Chemical aminoacylation of RNA by an intermolecular adenosine transfer reaction

Article abstract of DOI:10.1246/cl.2008.102

RNA aminoacylation, based on an adenosine transfer mechanism, was achieved by the intermolecular transfer of phenylalaninyl adenosine from a donor probe to an acceptor RNA. This method can be applied to a wide variety of unnatural amino acids for tRNA ami

Full text of DOI:10.1246/cl.2008.102

102
Chemistry Letters Vol.37, No.1 (2008)
Chemical Aminoacylation of RNA by an Intermolecular Adenosine Transfer Reaction
Mingzhe Liu, Hiroshi Jinmei, Hiroshi Abe,ꢀ and Yoshihiro Itoꢀ
Nano Medical Engineering Laboratory, RIKEN (The Institute of Physical and Chemical Research),
2-1 Hirosawa, Wako 351-0198
(Received September 10, 2007; CL-070986; E-mail: h-abe@riken.jp, y-ito@riken.jp)
RNA aminoacylation, based on an adenosine transfer mech-
amino acids. The transfer chemistry is a nucleophilic substitu-
tion reaction, which has formerly been used for the chemical li-
gation of nucleic acids.9–12 In the present study, we designed the
model system shown in Scheme 1, to confirm the transfer of ami-
noacylated adenosine 2 from the donor probe to the acceptor
RNA in the chemical reaction. When tRNA lacking a 30-terminal
adenosine is used as the acceptor, the aminoacylation of the
tRNA can be achieved.
anism, was achieved by the intermolecular transfer of phenylala-
ninyl adenosine from a donor probe to an acceptor RNA. This
method can be applied to a wide variety of unnatural amino acids
for tRNA aminoacylation.
tRNA aminoacylated with unnatural amino acids has be-
come a powerful tool for the site-specific incorporation of un-
natural amino acids into proteins. This has been used for various
applications, such as the investigation of protein–protein interac-
tions, conformational changes, and signal transduction.1–8 The
synthesis of aminoacylated tRNA has been reported by several
research groups. These approaches can be divided into two main
classes: enzymatic and nonenzymatic methods. Among the en-
zymatic methods, Hecht’s group first reported a general strategy
for the preparation of aminoacylated tRNA, in which T4 RNA
ligase attaches an aminoacylated pCpA derivative to tRNAs
lacking a 30-terminal dinucleotide.4 Suga’s group reported
RNA ribozymes that catalyze the aminoacylation of tRNAs with
phenylalanine derivatives as substrates.5 Mutated aminoacyl
tRNA synthetase catalyzing the aminoacylation of phenylala-
nine derivatives has been developed independently by Schultz’s
group and Yokoyama’s group.6,7 Although each of these tech-
niques has some advantages, they also have some restrictions
in their ability to selectively aminoacylate tRNA, or in their ap-
plicability to a wide range of substrate amino acids and tRNA.
Recently, Sisido’s group developed a novel nonenzymatic meth-
od for the aminoacylation of tRNA, which can be applied to a
wide variety of unnatural amino acids. The method is based on
an aminoacyl transfer mechanism using a peptide nucleic acid
(PNA) probe.8
The structures of the donor probe and the acceptor RNA
used are shown in Scheme 1. The donor consists of three parts:
an oligonucleotide (DNA), a linker, and an adenosine derivative
(R = 1 or 2). The donor probe binds to the acceptor to form a
DNA–RNA double strand before the transfer reaction. The ade-
nosine derivative is linked to the deoxyoligonucleotide by an
electrophilic linker containing a benzenesulfonyl group, which
makes the 50-hydroxy group of the adenosine (1 or 2) reactive
to nucleophilic substitution. The acceptor consists of an RNA
strand, except for deoxycytidine at the 30-terminus, which
has a phosphorothioate group. When the donor and acceptor
are hybridized, the phosphorothioate group of the acceptor
attacks the 50-carbon center of the adenosine in the donor probe.
The transfer of the adenosine derivative then proceeds from the
donor probe to the acceptor RNA.
First, the donor probe was synthesized according to
Scheme 2. Key compounds 3 and 4 were designed to contain
two electrophilic reactive centers, a bromoacetyl group and a
benzenesulfonyl group. The reactivity of the bromoacetyl group
in nucleophilic substitution is significantly higher than that of the
benzenesulfonyl group. Therefore, when the phosphorothioate
probe is mixed with compound 3 or 4, the bromoacetyl group se-
lectively reacts with the thioxide anion of the phosphorothioate
group, generating the donor probe (Scheme 2). The synthesis of
the protected adenosine derivative 3 and the phenylalanyl adeno-
sine derivative 4 is described in the Supporting Information.
Both compounds were synthesized from commercially available
Here, we propose a new nonenzymatic strategy for tRNA-
specific aminoacylation, which is based on an adenosine transfer
mechanism. This can be applied to a wide range of unnatural
O
R
O
S
donor 1: R = 1
donor 2: R = 2
dGdGdTdCdCdGdGdCdGdG-3'
O
O
dC rC rA rG rG rCrC rG rC rC rCrGrGrCrC-32P-5'
-S P O
O-
acceptor
NH2
N
HN Boc
N
N
N
N
N
N
N
O
Transfer of
O
O
dGdGdTdCdCdGdGdCdGdG-3'
adenosine derivative
HO
S
R-
=
O
O
O
O
O
O
dCrCrArGrGrCrCrGrCrCrCrGrGrCrC-32P-5'
product 1: R = 1
product 2: R = 2
O
S P O
R
O-
H2N
1
2
Scheme 1. Principle of intermolecular transfer of adenosine derivative from donor to acceptor.
Copyright ꢀ 2008 The Chemical Society of Japan

Chemistry Letters Vol.37, No.1 (2008)
103
ble between Product 2 and the acceptor. Therefore, the amount
of Product 2 formed was less than that of Product 1.18 To im-
prove the conversion of the transfer reaction, optimization of
the structure of the linker in the donor, as well as the reaction
conditions, is proceeding.19
In conclusion, the aminoacylation of RNA was achieved us-
ing an adenosine transfer method. The phenylalanyl-adenosine
transferred from the donor DNA to the acceptor RNA was con-
firmed. By using tRNA lacking a 30-terminal adenosine, instead
of the acceptor RNA, the chemical aminoacylation of tRNA by
unnatural amino acid is possible.
O
O
Br
3: R = 1
4: R = 2
R
N
H
S
O
O
+
O
P
-S
dGdGdTdCdCdGdGdCdGdG-3'
O
O-
R
O
O
S
O
O
O
HN
dGdGdTdCdCdGdGdCdGdG-3'
O
S
P
O-
This work was supported by Grants-in-Aid for Scientific
Research, the Riken Directors’ Fund from MEXT (Ministry of
Education, Culture, Sports, Science and Technology), NEDO
(the New Energy and Industrial Technology Development
Organization) (H. A.), and the Japan Society for the Promotion
of Science (M. L.).
donor 1: R = 1
donor 2: R = 2
Scheme 2. Synthesis of adenosine donor.
M
1
2
Product 2
Product 1
References and Notes
1
2
P. M. England, Biochemistry 2004, 43, 11623.
D. Kajihara, R. Abe, I. Iijima, C. Komiyama, M. Sisido, T.
Hohsaka, Nat. Methods. 2006, 3, 923.
3
4
5
6
7
T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel, Annu.
Rev. Biochem. 2004, 73, 147.
T. G. Heckler, L. H. Chang, Y. Zama, T. Naka, M. S.
Chorghade, S. M. Hecht, Biochemistry 1984, 23, 1468.
Y. Bessho, D. R. W. Hodgson, H. Suga, Nat. Biotechnol. 2002,
20, 723.
L. Wang, A. Brock, P. G. Schultz, J. Am. Chem. Soc. 2002,
124, 1836.
D. Kiga, K. Sakamoto, K. Kodama, T. Kigawa, T. Matsuda,
T. Yabuki, M. Shirouzu, Y. Harada, H. Nakayama, K. Takio,
Y. Hasegawa, Y. Endo, I. Hirao, S. Yokoyama, Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 9715.
Figure 1. Acidic nonnatural polyacrylamide gel electrophore-
sis analysis of the transfer of the adenosine derivative from the
donor DNA to the acceptor RNA (32P-labeled at the 50 end).
Lane M, acceptor RNA only; lane 1, transfer of adenosine deriv-
ative (1) from donor 1 to the acceptor; lane 2, transfer of amino-
acylated adenosine (2) from donor 2 to the acceptor. Reaction
conditions: [donor 1/2] = 10 mM, [acceptor] = 100 nM, [Tris-
borate pH 6.5] = 70 mM, [MgCl2] = 10 mM, [NaCl] = 100
mM, in 20 mL, at 35 ꢁC, for 2 h.
8
9
K. Ninomiya, T. Minohata, M. Nishimura, M. Sisido, J. Am.
Chem. Soc. 2004, 126, 15984.
S. M. Gryaznov, R. L. Letsinger, J. Am. Chem. Soc. 1993, 115,
3808.
10 M. K. Herrlein, J. S. Nelson, R. L. Letsinger, J. Am. Chem. Soc.
1995, 117, 10151.
20,30-O-isopropylideneadenosine.13 The coupling reaction of the
50 phosphorothioated oligonucleotide with 3 or 4 was carried out
at room temperature.14 After reaction for 1 h, the reaction mix-
ture was extracted twice with n-butanol, and then extracted with
chloroform to isolate the donor probe, which was used immedi-
ately in the transfer reaction without additional purification.15
The transfer reaction was carried out by hybridizing the do-
nor probe with the acceptor RNA and incubating them at 35 ꢁC
for 2 h (Scheme 1). The reaction was analyzed by acidic poly-
acrylamide gel electrophoresis.16 The acceptor RNA was labeled
with 32P for the trace. The results are shown in Figure 1. The
band in lane M shows the acceptor. Lane 1 contains donor 1
(R = 1). The band indicated by the dashed arrow indicates ade-
nosine derivative 1 successfully transferred from donor 1 to the
acceptor, resulting in the formation of Product 1. In lane 2, the
transfer of the phenylalanine-acylated adenosine 2 from donor
2 to the acceptor is also observed. The band indicated by the sol-
id arrow shows Product 2, with phenylalanine at its terminus.17
Although aminoacylation was achieved by the transfer reaction,
hydrolysis of the amino acid from Product 2 also occurred, re-
sulting in the formation of a hydrolyzed product, in the band visi-
11 H. Abe, E. T. Kool, J. Am. Chem. Soc. 2004, 126, 13980.
12 S. Sando, H. Abe, E. T. Kool, J. Am. Chem. Soc. 2004, 126,
1081.
13 Detail of synthesis is described in Supporting Information.19
14 Reaction was carried out under the following conditions:
20 mM 50-phosphorothioated oligonucleotide, 10 mM 3 or 4,
40% DMF in 160 mM triethylammonium acetate (TEAA)
buffer (pH 6.7) containing 1 mM dithiotheitol (DTT).
15 Donors 1 and 2 were characterized by MALDI-TOF mass
spectrometry. On the mass spectral chart for donor 2, apart
from a peak derived from donor 2 (Obsd: m=z 3833.2), a by-
product (Obsd: m=z 3686.0) was also detected. This indicates
that the phenylalanine was hydrolyzed partially from donor 2
(Figure S1).19
16 The transfer reaction was analyzed on 20% polyacrylamide
gel containing 7.5 M urea at 4 ꢁC in acidic buffer (pH 4.95)
containing 100 mM Tris-acetate and 2.5 mM EDTA.
17 Detailed analysis of transfer reaction indicated that Product 2
was aminoacylated transfer product (Figure S2).19
18 The yield of product 2 is about 5%.
19 Supporting Information is also available electronically on
the CSJ-Journal website, http://www.csj.jp/journals/chem-lett/
index.html.
Published on the web (Advance View) December 15, 2007; doi:10.1246/cl.2008.102