Supplementary Methods
Acid substrates and RNAs
All acid substrates were synthesized from the corresponding N-Boc protected
amino acids (Watanabe chemical, Japan). All CME substrates were synthesized same
procedure as previously described1. All DBE substrates were synthesized by coupling
with 3,5-dinitrobenzylchloride (Sigma). All CBT substrates were synthesized by coupling
with
4-chlrobenzyl
mercaptan
(TCI,
Japan)
using
N,N-bis(2-oxo-3-oxazolidiyl)phosphorodiamidic chloride2. Proton (1H NMR) magnetic
resonance spectra were recorded at 500 MHz on a Bluker AMX-500 spectrometer. The
data are reported as follows: multipicity (s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br = broad), coupling constant (Hz), integration. All oligonucleotides were
purchased from Operon (Japan). The constructs of tRNAAsnXXX (XXX = CUA, ACCC,
ACU, GUU or CUG) and microhelix RNA were synthesized using the same procedure as
previously described3 (see also ref. 8 in main text).
General procedure for synthesis of DBE substrates (valine 3,5-dinitrobenzyl
ester): A mixture of -N-Boc-valine (130 mg, 0.60 mmol), triethylamine (101 mg, 1.0
mmol) and 3,5-dinitrobenzyl chloride (108 mg, 0.50 mmol) in 0.1 mL of
dimethylformamide was stirred at room temperature for 12 h. After the reaction,
diethylether (9 mL) was added and the solution was washed with 0.5 M HCl (3 mL x 3),
4 % NaHCO3 (3 mL x 3) and brine (5 mL x1), and the organic layer was dried over
MgSO4 and concentrated under reduced pressure. The crude residue was dissolved in 2
mL of 4 M HCl/ethylacetate and incubated for 20 min at room temperarure. The solution
3
was concentrated under reduced pressue and the remained HCl was removed by repeating
the addition of diethylether (3 mL) and concentration under reduced pressue three times.
The product was precipitated by the addition of diethylether (3 mL) and the precipitants
were filtered in 62 % overall yield (123 mg, 0.369 mmol).; 1H NMR (DMSO-d6, 500
MHz)  8.83 (s, 1H), 8.70 (s, 2H), 8.44 (br, 3H), 5.56 (s, 2H), 4.07 (d, J = 4.6 Hz, 1H),
2.22 (m, 1H), 1.00 (d, J = 7.0 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H).
General procedure for synthesis of CBT substrates (valine 4-chlorobenzyl
thioester): A mixture of -N-Boc-valine (108 mg, 0.50 mmol), triethylamine (152 mg,
1.5 mmol), N,N-bis(2-oxo-3-oxazolidiyl)phosphorodiamidic chloride (114 mg, 0.45
mmol) and 4-chlrobenzyl mercaptan (79 mg, 0.5 mmol) in 5 mL of dichloromethane is
stirred at room temperature for 5 h. The solution was washed with 0.5 M HCl (3 mL x 3),
0.5 N NaOH (3 mL x 3) and brine (5 mL x1), and the organic layer was dried over
MgSO4 and concentrated under reduced pressure. The crude residue was dissolved in 2
mL of 4 N HCl/ethylacetate and incubated for 20 min at room temperarure. The solution
was concentrated under reduced pressue and the remained HCl was removed by repeating
the addition of diethylether (3 mL) and concentration under reduced pressure three times.
The product was precipitated by the addition of diethylether (3 mL) and the precipitants
were filtered in 53 % overall yield (78.7 mg, 0.267 mmol); 1H NMR (DMSO-d6, 500
MHz)  8.33 (br, 3H), 7.40 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 4.27 (d, J = 14
Hz, 1H), 4.25 (d, J = 14 Hz, 1H), 4.19 (br, 1H), 2.17 (m, 1H), 0.94 (d, J = 6.9 Hz, 3H),
0.89 (d, J = 6.9 Hz, 3H).
4
dFx was synthesized by using the following procedure. P3 was annealed with
dFxR46 (5'-ACCTA ACGCC ATGTA CCCTT TCGGG GATGC GGAAA TCTTT
CGATC C-3'), and extended by Taq DNA polymerase. The resulting product was diluted
10 times with PCR reaction buffer and amplified by using P4 (5'-GCATA TGTAA
TACGA CTCAC TATAG-3') and dFxR19 (5'-ACCTA ACGCC ATGTA CCCT-3') as
the 5'- and 3'-primers, respectively. The DNA product was transcribed by T7 RNA
polymerase and purified by 12% denaturing PAGE. eFx was synthesized by the same
procedure as dFx except that eFxR45 (5'-ACCTA ACGCT AATCC CCTTT CGGGG
CCGCG GAAAT CTTTC GATCC-3') and eFxR18 (5'-ACCT AACGC TAATC
CCCT-3') was used instead of dFxR46 and dFxR19. The tRNA, dFx or eFx was
independently dissolved in water and its concentration was adjusted to 200 µM.
Acylation of tRNA and microhelix
Reactions were carried out under the following conditions: 5 µL of 20 µM dFx
or eFx, 20 µM tRNAAsnCUA, and 5 mM acid substrate in 0.1 M Hepes-K buffer pH 7.5,
0.1 M KCl, 600 mM MgCl2 and 20 % DMSO on ice. The procedure was as follows: 40
µM tRNAAsnCUA in 0.2 M Hepes-K buffer pH 7.5, 0.2 M KCl (2.5 µL) was heated at 95
˚C for 3 min and cooled to 25 ˚C over 5 min. MgCl2 (3 M, 1 µL) and dFx or eFx (200
µM, 0.5 µL) were added and the mixture was stored at 25 ˚C for 5 min. The reaction was
initiated by addition of 1 µL of 25 mM of the substrate in DMSO and incubated on ice for
the listed times (Supplementary Table 1 online).
5
Analysis of acylation
After the acylation of tRNAAsnCUA, the reaction was stopped by addition of 15
µL of 0.6 M sodium acetate pH 5 (with 5 mM DTT for Cys). The RNA was recovered by
ethanol precipitation. The pellet was rinsed with 70 % ethanol containing 0.1 M NaCl,
and then dissolved in 3.5 µL of 7.5 mg/mL sulfosuccinimidyl-D-biotin (Dojin, Japan) in
0.4 M Hepes-K pH 8.0. The biotinylation reaction was carried out on ice for 1 hour, and
stopped by addition of 6.5 µL of 0.6 M sodium acetate pH 5 (with 5 mM DTT for Cys)
followed by precipitation with ethanol. The pellet was rinsed twice with 70 % ethanol
containing 0.1 M NaCl and dissolved in 100 µL of 10 mM sodium acetate pH 5 (with 5
mM DTT for Cys). 0.5 µL of this solution was mixed with 1.5 µL of loading buffer (0.2
mg/mL streptavidin in 37 mM piperazine pH 6.1, 37 mM EDTA and 6 M urea), and
analyzed by 12 % denaturing PAGE (6 M urea). The RNA was stained with Syber Green
II (Molecular Probe) and analyzed by FLA-5100 (Fuji, Japan).
Acid PAGE analysis was carried out by the following procedure. After the
acylation of microhelix RNA, the reaction was stopped by addition of 15 µL of 0.6 M
sodium acetate pH 5 (with 5 mM DTT for Cys). Ethanol-precipitation followed by
rinsing with 70% ethanol gave pellets which were then dissolved in 2.5 µL of 10 mM
sodium acetate pH 5 (with 5 mM DTT for Cys). 1 µL of the solution was mixed with 1
µL of loading buffer (50 mM sodium acetate, 8 M urea) and analyzed by 20 % denaturing
acid PAGE (50 mM sodium acetate, 6 M urea). The RNA was stained with ethidium
bromide and analyzed by FLA-5100 (Fuji, Japan).
6
Calculation of suppression efficiency for protein site-specific mutagenesis
The suppression efficiency was calculated based on the radioactivity of bands I
and II, expressed as I/(I+IIx5/3); the intensity of band II was normalized in order to
reflect the number of Met residues, based on the consideration that full-length and
truncated proteins contain 5 and 3 Mets, respectively. We excluded band III from the
calculation of suppression efficiencies, since this truncation might occur due to an
abnormal termination, such as falling-off-termination of peptidyl-tRNAs from the
ribosome.
Preparation of mRNA for peptide synthesis
Template DNA for peptide synthesis was prepared by the following procedure.
(5'-GTAAT ACGAC TCACT ATAGG GTGAT CCAAC TTTAA TAAGG AGGTA
TACCA ATG –3') was annealed with (5'-TCATC TTTAT AATCG GTGGT CTGCT
GGTTG TTACT ACTCA TTGGT ATACC TCCTT ATT-3') and extended by Taq DNA
polymerase. The resulting product was diluted 10 times with PCR reaction mixture and
amplified using (5'-GTAATACGACTCACTATAGGG –3') and (5'- CGAAG CTTAT
TTATC ATCAT CATCT TTATA ATCGG TGGT-3') as 5'- and 3'-primers, respectively.
The DNA product was transcribed by T7 RNA polymerase and purified by 8 %
denaturing PAGE.
7
Translation
Acyl-tRNAAsnCUA was prepared by the following procedure. 40 µM of
tRNAAsnCUA in 0.2 M Hepes-K buffer pH 7.5, 0.2 M KCl (12.5 µL) was heated at 95 ˚C
for 3 min and cooled to 25 ˚C over 5 min. MgCl2 (3 M, 5 µL) and dFx or eFx (200 µM,
2.5 µL) were added and the solution was stored at 25 ˚C for 5 min. The reaction was
initiated by addition of 5 µL of 25 mM substrate in DMSO and incubated on ice for the
indicated times (Supplementary Table 1 online). The reaction was stopped by addition
of 75 µL of 0.6 M sodium acetate pH 5 (with 5 mM DTT for Cys), and the RNA was
recovered by ethanol precipitation. The pellet was rinsed twice with 70 % ethanol with
0.1 M sodium acetate pH 5.0, and once with 70 % ethanol. The pellet was dried and
stored at –80 ˚C. The acyl-tRNA was dissolved in 3.5 µL of 1 mM sodium acetate and
used for 3 rounds of translation. The batch translation (5 µL) was carried out using the
rapid translation system, RTS-100® (Roche Diagnostics), in the presence of 35S-Met and
analyzed by SDS-PAGE according to previous report (see ref. 9 in main text). Similarly,
batch
translation
was
performed
using
the
PURE
system
(custom-made
PURESYSTEM®, Post Genome Institute Co., ltd., Japan).
The protein expression efficiency was determined based on our previous protocols
and data published elsewhere9. The yields of the mutants under the optimal conditions
was calculated by the equation of (suppression efficiency for each acid)/ (suppression
efficiency for Iph) x 76 µg/mL. The suppression efficiency for each amino acid were
shown in graphs Supplementary Figure 6a–d. We generally obtained the suppression
efficiency of Iph to be 0.8, as seen in Supplementary Figure 6c, lane 31, a 76 µg/mL
8
expression efficiency was determined based on the quantification of Iph-GFP after the
His-tag isolation.
The translation of nonnatural peptides was carried out using the PURE system
with 0.4 µM of mRNA and 3 mM EDTA. 200 µM of Met, Thr, Tyr, Lys, Ser, Asn, Gln
and 50 µM of [14C]-Asp were added for natural peptide synthesis. 50 µM of each
Aly-tRNAAsnACU, Cit-tRNAAsnGUU and Iph-tRNAAsnCUG were added instead of Ser, Asn
and Gln for nonnatural peptide synthesis. The reaction was carried out in 2.5 µL at 37 ˚C
for 1 hour and the products were analyzed by Tricine-SDS-PAGE.
Mass spectra measurements of peptides
For mass spectra analysis, the reactions (L) were performed in the absence of
[14C]-Asp. The products were precipitated with 50 µL of acetone, dissolved in 2.5 µL of
water, and then immobilized with FLAG-M2 agarose (Sigma). After the resin was
washed with 50 µL of W buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) twice, the
immobilized peptides were eluted with 2.5 µL of 0.2 % TFA, desalted with C18 Zip tips
(Millipore), and eluted with 1.5 µL of a 50% acetonitrile, 0.1% TFA solution saturated
with the matrix (R)-cyano-4-hydroxycinnamic acid. Mass measurements were performed
using MALDI-TOF (Autoflex®, BRUKER) in the positive mode and externally
calibrated with Substance P (ave. 1,348.66 Da), Bombesin (ave. 1,620.88 Da), ACTH
clip 1-17, (ave. 2,094.46 Da), Somatostatin 28, (ave. 3,149.61 Da) as standards.
9
References
1.
2.
3.
Saito, H., Kourouklis, D. & Suga, H. EMBO J. 20, 1797-806 (2001).
Diago-Meseguer, J., Palomo-Coll, A.L., Fernández-Lizarbe, J.R. &
Zugaza-Bilbao, A. Synthesis 1980, 547-551 (1980).
Kourouklis, D., Murakami, H. & Suga, H. Methods 36, 239-44 (2005).
10