP was
incorporated into RNA by the pairing between the
substrate of Ph-y and s in the template.
this assay, the composition of Cp was relatively small in
comparison to the theoretical number. However, this
did not result from the s–y pairing in transcription,
because this tendency also appeared in the natural
transcription, using the control template in the absence
of Ph-yTP (Table 1, entry 3).
Incorporations of nucleotide analogs containing a
hydrophobic propynyl or phenyl group into nucleic
acids increase the thermal stabilities of their duplex
structures.6–8 In RNA–protein complexes, stacking
interactions between the aromatic groups on the b-
sheets of proteins and the base moieties of RNAs are a
common structural feature.9 Thus, the introduction of
Ph-y to appropriate sites in RNAs would strengthen the
stacking interactions and stabilize the tertiary structures
of RNA and RNA–other molecule complexes.
To confirm the specificity of the Ph-yTP incorporation,
we performed a nucleotide composition analysis4 of the
transcripts. In the transcripts, the nucleotides that
became the 50-neighbor of A were labeled at their 30-
phosphates, because [a-32P]ATP was used for internal
labeling in transcription (Fig. 2a). Then, the transcripts
were digested with RNase T2, and the resulting labeled
nucleoside 30-monophosphates, including the nucleotide
opposite s, were analyzed by 2D thin-layer chromato-
graphy (2D-TLC) (Fig. 2c and Table 1). On the 2D-
TLC, a spot corresponding to the nucleotide of Ph-y
(Ph-yp) appeared only from the transcription using the
template containing s (Fig. 2c, N ¼ s). In contrast, the
transcription using the control template containing A
instead s gave a spot corresponding to Up, instead of
Ph-yp (Fig. 2c, N ¼ A). To detect the Ph-yTP mis-
incorporation opposite A and G, we added one A and
one G before T in the control template, and confirmed
that no spot corresponding to Ph-y was detected by the
analysis of the transcript from the control template (Fig.
2c, N ¼ A). Quantification of the spots on the 2D-TLC
also confirmed the site-specific incorporation of Ph-yTP
opposite s (Table 1, entry 1); in the transcription in the
presence of 1 mM Ph-yTP and natural NTPs, Ph-yp was
incorporated into RNA with 98% specificity. Although
Ph-yp showed a large mobility shift on the 2D-TLC
because of its increased hydrophobicity, it was insig-
nificant for the quantification. In the transcript obtained
from the natural template in the presence of Ph-yTP,
Ph-yp was scarcely detected (0.4%) (Table 1, entry 2). In
To assess the utility of the specific Ph-y incorporation
into RNA, we introduced Ph-y or y into a theophylline-
binding RNA aptamer,15–17 in place of the uridine at
position 24 in an internal loop in the aptamer, and
compared the thermal stability of these aptamers with
the original aptamer. The sequence of the original
aptamer is shown in Figure 3, and the uridine at position
24 was replaced by Ph-y or y. Transcription was carried
out by T7 RNA polymerase, with 1 mM natural NTPs,
Ph-yTP (1 mM) or yTP (1 mM), and a double-stranded
DNA template containing s, at 37 ꢁC for 6 h. The
thermal stability of each RNA aptamer (1.3–1.6 lM)
was examined in 10 mM sodium phosphate (pH 7.0),
50 mM NaCl, and 0.1 mM EDTA using a SHIMADZU
spectrophotometer, UV-2450, at a heating rate of 0.5 ꢁC/
min. The stability of the aptamer containing Ph-y at
position 24 (Ph-y24: Tm ¼ 67.0 ꢁC) was greatly
improved in comparison to that of the original aptamer
(U24: Tm ¼ 63.3 ꢁC). In contrast, the stability of the
aptamer containing y at position 24 (y24: Tm ¼ 63.3 ꢁC)
was as low as that of the original aptamer. These results
show the importance of the phenylethynyl group of y for
the stabilization of the RNA aptamer. The binding
efficiency of this Ph-y modified aptamer with theophil-
line is being investigated.

2596
M. Endo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2593–2596
Ministry of Education, Culture, Sports, Science, and
Technology.
A A
L1
L4
A
G
15
16
C
G
U
C
G
C
A
G
C
G
References and notes
C
U
1. Benner, S. A.; Burgstaller, P.; Battersby, T. R.; Jurczyk, S.
In The RNA World; Gesteland, R. F., Cech, T. R., Atkins,
J. F., Eds.; Cold Spring Harbor Laboratory: Cold Spring
Harbor, New York, 1999; pp 163–181.
2. Tor, Y.; Dervan, P. B. J. Am. Chem. Soc. 1993, 115, 4461.
3. Ohtsuki, T.; Kimoto, M.; Ishikawa, M.; Mitsui, T.; Hirao,
I.; Yokoyama, S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
4992.
4. Hirao, I.; Ohtsuki, T.; Fujiwara, T.; Mitsui, T.; Yokog-
awa, T.; Okuni, T.; Nakayama, H.; Takio, K.; Yabuki, T.;
Kigawa, T.; Kodama, K.; Yokogawa, T.; Nishikawa, K.;
Yokoyama, S. Nat. Biotechnol. 2002, 20, 177.
5. Kimoto, M.; Endo, M.; Mitsui, T.; Okuni, T.; Hirao, I.;
Yokoyama, S. Chem. Biol. 2003, 11, 47.
C
U
24
10
A
U24: Tm = 63.3 C
y24: Tm = 63.3 C
Ph-y24: Tm = 67.0 C
C
C
G
G
A
C
6
28
A
U
A
G
G
C
G
A
G
G
G
C
G
C
U
C
33
1
6. McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P.
G.; Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585.
7. Mitsui, T.; Kimoto, M.; Sato, A.; Yokoyama, S.; Hirao, I.
Bioorg. Med. Chem. Lett. 2003, 13, 4515.
Figure 3. Site-specific incorporation of Ph-y into a theophylline-
binding RNA aptamer. Unnatural components, y and Ph-y, were
introduced at position 24, in place of the uridine of the original apt-
amer (U24). The numbering of the residues corresponds to the original
duplex aptamer.16;17
8. Nakano, S.; Uotani, Y.; Nakashima, S.; Anno, Y.; Fujii,
M.; Sugimoto, N. J. Am. Chem. Soc. 2003, 125, 8086.
9. Mattaj, I. W.; Nagai, K. Nat. Struct. Biol. 1995, 2, 518.
10. Brown, L. J.; May, J. P.; Brown, T. Tetrahedron Lett.
2001, 42, 2587.
11. 5-Phenylethynyl-3-(b-D-ribofuranosyl)pyridin-2-one (3):
1H NMR (270 MHz, DMSO-d6) d 3.50 (m, 1H), 3.65 (m,
1H), 3.83 (m, 3H), 4.68 (d, 1H, J ¼ 4:1 Hz), 4.73 (d, 1H,
J ¼ 5:1 Hz), 4.96 (t, 1H, J ¼ 6:1 Hz), 5.11 (br s, 1H), 7.39
(m, 3H), 7.48 (m, 2H), 7.70 (s, 1H), 7.72 (s, 1H), 12.11 (s,
1H); 13C NMR (68 MHz, DMSO-d6) d 61.17, 70.57, 74.57,
80.37, 83.47, 86.10, 88.71, 100.17, 122.32, 128.30, 128.54,
130.89, 131.16, 137.68, 138.49, 160.24. HR-MS (FAB, 3-
NBA matrix) calcd for C18H18NO5 (Mþ1) 328.1185,
found: 328.1188.
By the specific transcription involving the unnatural s–y
pair, the hydrophobic Ph-y can be incorporated into
large RNA fragments at desired positions. The DNA
templates containing s are easily prepared by PCR
amplification with a 30-primer containing s.5 Thus, this
specific transcription mediated by unnatural base pairs
is more useful than chemical RNA synthesis especially
for the preparation of long RNA fragments. Efficient
sites in RNA molecules for the incorporation of
unnatural components can be predicted from the 3D
structures of the RNA molecules. Uridine residues in
single-stranded regions, G–U pairs, and certain base
triplets18 in the RNA molecules might be suitable Ph-y
incorporation sites for the thermal stabilization of RNA
molecules.
12. 5-Phenylethynyl-3-(b-D-ribofuranosyl)pyridin-2-one 5-tri-
1
phosphate (4): H NMR (270 MHz, D2O) d 1.11 (t, 27H,
J ¼ 7:3 Hz), 3.03 (q, 18H, J ¼ 7:3 Hz), 4.09 (m, 5H), 4.86
(d, 1H, J ¼ 3:7 Hz), 7.27 (m, 3H), 7.43 (m, 2H), 7.60 (s,
1H), 7.81 (s, 1H). 31P NMR (109 MHz, D2O) d ꢀ22.83 (t,
1H, J ¼ 19:5 Hz, 21.4 Hz), ꢀ10.72 (d, 1H, J ¼ 19:5 Hz),
ꢀ10.15 (d, 1H, J ¼ 21:4 Hz). ESI-MS calcd for
C18H19NO14P3 (Mꢀ1) 566.00, found: 565.50.
€
ꢀ
€
13. Kovacs, T.; Otvos, L. Tetrahedron Lett. 1988, 29, 4525.
14. Mitsui, T.; Kitamura, A.; Kimoto, M.; To, T.; Sato, A.;
Hirao, I.; Yokoyama, S. J. Am. Chem. Soc. 2003, 125,
5298.
Acknowledgements
15. Jenison, R. D.; Gill, S. C.; Pardi, A.; Polinsky, B. Science
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C. L.; Pardi, A. Biochemistry 1998, 37, 9186.
17. Zimmermann, G. R.; Wick, C. L.; Shields, T. P.; Jenison,
R. D.; Pardi, A. RNA 2000, 6, 659.
This work was supported by the RIKEN Structural
Genomics/Proteomics Initiative (RSGI), the National
Project on Protein Structural and Functional Analyses,
Ministry of Education, Culture, Sports, Science and
Technology of Japan, and by a Grant-in-Aid for
Scientific Research (KAKENHI 15350097) from the
18. Walberer, B. J.; Cheng, A. C.; Frankel, A. D. J. Mol. Biol.
2003, 327, 767.