3646–3651 Nucleic Acids Research, 2001, Vol. 29, No. 17
© 2001 Oxford University Press
Five-base codons for incorporation of nonnatural
amino acids into proteins
Takahiro Hohsaka, Yuki Ashizuka, Hiroshi Murakami and Masahiko Sisido*
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka,
Okayama 700-8530, Japan
Received April 24, 2001; Revised June 12, 2001; Accepted July 13, 2001
ABSTRACT
Extension of the genetic code for the introduction of
nonnatural amino acids into proteins was examined
by using five-base codon–anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the
Tyr54 position and a tRNAUACCG chemically aminoacylated with a nonnatural amino acid were added to
an Escherichia coli in vitro translation system.
Western blot analysis indicated that the CGGUA
codon is decoded by the aminoacyl-tRNA containing
the UACCG anticodon. HPLC analysis of the tryptic
fragment of the translation product revealed that the
nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the
reading frame adjacent to the CGGUA codon.
Another 15 five-base codons CGGN1N2, where N1 and
N2 indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the
complementary five-base anticodons. These results
provide a novel strategy for nonnatural mutagenesis
as well as a novel insight into the mechanism of
frameshift suppression.
INTRODUCTION
Extension of the genetic code is an important step to introduce
nonnatural amino acids into proteins. Extension was first
achieved using a nonsense UAG codon that could be decoded
by a chemically aminoacylated nonsense suppressor tRNACUA
(1,2). This methodology has been widely used to study structure and function of proteins (3–7) and the mechanism of translation (8–10). However, the nonsense suppression strategy is
not highly efficient because of competition with a release
factor, although some modifications have been reported (11).
In addition, more than two different nonnatural amino acids
cannot be introduced into a single protein, because of the
limited types of stop codons. To make nonnatural mutagenesis
more useful and more practical, we need to develop an alternative strategy for extension of the codon system that can achieve
efficient and multiple incorporation of nonnatural amino acids.
Bain et al. reported a nonnatural codon–anticodon pair
containing isoC and isoG as a candidate for the extended
codon–anticodon pair (12). However, the mRNA and tRNA
containing the nonnatural bases must be synthesized chemically.
Recently we reported that four-base codons such as AGGU
and CGGG are decoded by chemically aminoacylated tRNAs
containing complementary four-base anticodons (13–15).
When a tRNACCCG chemically aminoacylated with a nonnatural
amino acid was added to an Escherichia coli or rabbit
reticulocyte in vitro translation system together with a mRNA
containing a CGGG codon, the nonnatural amino acid was
successfully introduced into the directed position of the protein
(15). Moreover, we have achieved incorporation of two
different nonnatural amino acids into a single protein by using
two independent four-base codons (16). In this article we
report that even five-base codons can be decoded by aminoacylated tRNAs containing five-base anticodons. This fivebase codon system has the advantage that up to 16 five-base
codons may be derived from a single triplet codon. In addition,
investigation of five-base decoding may provide a novel
insight into the mechanism of frameshifting.
Five-base codon–anticodon pairing has not yet been
proposed, but two unusual findings related to extended base
pairing have been reported. First, decoding of five consecutive
bases as a single codon was observed in an E.coli hopR mutant,
in which GUGUG was translated into valine by a mutant
tRNAVal containing a three-base anticodon (17). This phenomenon is explained in terms of tRNA hopping. Secondly,
tRNAs having nine-base anticodon loops were synthesized by
inserting one base on either the 5′- or 3′-side of the four-base
anticodon and some of them could decode the four-base codon
in vivo (18). This report shows the ability of the ribosome to
accept a nine-base anticodon loop.
MATERIALS AND METHODS
Plasmid
A synthetic streptavidin gene was purchased from R&D
Systems Europe. The streptavidin gene was cloned into a
pGEMEX-1 (Promega)-derived vector, which was constructed
for a previous study (15). The resulting plasmid contained
the streptavidin gene fused with a T7 tag and a His tag at the
N- and C-termini, respectively, for western blotting and metal
chelation affinity purification. Substitution of the Tyr54 position of streptavidin by five-base codons CGGXX was carried
out by PCR using oligonucleotides CAGTCAGTACYYCCGGCGGGA, where YY is the complementary sequence of XX.
*To whom correspondence should be addressed. Tel: +81 86 251 8218; Fax: +81 86 251 8219; Email: [email protected]
Nucleic Acids Research, 2001, Vol. 29, No. 17 3647
A synthetic yeast tRNAPhe gene containing a CCCG anticodon
was previously constructed in pUC18 (15). Substitution of
CCCG by five-base anticodons XXCCG was carried out in a
similar manner using oligonucleotides GCCAGACTXXCCGAATC.
In vitro translation
The mRNA and nitrophenylalanyl-tRNA were prepared as
previously reported (15). The reaction mixture for in vitro
translation (10 µl) contained 2 µl of E.coli S30 extract
(Promega for linear template), 12 mM magnesium acetate,
0.1 mM each of amino acids except arginine, 0.01 mM
arginine, 16 µg of mRNA and 6.25 µg of aminoacyl-tRNA
unless stated otherwise. The reaction mixture was incubated at
37°C for 1 h, then cooled to 0°C.
Western blot analysis
The reaction mixture for in vitro translation (2 µl) was mixed
with gel loading buffer (38 µl) and 5 µl of the resulting solution
was analyzed by 15% SDS–PAGE. Western blotting was
carried out on a PVDF membrane (Bio-Rad) using an anti-T7
tag monoclonal antibody (Novagen) and ProtoBlot II AP
system (Promega). The efficiency of five-base decoding was
estimated by comparing the band intensity of the full-length
protein with those of serial dilutions of in vitro translated wildtype streptavidin.
Dot blot analysis
The reaction mixture (0.5 µl) was diluted in 200 µl of TBS
(20 mM Tris–HCl, 150 mM NaCl, pH 7.5) and spotted onto a
nitrocellulose membrane using a vacuum blotter. After
washing twice for 5 min with TBS, the membrane was incubated with 3% gelatin in TBS at 37°C for 30 min and then with
2 µg/ml biotinylated alkaline phosphatase (Zymed) in 1%
gelatin in TBS for 30 min. After washing three times with
TBST (TBS containing 0.02% Tween-20) for 5 min and once
with TBS briefly, the membrane was incubated in NBT/BCIP
solution at 37°C for 30 min, then washed with water and dried.
HPLC analysis of tryptic fragment
A tRNA aminoacylated with ε-nitrobenzoxadiazolyl-L-lysine
[Lys(NBD)] (see Fig. 2B) was prepared as described (16).
Translation of the CGGUA mutant in the presence of
Lys(NBD)-tRNAUACCG was carried out as described above on a
20 µl scale. The full-length product was purified on a Ni–NTA
affinity column (Qiagen) under denaturing conditions. The
eluate (50 mM phosphate buffer, pH 5.0, containing 8 M urea)
was diluted with 7 vol of 50 mM Tris–HCl pH 7.5, 1 mM
CaCl2, then incubated with 1 µg/ml trypsin (Promega) at 37°C
for 30 min. Reverse phase HPLC analysis was on an ODS
column (µBondasphere 5µ 100 Å; Waters) with a linear
gradient from 0.1% TFA to acetonitrile over 50 min and a flow
rate of 1 ml/min. The eluate was monitored with a fluorescence
detector (λex 470 nm, λem 530 nm) and the fluorescent fraction
was collected. After evaporation of the solvent, the fraction
was subjected to MALDI-TOF mass spectrometry (Voyager
DE-Pro; PE Biosystems).
The chemical synthesis of Lys(NBD)-Val-Leu-Thr-Gly-Arg
was carried out as follows. NBD-Cl (0.4 mg) and α-Fmoc-LysVal-Leu-Thr-Gly-Arg (0.4 mg) synthesized on a peptide
synthesizer (Pioneer, PE Biosystems) were added to a mixture
Figure 1. Nucleotide and amino acid sequence of the 53–61 region of a
mutated streptavidin. (A) A tRNAUACCG aminoacylated with a nonnatural
amino acid (Xaa) decodes a CGGUA five-base codon and the correct reading
frame is maintained. (B) When the aminoacyl-tRNAUACCG does not work, then
the CGG triplet will be read by an endogenous arginyl-tRNACCG and
translation will be stopped at a UAG codon. In the cases of other five-base
codons, translation will be terminated at a UGA codon three triplets downstream of the CGG.
of 50 µl of methanol and 10 µl of 4% NaHCO3. After incubation at 55°C for 1 h, Fmoc-Lys(NBD)-Val-Leu-Thr-Gly-Arg
was isolated by reverse phase HPLC. Removal of the Fmoc
group was by incubation in 20% piperidine/DMF at 37°C for 2 h,
then the desired product was purified by reverse phase HPLC.
Mass spectrum (FAB) calculated for C35H58N13O11 (MH+)
836.4379, found 836.4391.
RESULTS AND DISCUSSION
Design of the expression system
We employed a streptavidin expression system to evaluate
five-base decoding, as previously reported for a four-base
codon system (13,15). At first, a combination of a CGGUA
codon and a UACCG anticodon was investigated. Since CGG
is a minor codon in the E.coli translation system, the use of
CGG-derived five-base codons will avoid serious competition
with endogenous arginyl-tRNACCG. The CGGUA codon was
introduced into the Tyr54 position of the streptavidin gene and
the UACCG anticodon was introduced into the anticodon of a
synthetic yeast tRNAPhe gene. As shown in Figure 1, when the
CGGUA codon is recognized by the aminoacyl-tRNAUACCG,
the five-base codon will be translated into the nonnatural
amino acid and a correct reading frame will be maintained.
However, when the first three bases of the CGGUA sequence
are read as a triplet codon by endogenous arginyl-tRNACCG,
translation will be terminated at a subsequent stop codon. As a
result, five-base decoding will yield full-length streptavidin
with the correct amino acid sequence except that the nonnatural
amino acid corresponding to the CGGUA codon is incorporated,
whereas failure of five-base decoding will yield a truncated
protein. These two products are easily distinguishable by gel
electrophoresis.
p-Nitrophenylalanine (nitroPhe, Fig. 2A) was used as the
nonnatural amino acid, because it had been incorporated into
streptavidin in good yield (14,15) and mutated streptavidin
containing nitroPhe at the Tyr54 position was found to be
active in biotin binding (14).
3648 Nucleic Acids Research, 2001, Vol. 29, No. 17
Figure 2. Structures of nonnatural amino acids. (A) Nitrophenylalanine
(nitroPhe). (B) ε-Nitrobenzoxadiazolyl-L-lysine [Lys(NBD)].
Figure 4. HPLC analysis of a tryptic fragment of a mutated streptavidin synthesized in the presence of Lys(NBD)-tRNAUACCG. The eluate was monitored
with a fluorescence detector (λex 470 nm, λem 530 nm). (Inset) MALDI-TOF
mass spectra of the fluorescent fraction. (A) Tryptic fragment of the mutated
streptavidin. (B) Chemically synthesized peptide Lys(NBD)-Val-Leu-Thr-Gly-Arg.
Figure 3. Incorporation of a nonnatural amino acid into the Tyr54 position of
streptavidin under direction of the CGGUA codon. (A) Western blot and
(B) dot blot analysis of translation of the mutated streptavidin mRNA containing a
CGGUA codon at the Tyr54 position in the presence of a tRNAUACCG aminoacylated with nitroPhe. Translation products were detected using an anti-T7
tag antibody and an alkaline phosphatase-labeled anti-mouse IgG for western
blotting. Biotin-binding activity was evaluated with biotinylated alkaline phosphatase for dot blotting.
Incorporation of nonnatural amino acids using a CGGUA–
UACCG codon–anticodon pair
The mutated mRNA containing the CGGUA codon at the
Tyr54 position and the tRNAUACCG chemically aminoacylated
with nitroPhe were added to the E.coli in vitro translation
system. Then, the reaction mixture was analyzed by SDS–PAGE,
followed by western blot analysis using an anti-T7 tag antibody to detect translation products. As shown in Figure 3A, in
the absence of tRNAUACCG and in the presence of nonaminoacylated tRNAUACCG, only a truncated product was
observed instead of full-length streptavidin. This truncated
product would result from three-base decoding at the Tyr54
position followed by termination of translation. In the presence
of tRNAUACCG aminoacylated with nitroPhe, however, fulllength streptavidin was successfully synthesized. The yield of
full-length streptavidin increased with increasing concentration of
aminoacyl-tRNAUACCG. These results suggest that the reading
frame of the mutated mRNA is correctly maintained by addition of aminoacylated tRNAUACCG and, as a consequence, the
nonnatural amino acid corresponding to the CGGUA codon is
successfully incorporated. The ineffectiveness of nonaminoacylated tRNAUACCG supports the idea that the tRNA
cannot be aminoacylated by any endogenous aminoacyl-tRNA
synthetases. The biotin-binding activity of the translation
product was evaluated by dot blot analysis using biotinylated
alkaline phosphatase. As shown in Figure 3B, biotin-binding
activity was observed only when nitroPhe-tRNAUACCG was
added to the translation reaction. Because the binding activity
of mutated streptavidin containing nitroPhe at the Tyr54 position was unaffected (14), the dot blotting results strongly
suggest that nitroPhe is incorporated at the directed position
without any alteration of the following amino acid sequence.
Furthermore, incorporation of the nonnatural amino acid was
directly confirmed using a fluorescently labeled amino acid,
Lys(NBD) (Fig. 2B; 16). Mutated mRNA containing the
CGGUA codon at the Tyr54 position and tRNAUACCG aminoacylated with Lys(NBD) were added to the translation mixture.
Full-length streptavidin was purified on a Ni–NTA affinity
column under denaturing conditions, then digested with
trypsin to generate a 54–59 amino acid fragment (Fig. 1A). The
trypsin digestion reaction mixture was applied to a reverse
Nucleic Acids Research, 2001, Vol. 29, No. 17 3649
Figure 5. Effect of mismatch at the fifth letter of five-base codons in five-base
decoding. Each pair of mRNA containing a CGGUX (X indicates one of four
nucleotides) codon at the Tyr54 position and a nitroPhe-tRNA containing a
YACCG (Y indicates one of four nucleotides) anticodon was added to the
in vitro translation. Full-length streptavidin was visualized by western blotting.
phase HPLC column equipped with a fluorescence detector
(λex 470 nm, λem 530 nm). As shown in Figure 4, a single peak
was observed and its retention time was found to be identical to
that of the chemically synthesized peptide Lys(NBD)-ValLeu-Thr-Gly-Arg. In addition, the mass spectrum of the
fluorescent peptide fragment was identical to that of the chemically synthesized peptide (Fig. 4). These results demonstrate
that the nonnatural amino acid is incorporated corresponding
to the CGGUA codon and exclude other possible mechanisms,
such as a –1 frameshift and two consecutive +1 frameshifts.
Effect of a mismatch at the fifth letter of the five-base
codon
Previous studies have shown that some tRNAs containing
four-base anticodons are insensitive to mismatches at the
fourth letter of four-base codons (19,20). Bossi and Smith
proposed that the first letters of the four-base anticodons of
such tRNAs sterically interfere with reading the adjacent inframe codon by the next tRNA (21). Therefore, it is likely that
the fifth letter of the five-base codon is not involved in codon–
anticodon pairing. To test this possibility, single base substitutions at the fifth letter of the five-base codon were introduced
(Fig. 5). When mRNA containing a CGGUG or CGGUC
codon was added to the translation mixture together with
nitroPhe-tRNAUACCG, no full-length streptavidin was synthesized. In the case of the CGGUU codon, however, a small
amount of full-length streptavidin was produced. In a similar
manner, single base substitutions at the first letter of the fivebase anticodon were introduced. When mRNA containing the
CGGUA codon was added to the translation mixture together
with the nitroPhe-tRNA containing a CACCG or AACCG
anticodon, a negligible amount of full-length streptavidin was
synthesized. In the case of the GACCG anticodon, however, a
small amount of full-length streptavidin was produced. These
results indicate that the fifth letter of the five-base codons is
recognized by the first letter of the five-base anticodons. U–U
and A–G pairs, however, suggest that the recognition is
partially loose.
Other five-base codon–anticodon pairs
The five-base decoding system may derive up to 16 extended
codons from a single three-base codon. Mutated mRNAs
containing five-base codons CGGN1N2, where N1 and N2 indicate
one of four bases, at the Tyr54 position were added to the
translation mixture together with nitroPhe-tRNAs containing
the complementary five-base anticodons. Western blot analysis
showed that all combinations of complementary five-base
codon–anticodon pairs gave full-length streptavidin (Fig. 6A).
Figure 6. Western blot analysis of translation of a mutated streptavidin mRNA
containing a CGGN1N2 codon (N1 and N2 indicate one of four nucleotides) at
the Tyr54 position. (A) In the presence of a nitroPhe-tRNA with a complementary
anticodon. (B) In the presence of a non-aminoacylated tRNA with a complementary anticodon.
Table 1. Efficiency (%) of five-base decoding by nitroPhe-tRNAs containing
complementary five-base anticodons
Fourth letter
Fifth letter
U
G
C
A
U
18
14
8
16
G
19
9
24
5
C
21
15
29
34
A
30
12
42
13
The values are expressed as yields of full-length streptavidin relative to that of
wild-type streptavidin.
On the other hand, negligible amounts of full-length streptavidin
were produced in the presence of non-aminoacylated tRNAs
(Fig. 6B). These results suggest that the 16 five-base codons
are successfully decoded by aminoacylated tRNAs containing
the complementary five-base anticodons. The fact that any
bases are allowed for N1 and N2 indicates that five-base
decoding is effective even when the CGG triplet is followed by
a stop codon (UAG), minor codons (AGG, CGG and GGG) or
major codons.
Efficiency of five-base decoding was estimated by densitometric analysis of western blots and is summarized in Table 1.
The highest yield of full-length streptavidin was observed in
the case of the CGGAC codon (42% relative to wild-type
streptavidin). This efficiency is much higher than that of amber
suppression (12%; 1), but rather lower than that of the CGGG
four-base codon (70%; 15).
Mechanism of five-base decoding
The above results suggest that the ribosome moves by five
bases along the five-base codon–anticodon pair. This phenomenon might be interpreted in terms of a single-step quintuplet
translocation. However, codon–anticodon pairing by five
consecutive bases would cause severe deformation of the anticodon loop, resulting in failure of the translocation process.
3650 Nucleic Acids Research, 2001, Vol. 29, No. 17
Figure 7. Western blot analysis of translation of a mutated streptavidin mRNA
containing a CGGU codon at the Tyr83 position in the presence of nitroPhetRNAs containing four- or five-base anticodons. The partial sequence of the
mutated mRNA is AAC-AAC-CGGU-CGU-AAU-GCG-CAC-AGC-GCCACU-ACG-UGG-UCU-GGC-CAA-UAC-GUU-GGC-GGU-GCU-GAGGCU-CGU-AUC-AAC-ACU-CAG-UGG-CUG-UUA-ACA. The first underlined UAA appears when the CGG is decoded as a three-base codon by an
endogenous arginyl-tRNACCG. The second underlined UAA appears when
CGGUC is decoded as a five-base codon by the nitroPhe-tRNAGACCG.
Farabaugh and co-workers proposed a model for +1 frameshift
suppression (22,23), arguing against a classical quadruplet
translocation model. They proposed that a +1 frameshift
occurs in the P site after triplet translocation, even though
frameshift suppressor tRNA contains a four-base anticodon.
The idea that frameshifting occurs not in the A but in the P site
is supported by the finding that binding of peptidyl-tRNA in
the P site is weaker than in the A site (24).
Considering the Farabaugh and Björk model for +1
frameshift suppression, it is expected that five-base decoding is
caused by an additional +1 frameshift in the P site after the
initial +1 frameshift. To examine this possibility, four-base
decoding by tRNAs containing five-base anticodons was
investigated. mRNA containing a four-base CGGU codon at
the Tyr83 position of streptavidin (15) and aminoacylated
tRNAs containing XACCG anticodons were added to the
translation mixture. As shown in Figure 7, full-length streptavidin
was successfully synthesized when the four-base codon
(CGGU) and five-base anticodon (NACCG) pairs were used,
as well as the four-base codon (CGGU) and four-base anticodon (ACCG) pair. This result indicates that the CGGU
codon can be decoded as a single four-base codon by tRNAs
containing XACCG anticodons. Addition of nitroPhetRNA GACCG, however, yielded another truncated product of
∼10 kDa. This product could be generated through five-base
decoding of the four-base codon CGGU plus the first letter C
of the next CGU codon. Such five-base decoding gives a truncated product terminated at a UAA codon 27 triplets downstream of the CGGUC. It should be noted that the four-base
decoding product in the cases of five-base codons at the Tyr54
position might be terminated at a UGA codon 7 triplets downstream of the CGGU (Fig. 1A), but it could not be distinguished from the three-base decoding product by western
blotting.
Here we propose a model for five-base decoding by tRNA
containing a five-base anticodon (Fig. 8). At first, the
peptidyl-tRNA containing the five-base anticodon binds to the
P site with the reading frame shifted by one base (Fig. 8A).
According to the Farabaugh and Björk model for the sufJ
Figure 8. A model for five-base decoding. A peptidyl-tRNA containing a fivebase anticodon binds to the P site with the reading frame shifted by one base
(A). Binding of Ser-tRNAGCU to the empty ribosomal A site results in four-base
decoding (B). On the other hand, base pairing of the fifth letter of the five-base
codon with the first letter of the five-base anticodon leads to an additional +1
frameshift (C). Binding of Val-tRNAGAC to the empty ribosomal A site results
in five-base decoding (D).
suppressor (22), this intermediate would be produced not by
quadruplet translocation but by triplet translocation followed by
a +1 frameshift through isomerization of the codon–anticodon
complex. At this stage it is uncertain whether the first letter of
the five-base codon interacts with the fifth letter of the fivebase anticodon.
If the next Ser-tRNAGCU binds to the empty A site of this
intermediate ribosome complex, four-base decoding will be
observed (Fig. 8B). On the other hand, if the fifth letter of the
five-base codon pairs with the first letter of the five-base anticodon prior to binding of Ser-tRNAGCU, the additional +1
frameshift will occur in the P site (Fig. 8C). At this stage base
pairing of the first and the second letters of the five-base codon
with the fifth and the fourth letters of the five-base anticodon,
respectively, is uncertain. Then, the next Val-tRNAGAC binds
to the empty ribosomal A site to yield the five-base decoding
product (Fig. 8D). The present model implies that five-base
decoding is intrinsically inefficient because the additional +1
frameshift competes with binding of an aminoacyl-tRNA to
the empty A site (Fig. 8B). The lower efficiency of five-base
decoding than that of four-base decoding may be accounted for
by this model. Moreover, this model may explain the accurate
recognition of the first letter of the five-base anticodon
observed in Figure 5. Although U as the first letter of triplet
anticodons can pair with all four bases, U as the first letter of a
five-base anticodon can only pair with A (or U to a lesser
extent). In our model pairing of the first letter of the five-base
anticodon occurs in the ribosomal P site in competition with
binding of the next aminoacyl-tRNA, whereas that of triplet
anticodons occurs in the A site without competition. These
differences may explain the different specificities of the first
letters of three-base and five-base anticodons.
Nucleic Acids Research, 2001, Vol. 29, No. 17 3651
CONCLUSIONS
For the first time we have demonstrated five-base decoding by
using aminoacyl-tRNAs containing five-base anticodons in the
E.coli in vitro translation system. This novel frameshift
strategy will find wide application, such as multiple incorporation of nonnatural amino acids into proteins. The results in
Figure 5 indicate that the CGGUA and CGGUG codons can be
used independently, because the CGGUA codon cannot be recognized by tRNACACCG and the CGGUG codon cannot be
recognized by tRNAUACCG. Such independent five-base codons
will enable us to introduce various kinds of nonnatural amino
acids into a single protein.
The present study also demonstrates the feasibility and
utility of the application of chemically aminoacylated
frameshift suppressor tRNA. In previous studies of frameshift
suppression, suppressor tRNAs must be aminoacylated by any
of aminoacyl-tRNA synthetases. Most aminoacyl-tRNA
synthetases, however, recognize anticodons as identity determinants, which limits mutation of the anticodon loop of
frameshift suppressor tRNA. Since the chemical aminoacylation method is free from enzymatic recognition, it will serve to
probe structure–function relationships of frameshift suppressor
tRNAs.
ACKNOWLEDGEMENTS
We thank Prof. Shigeyuki Yokoyama (The University of
Tokyo) for his important suggestions and Prof. Shinji Toyota
(Okayama University of Science) for his measurement of
the FAB mass spectrum. This research was supported by a
Grant-in-Aid for Specially Promoted Research from the
Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 11102003).
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