Translation Initiation with Initiator tRNA Charged with Exotic Peptides
Goto et al.
Supporting information for:
Translation Initiation with Initiator tRNA Charged with Exotic
Peptides
Yuki Goto, Hiroaki Suga*
Research Center for Advanced Science and Technology, Department of
Advanced Interdisciplinary Studies, The University of Tokyo,
4-6-1, Komaba, Meguro-ku, Tokyo, 153-8904, Japan
Contact: [email protected]
Supplementary Figures
Figure S1
Chemical structures of exotic peptides and their activating groups. (A) Structure of the
exotic peptides exploited as a reprogrammed initiator. (B) Structure of activating groups.
Peptide 1–7 as well as 9–11 were activated with CME group, while 8 was activated with
CBT for the accessibility to flexizyme system.1
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Translation Initiation with Initiator tRNA Charged with Exotic Peptides
Goto et al.
Figure S2
Acid PAGE analysis of the RNAs charged with the exotic peptides shown in Figure S1.
A microhelix RNA1 was incubated with each exotic peptide in the presence of
flexizyme and analyzed by denaturing acid PAGE. “NC” indicates the lane in which the
untreated microhelix RNA was analyzed. Labels of bands (F, P, M) are as follows: F,
flexizyme; P, Xpep-microhelix RNA; R, microhelix RNA. Acylation yields shown in
the figure were calculated based on the band intensity of Xpep-RNA (P) and microhelix
RNA (M), presented as (P)/[(P)+(M)].
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Translation Initiation with Initiator tRNA Charged with Exotic Peptides
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Figure S3
MALDI-TOF mass spectra of the respective Xpep-peptides. The calculated mass (C)
and observed mass (O) are shown in each spectrum.
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Translation Initiation with Initiator tRNA Charged with Exotic Peptides
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Supplementary Methods
Materials
Chemicals were purchased from Watanabe Chemical Industries, Nacalai Tesque,
Kanto Chemical, Sigma-Aldrich Japan, or Wako Pure Chemical Industries unless noted
otherwise and used without further purification. All oligonucleotides were purchased
from Operon Biotechnologies. Flexizyme, tRNAfMetCAU, and template DNA for
translation were synthesized using the same procedure as previously described.1,2,3
General methods for the synthesis of exotic peptides activated with the proper
leaving groups.
Each peptide was assembled by solid-phase peptide synthesis using standard
Fmoc-protocols.4
General procedure for the synthesis of Boc-Xpep-OH: A mixture of exotic peptide
whose side chain groups were properly protected (0.10 mmol), di-tert-butyl dicarbonate
(0.12 mmol) and NaHCO3 (0.30 mmol) in 0.2 mL of 50% 1,4-dioxane (aq.) was stirred
at room temperature for 1 h. After the reaction, the mixture was evaporated to remove
1,4-dioxane and the solution was washed by ethyl acetate (3 mL x 2). The water layer
was acidified by 1M HCl, and the solution was then extracted with ethyl acetate (3 mL
x 2). The combined organic layer was dried over MgSO4 and concentrated under
reduced pressure. The obtained residue was directly used for the next step (activation of
the C-terminal carboxyl group).
General procedure for the synthesis of Boc-Xpep-CME: Each Boc-Xpep-OH (0.10
mmol) was mixed with Et3N (0.12 mmol) and chloroacetonitrile (0.1 mL) in 0.2 mL of
DMF, and the reaction mixture was stirred at room temperature for 12 h. After the
reaction, Et2O (6 mL) was added and the solution was washed with 1 M HCl (3 mL x 3),
sat. NaHCO3 (3 mL x 3) and brine (5 mL x 1), and the organic layer was dried over
MgSO4 and concentrated under reduced pressure. The crude residue was roughly
purified by silica gel column chromatography and then used for the next step
(deprotection of the Boc group).
General procedure for the synthesis of Boc-Xpep-CBT: 1,3-Dicyclocarbodiimide
(0.12 mmol) in 0.2 ml of ethyl acetate was added to a mixture of Boc-Xpep-OH (0.10
mmol) and 4-chlorobenzylmercaptan (0.12 mmol) in 0.4 mL of 1,4-dioxane. The
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Goto et al.
reaction mixture was stirred at room temperature for 3 h. After the reaction, the
precipitate was removed by filtration, and the filtrate was evaporated under reduced
pressure. The crude residue was roughly purified by silica gel column chromatography
and then used for the next step (deprotection of the Boc group).
General procedure for the deprotection of the N-terminal Boc group: The resulting
Boc-Xpep-R (R = CME or CBT) was dissolved in CH2Cl2 (1 mL) and then added ethyl
acetate containing 4 M HCl (2 mL). The reaction mixture was stirred at room
temperature for 15 min. The solvent was evaporated to dryness and the remained HCl
was removed by repeating the addition of Et2O (5 mL) and concentration under reduced
pressure three times. The residue was dissolved in MeCN (0.3 mL), and then the
product was precipitated by the addition of Et2O (3 mL). The precipitant was purified
using reverse-phase HPLC to give the activated exotic peptide.
Exotic peptide 1 (DPhe-LPhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ = 9.19 (d, 1H,
J = 8.6 Hz), 8.10 (br, 3H), 7.30–7.24 (m, 10H), 5.07–4.50 (m, 2H), 4.77 (m, 1H), 4.04
(br, 1H), 3.25–3.20 (m, 2H), 2.91–2.85 (m, 2H); ESI-MS, m/z 352 [(M + H)+]; HRMS
calcd. for C20 H22 O3 N3 [(M + H)+] 352.1661, found 352.1643.
Exotic peptide 2 (DPhe-DPhe-LPhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ =
8.98–8.83 (m, 2H), 8.10 (br, 3H), 7.32–7.07 (m, 15H), 5.04–4.50 (m, 2H), 4.62 (m, 2H),
3.99 (br, 1H), 3.24–3.11 (m, 2H), 2.90 (m, 2H), 2.72–2.56 (m, 2H); ESI-MS, m/z 499
[(M + H)+]; HRMS calcd. for C29 H31 O4 N4 [(M + H)+] 499.2345, found 499.2360.
Exotic peptide 3 (DPhe-4Abz-LPhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ = 8.89
(m, 1H), 8.44 (br, 3H), 7.80 (d, 2H, J = 7.1 Hz), 7.65 (d, 2H, J = 7.1 Hz), 7.44–7.19, (m,
11H), 5.02–4.48 (m, 2H), 4.77 (m, 1H), 4.30 (br, 1H), 3.30–3.11 (m, 4H); ESI-MS, m/z
471 [(M + H)+]; HRMS calcd. for C27 H27 O4 N4 [(M + H)+] 471.2032, found 471.2076.
Exotic peptide 4 (DPhe-βAla-LPhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ =
8.64–8.48 (m, 2H), 8.23 (br, 3H), 7.36–7.16 (m, 10H), 4.97–4.40 (m, 2H), 4.58 (m, 1H),
3.92 (br, 1H), 3.25–2.90 (m, 6H), 2.20 (t, 2H, J = 6.2 Hz); ESI-MS, m/z 423 [(M + H)+];
HRMS calcd. for C23 H27 O4 N4 [(M + H)+] 423.2032, found 423.2029.
Exotic peptide 5 (DPhe-MeGlu-LPhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ = 8.47
(d, 2H, J = 7.1 Hz), 8.15 (br, 3H), 7.37–7.20 (m, 10H), 4.98 (s, 2H), 4.86 (m, 1H), 4.59
(br, 1H), 4.49 (m, 1H), 307–2.95 (m, 4H), 2.72 (s, 3H), 2.08–2.01 (m, 3H), 1.69 (m,
1H); ESI-MS, m/z 495 [(M + H)+]; HRMS calcd. for C26 H31 O6 N4 [(M + H)+] 495.2243,
found 495.2230.
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Exotic peptide 6 (DPhe-βAla-DPhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ = 8.53
(d, 1H, J = 7.9 Hz), 8.42 (m, 1H), 8.18 (br, 3H), 7.35–7.18 (m, 10H), 4.97–4.44 (m, 2H),
4.58 (m, 1H), 3.90 (br, 1H), 3.28–2.90 (m, 6H), 2.24 (m, 1H), 2.13 (m, 1H); ESI-MS,
m/z 423 [(M + H)+]; HRMS calcd. for C23 H27 O4 N4 [(M + H)+] 423.2032, found
423.2021.
Exotic peptide 7 (DPhe-βAla-MePhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ = 8.42
(m, 1H), 8.22 (br, 3H), 7.33–7.20 (m, 10H), 4.99–4.42 (m, 2H), 4.62 (m, 1H), 3.92 (br,
1H), 3.31–2.90 (m, 6H), 2.73 (s, 3H), 2.25–2.11 (m, 2H); ESI-MS, m/z 437 [(M + H)+];
HRMS calcd. for C24 H29 O4 N4 [(M + H)+] 437.2189, found 437.2166.
Exotic peptide 8 (DPhe-βAla-LMet-CME): 1H NMR (DMSO-d6, 500 MHz) δ =
8.54–8.46 (m, 2H), 8.19 (br, 3H), 7.33–7.19 (m, 5H), 4.98–4.45 (m, 2H), 4.40 (m, 1H),
3.92 (br, 1H), 3.30–3.19, (m, 2H), 3.02 (m, 1H), 2.94 (m, 1H), 2.55 (m, 2H), 2.27 (m,
2H), 2.02 (s, 3H), 2.02–1.87 (m, 2H); ESI-MS, m/z 407 [(M + H)+]; HRMS calcd. for
C19 H27 O4 N4 S1 [(M + H)+] 407.1753, found 407.1744.
Exotic peptide 9 (DPhe-βAla-LGln-CBT): 1H NMR (DMSO-d6, 500 MHz) δ = 8.64
(d, 1H, J = 7.6 Hz), 8.52 (m, 1H), 8.19 (br, 3H), 7.37–7.23 (m, 9H), 6.82 (br, 2H), 4.36
(m, 1H), 4.03 (m, 2H), 3.94 (br, 1H), 3.37–3.19 (m, 2H), 3.03 (m, 1H), 2.95 (m, 1H),
2.36–2.27 (m, 2H), 2.13 (t, 2H, J = 7.4 Hz), 1.75 (m, 2H); ESI-MS, m/z 505 [(M + H)+];
HRMS calcd. for C24 H30 O4 N4 S1 Cl1 [(M + H)+] 505.1682, found 505.1664.
Exotic peptide 10 (DGlu-DLys-LPhe-CME): 1H NMR (DMSO-d6, 500 MHz) δ = 8.73
(d, 1H, J = 7.9 Hz), 8.53 (d, 1H, J = 7.9 Hz), 7.87 (br, 3H), 7.31–7.22 (m, 5H), 5.02 (m,
2H), 4.59 (m, 1H), 4.28 (m, 1H), 3.77 (br, 1H), 3.15 (m, 1H), 2.92 (m, 1H), 2.66 (m,
2H), 2.34 (t, 2H, J = 8.0 Hz), 1.91 (m, 2H), 1.44–1.25 (m, 4H), 1.06 (m, 2H); ESI-MS,
m/z 462 [(M + H)+]; HRMS calcd. for C22 H32 O6 N5 [(M + H)+] 462.2353, found
462.2377.
Exotic peptide 11 (DGlu-DLys-DGlu-DLys-LPhe-CME): 1H NMR (DMSO-d6, 500
MHz) δ = 8.58 (d, 1H, J = 7.9 Hz), 8.55 (d, 1H, J = 7.4 Hz), 8.20 (d, 1H, J = 7.1 Hz),
7.91 (d, 1H, J = 8.1 Hz), 7.77 (br, 3H), 7.31–7.22 (m, 5H), 5.02 (m, 2H), 4.58 (m, 1H),
4.26–4.18 (m, 3H), 3.79 (br, 1H), 3.13 (m, 1H), 2.92 (m, 1H), 2.76 (t, 2H, J = 7.6 Hz),
2.66 (t, 2H, J = 7.9 Hz), 2.35 (m, 2H), 2.24 (m, 2H), 1.92 (m, 3H), 1.69 (m, 2H), 1.52
(m, 3H), 1.41–1.25 (m, 6H), 1.06 (m, 2H); ESI-MS, m/z 719 [(M + H)+]; HRMS calcd.
for C33 H51 O10 N8 [(M + H)+] 719.3728, found 719.3723.
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Translation Initiation with Initiator tRNA Charged with Exotic Peptides
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Acylation of tRNAfMetCAU and microhelix RNA with exotic peptides by means of
flexizyme system
Reactions were carried out under the following conditions: 5 µL of 20 µM
flexizyme, 20 µM tRNAfMetCAU or microhelix RNA, and 5 mM exotic peptide 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 tRNAfMetCAU or microhelix RNA in 0.2 M
HEPES-K buffer pH 7.5, 0.2 M KCl (7.5 µL) was heated at 95 ˚C for 3 min and cooled
to 25 ˚C for 5 min. MgCl2 (3 M; 3 µL) and flexizyme (200 µM; 1.5 µL) were added and
the mixture was incubated at 25 ˚C for 5 min. The reaction was initiated by addition of
3 µL of 5 mM exotic peptide substrate in DMSO and incubated on ice for 2–6 hours.
Analysis of acylation by acid PAGE analysis
After the acylation of microhelix RNA, the reaction was stopped by addition of 45
µL of 0.6 M sodium acetate (pH 5) and the RNA was recovered by ethanol precipitation.
The pellet was then dissolved in 6 µL of 10 mM sodium acetate (pH 5). 0.5 µL of the
solution was mixed with 1.5 µL of loading buffer (150 mM sodium acetate, 10 mM
EDTA in 90% formamide) 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).
Initiation reprogramming with exotic peptidyl-tRNAs.
After acylation, the reaction was stopped by addition of 45 µL of 0.6 M sodium
acetate (pH 5) and the RNA was recovered by ethanol precipitation. The pellet was
rinsed twice with 100 µL of 70 % ethanol with 0.1 M sodium acetate (pH 5), and once
with 30 µL of 70 % ethanol. The Xpep-tRNAfMetCAU was dissolved in 0.5 µL of 1 mM
sodium acetate just before adding to translation mixture.
The wPURE system containing all necessary components for translation except for
methionine and aspartic acid was used in this study.2 Translation was carried out using
wPURE system in the presence of template DNA (0.04 µM), respective
Xpep-tRNAfMetCAU molecules (120 µM), and [14C]-Asp (50 µM). The wild-type
expression was carried out with wPURE system with template DNA (0.04 µM), Met
(200 µM), and [14C]-Asp (50 µM). The translation mixture (2.5 µL) was incubated at 37
˚C for 1 hour and analyzed by tricine-SDS PAGE and autoradiography (FLA-5100, Fuji,
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Japan).
Mass spectrometry of the expressed peptides.
For MALDI-TOF analysis, the translation reaction (5 µL) was performed in the
presence of Asp, instead of [14C]-Asp. The translation product was immobilized with
FLAG-M2 agarose (Sigma). After washing the resin with 30 µL of W buffer (50 mM
Tris-HCl pH 8.0, 150 mM NaCl), the immobilized peptides were eluted with 10 µL of
0.2 % TFA. The purified peptide was desalted with ZipTipµ-C18 (Millipore), and eluted
with 1 µL of a 50% MeCN, 0.1% TFA solution saturated with the matrix
(R)-cyano-4-hydroxycinnamic acid onto MTP 384 target plate ground steel T F
(BRUKER DALTONICS). MALDI mass measurement was performed using autoflex II
under the linear/positive mode and externally calibrated with peptide calibration
standard II (BRUKER DALTONICS).
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References
(1)
Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H. Nat. Methods 2006, 3, 357-359.
(2)
Goto, Y.; Ohta, A.; Sako, Y.; Yamagishi, Y.; Murakami, H.; Suga, H. ACS
Chem. Biol. 2008, 3, 120-129.
(3)
Goto, Y.; Murakami, H.; Suga, H. RNA 2008, 14, 1390-1398.
(4)
Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214.
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