SUEMENTARY INRMATIN - Nature · SUEMENTARY INRMATIN.10/.2517 1 ... MS spectra were obtained on an Agilent 1200 Series HPLC system with LC-MS ... or Shimadzu LCMS-IT/ TOF

NATURE CHEMISTRY | www.nature.com/naturechemistry 1

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2517

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Supplementary Information

A synthetic molecular system capable of mirror-image

genetic replication and transcription

Zimou Wang1,3, Weiliang Xu2,3, Lei Liu2*, Ting F. Zhu1*

1School of Life Sciences, Center for Synthetic and Systems Biology, Ministry of Education

Key Laboratory of Bioinformatics, Collaborative Innovation Center for Diagnosis and

Treatment of Infectious Diseases, Tsinghua University, Beijing 100084, China 2Tsinghua-Peking Center for Life Sciences, Ministry of Education Key Laboratory of

Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry,

Tsinghua University, Beijing 100084, China 3These authors contributed equally to this work.

*To whom correspondence should be addressed. E-mail: [email protected]

(T.F.Z.); [email protected] (L.L.) Table of Contents

Page Materials 2 Supplementary Methods 3 Supplementary Figures S1-S9 5 Supplementary Tables S1-S2 15 Supplementary References 17



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Materials The HPLC purified L-DNA oligonucleotides, L-dNTPs, and L-NTPs were purchased from ChemGenes (MA, U.S.). D-DNA oligonucleotides were ordered from Tsingke Biotech (Beijing, China) and Sangon Biotech (Shanghai, China). Modified DNA oligonucleotides were purified by HPLC, while the others were purified by PAGE. The ProteoSilver Plus Silver Stain Kit was purchased from Simga-Aldrich (MI, U.S.). Protein ladder and the pEASY T3 Cloning Kit were purchased from Transgen Biotech (Beijing, China). RNase A was purchased from Fermentas (MA, U.S.). The PAGE DNA Purification Kit was purchased from Tiandz Inc (Beijing, China). Phenol:chlorophorm:isopentanol (25:24:1, pH >7.8) was purchased from Solarbio Life Sciences (Beijing, China). Glycogen was purchased from Aidlab Biotechnologies (Beijing, China). Recombinant RNase Inhibitor was purchased from TaKaRa Bio-Technology (Dalian, China). Fmoc-D-amino acids and Fmoc-L-amino acids were purchased from CS Bio or GL Biochem (Shanghai, China). O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazole (HOAT)1-hydroxy-benzotriazole (HOBT) were purchased from GL Biochem (Shanghai, China). N,N’-Diisopropyl-carbodiimide (DIC), 1,2-ethanedithiol and 4-mercaptophenylacetic acid (MPAA) were purchased from Alfa Aesar. Acetonitrile (HPLC grade) was purchased from J. T. Baker (NJ, U.S.). Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), potassium chloride (KCl), magnesium acetate tetrahydrate (Mg(AcO)2·4H2O), ethylenediamine tetraacetic acid (EDTA), glycerol, sodium dihydrogen phosphate (NaH2PO4), guanidine hydrochloride (Gn·HCl), diethyl ether anhydrous (Et2O), and 85% hydrazine hydrate were purchased from Sinopharm Chemical Reagent. L-glutathione (reduced), thioanisole and trifluoroacetic acid (TFA) (HPLC grade) were purchased from J&K Scientific (Beijing, China). Triisopropylsilane (TIPS), and N,N-diisopropylethylamine (DIEA) were purchased from Ouhe Technology (Beijing, China). Sodium hydroxide (NaOH), N,N-Dimethylformamide (DMF), dichloromethane (DCM), hydrochloric acid (HCl), and sodium nitrite (NaNO2) were purchased from Beijing Chemical Works (Beijing, China). 2-chlrotrityl chloride(2CTC) resin (loading = 0.53 mM/g) was purchased from NanKai HeCheng (Tianjin, China). β-mercaptoethanol was purchased from ZhongKeTuoZhan (Beijing, China). VA-044 was purchased from KeXin New Materials (Qingdao, China).



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Supplementary Methods

Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS)

Peptides containing the C-terminal region of ASFV pol X were synthesized on 2-chlorotrityl chloride (2CTC) resin to generate C-terminal peptide carboxylates. All other peptides were synthesized on hydrazine 2CTC resin to generate C-terminal peptide hydrazides. Fmoc-D-amino acids and Fmoc-L-amino acids were purchased from CS Bio. The peptide chain elongation was manually carried out to avoid cross contamination by residual amino acids and peptides of the opposite chirality. The Acetamidomethyl (Acm) group was used to protect the N-terminal D-Cys86 in order to avoid side reactions such as cyclization or oligomerization during native chemical ligation1. The resin was swelled in DCM/DMF before use. The coupling was carried out using a solution of 4 eq. Fmoc-amino acid, 3.8 eq. HATU, 3.8 eq. HOAT, and 8 eq. DIEA in DMF at 30 °C. Each coupling step required 1 h and the resin was washed by DMF and DCM before Fmoc-deprotection. Double coupling was carried out when needed. In the case of 2CTC resin, the first amino acid was coupled with 4 eq. Fmoc-amino acid, 8 eq. DIEA in DMF-DCM (1:5) overnight and the resin was capped with methanol to quench the remaining 2-chlorotrityl chloride resin. The Fmoc group was removed by treatment with 20% piperidine in DMF twice (5 min and 10 min) followed by DMF and DCM wash. After the assembly of all peptide chains, the N-terminal Fmoc group was removed. After wash, the cleavage step was carried out by treating the resin with cleavage cocktail K (trifluoroacetic acid /phenol/water/thioanisole/1,2-ethanedithiol, 82.5:5:5:5:2.5) for 3 h. In the next step, the cleavage solution was collected and concentrated by blowing pure N2. After precipitation by cold diethyl ether and centrifugation, the crude peptides were obtained. Purification and analysis were carried out using analytical and semi-preparative HPLC and ESI-MS, and the desired peptides were obtained after lyophilization.

Native chemical ligation (NCL)

The C-terminal peptide hydrazide was dissolved in acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.2 M Na2HPO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15oC), and 15 eq. NaNO2 in acidified ligation buffer was added. The activation reaction system was kept in ice-salt bath with stirring for 30 min, after which 40 eq. MPAA in acidified ligation solution with 1.2 eq. N-terminal Cys peptide was added. The mixture was kept in ice-salt bath for 5 min under stirring, after which the pH of the solution was adjusted to 6.8 at room temperature. After 8 h, 80 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in a ligation buffer (pH 7.0) was added to dilute the system twice and the reaction system was kept for 1 h under stirring. Finally, the ligation product was analyzed by HPLC and ESI-MS, and purified by semi-preparative HPLC.

HPLC, mass spectrometry (MS), and circular dichroism (CD) spectroscopy

Reversed phase HPLC was performed on a Shimadzu Prominence HPLC system with LC-20AT as the solvent delivery unit. For analyzing the peptides, Vydac C18 (4.6 × 250 mm) was used at a flow rate of 1.0 ml/min. For peptide purification, Vydac C18 (10 × 250 mm and 22 × 150 mm) was used at flow rates of 4-6 ml/min. The UV absorption at 214 nm and 254



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nm were monitored during the injections. Water with 0.1% TFA and acetonitrile with 0.1% TFA were used as solvents, the gradients of which were optimized for each peptide. The ESI-MS spectra were obtained on an Agilent 1200 Series HPLC system with LC-MS (using Agilent 6340 ion trap as the mass spectrometer) or Shimadzu LCMS-IT/ TOF. The CD spectra were obtained on an Applied Photophysics Pistar-180 CD spectrometer.

Peptide sequencing by mass spectrometry

The synthetic L-ASFV pol X was purified by 15% SDS-PAGE, reduced by the addition of 10 mM DTT, and alkylated by 55 mM iodoacetamide. In-gel digestion was performed by adding trypsin and Glu-C in 25 mM NH4HCO3 at 37 °C overnight, and the peptides were extracted and analyzed by LC-MS. Only the synthetic L-ASFV pol X was sequenced to validate the synthetic route, because the synthetic D-ASFV pol X could not be effectively digested by trypsin. The samples were separated by gradient elution at a flow rate of 0.3 μl/min by the UltiMate 3000 RSLCnanoUHPLC system, connected to a Thermo LTQ-Orbitrapvelos mass spectrometer. The LTQ-Orbitrap mass spectrometer was operated under the data-dependent acquisition mode using Xcalibur 2.2., with full-scan mass spectrum in the Orbitrap (350-20000 m/z, 60 000 resolution) followed by 3 MS/MS scans in the quadruple collision cell using collision-induced dissociation (CID), with the precursor mass selection window set to 2 Da.

L-DNA sequencing by ESI-MS

The full-length L-DNA product was prepared in a large reaction system of 200 μl in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl, containing 12 μg D-ASFV pol X, 20 μM L-primer12 (without modification), 20 μM L-template18, and 0.4 mM L-dNTPs (each), incubated at 37 °C. The full-length product (18 nt) was separated from the intermediate bands (13-17 nt) and the unextended primer (12 nt) by 20% PAGE in 8 M urea, purified, and resuspended in 20 μl ddH2O for ESI-MS analysis under the negative mode. The intermediate bands were also purified, but their quantities were not sufficient for ESI-MS. The elution buffer contained 50% 10 mM ammonia acetate aqueous solution (pH 7.0) and 50 % acetonitrile, with the flow rate set to 0.8 ml/min.



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Supplementary Figures

Supplementary Figure S1 | Synthetic route for synthesizing D-ASFV pol X. The synthesis was achieved by assembling 3 peptide segments in the C- to N-terminus direction using hydrazides as thioester surrogates.



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Supplementary Figure S2 | Ligation of peptide segments 1 and 2. a, The high hydrophobicity of peptide segment 2 makes it difficult to synthesize, purify, and dissolve in Gn·HCl solution. The isoacyl peptide strategy facilitates the peptide synthesis and purification, and improves its solubility in Gn·HCl solution. In addition, the incorporation of isoacyl dipeptide works as a traceless modification due to the rapid O-to-N acyl shift at pH 7. Thus the isoacyl dipeptide (D-Lys95-D-Thr96) was incorporated in segment 2. We dissolved 5 μmol of segment 2 (12.7 mg) in 1.5 ml acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.2 M Na2HPO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15oC), and a 150 μl solution with 0.25 M NaNO2 in acidified ligation buffer was added to the mixture. The activation reaction was kept and stirred in ice-salt bath for 30 min, after which 1 ml solution of 0.2 M MPAA (in 6 M Gn·HCl and 0.2 M Na2HPO4, pH 6.0) with 6 μmol segment 1 (49.2 mg) was added. The mixture was stirred in ice-salt bath for 5 min. Then the pH of the solution was adjusted to 6.8 at room temperature. After 8 h, 3 ml 80 mM TCEP in ligation buffer (pH 7.0) was added and the reaction system was kept on stirrer for 1 h. The ligation product 3 was validated by HPLC and ESI-MS and purified by semi-preparative HPLC. After lyophilization, ligation product 3 was obtained with a 23% yield (12 mg). b, Analytical HPLC chromatogram of the ligation product 3 (λ=214 nm). The HPLC analysis was carried out with a Vydac C18 (4.6 × 250 mm) column by the gradient method (20-70% acetonitrile with 0.1% TFA over 30 min). c, ESI-MS spectrum of the ligation product 3, with an observed M.W. of 10717.2 Da (calculated M.W. 10716.8 Da).



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Supplementary Figure S3 | Desulfurization of product 3. a, A quantity of 1 μmol product 3 (10.7 mg) was dissolved in 2.5 ml 200 mM TCEP solution (6 M Gn·HCl and 0.2 Na2HPO4, pH 6.9), added with 50 μmol (16 mg) VA-044 and 100 μmol (30.7 mg) L-glutathione (reduced). The mixture was stirred at 37 oC overnight. Finally, the desulfurization product 4 was validated by HPLC and ESI-MS and purified by semi-preparative HPLC. After lyophilization, product 4 was obtained with a 50% yield (5.0 mg). b, Analytical HPLC chromatogram of the desulfurization product 4 (λ=214 nm). The HPLC analysis was carried out with a Vydac C18 (4.6 × 250 mm) column by the gradient method (20-70% acetonitrile with 0.1% TFA over 30 min). c, ESI-MS spectrum of the desulfurization product 4, with an observed M.W. of 10684.8 Da (calculated M.W. 10684.7 Da).



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Supplementary Figure S4 | Acm removal of product 4. a, A quantity of 0.5 μmol product 4 (5.0 mg) was dissolved in 1 ml AcOH/H2O (1:1), before AgOAc (25 µmol, 5 mg) was added. The reaction was carried out overnight at 30 oC on stirrer, after which 2.5 mmol β-mercaptoethanol dissolved in 6 M Gn·HCl aqueous solution was added to quench the reaction, and the reaction solution was stirred for another 1 h. After centrifugation, the supernatant was purified by semi-preparative HPLC. After lyophilization, the Acm removed product 5 was obtained with a 64% yield (3.2 mg). b, Analytical HPLC chromatogram of the Acm removed product 5 (λ=214 nm). The HPLC analysis was carried out with a Vydac C18 (4.6 × 250 mm) column by the gradient method (20-70% acetonitrile with 0.1% TFA over 30 min). c, ESI-MS spectrum of the Acm removed product 5, with an observed M.W. of 10613.9 Da (calculated M.W. 10613.7 Da).



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Supplementary Figure S5 | Ligation of product 5 and segment 6. a, To improve the purity of segment 6 during SPPS, D-Leu-D-Ser(ψMe,Me Pro)-OH and Gly-D-Ser(ψMe,Me Pro)-OH pseudoproline dipeptides were incorporated at relevant positions. A quantity of 1 µmol segment 6 (9.7 mg) was dissolved in 0.1 ml acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.2 M Na2HPO4, pH 3.0). The mixture was cooled in ice-salt bath (-15oC), and a 60 µl solution of 0.25 M NaNO2 in acidified ligation buffer was added to the mixture. The activation reaction was kept in ice-salt bath with stirring for 30 min, before a 0.1 ml solution of 0.4 M MPAA (in 6 M Gn·HCl and 0.2 M Na2HPO4, pH 6.0) with 1 µmol product 5 (10.6 mg) was added. The mixture was kept in ice-salt bath and stirred for another 5 min, after which the pH of the solution was adjusted to 6.5 at room temperature. After 12 h, 0.3 ml 80 mM TCEP in ligation buffer (pH 7.0) was added and the reaction solution was stirred for 1 h. The ligated product 7 (full-length D-ASFV pol X) was validated by HPLC and ESI-MS, and purified by semi-preparative HPLC. After lyophilization, product 7 was obtained with a 25% yield (5.1 mg). b, Analytical HPLC chromatogram of the ligation product 7 (D-ASFV pol X) (λ=214 nm). The HPLC analysis was carried out with a Vydac C18 (4.6 × 250 mm) column by the gradient method (20-70% acetonitrile with 0.1% TFA over 30 min). c, ESI-MS spectrum of the ligation product 7, with an observed M.W. of 20315.9 Da (calculated M.W. 20316.5 Da).



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Supplementary Figure S6 | Characterization of D-ASFV pol X after folding. a, Analytical HPLC chromatogram of folded D-ASFV pol X (λ=214 nm). The HPLC analysis was carried out with a Vydac C18 (4.6 × 250 mm) column by the gradient method (20-70% acetonitrile with 0.1% TFA over 30 min). b, ESI-MS spectrum of folded D-ASFV pol X with an observed M.W. of 20313.8 Da (calculated M.W. 20314.5 Da).



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Supplementary Figure S7 | Purity and chirality of L-dNTPs, L-DNA primers and templates were assessed by HPLC and CD. a, RP-HPLC profiles of L-dNTPs (data provided by ChemGenes). b, c, CD spectra of D- and L-primers, and D- and L-templates, respectively.



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Supplementary Figure S8 | Characterization and polymerization of D- and L-DNAs. a, L-DNA primers could not be efficiently radiolabeled through phosphorylation by T4 polynucleotide kinase (PNK). The PNK catalyzed 32P labeling of L- and D-DNA primers was carried out in a 50 μl system containing 25 μmol of L- or D-primer15, [γ-32P]ATP (3000 Ci/mmol), and 50 units of T4 PNK (NEB, U.S.), incubated at 37 oC for 3 h. The



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oligonucleotide was purified from the unreacted [γ-32P]ATP using a G25 MicroSpin Column. The labeled primer was analyzed by 20% PAGE in 8 M urea and visualized by autoradiography. We estimated an at least 100-fold lower radiolabeling efficiency of the L-primer as compared with that of the D-primer. b, Mirror-image DNA polymerization with a different set of template and primer sequences. The reaction was carried out in 50 mM Tris-HCl pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl, containing 2.5 μM of 15-nt L-primer15 (without modification), 2.5 μM of 21-nt L-template21, 0.2 mM L-dNTPs (each), and 1.4 μg D-ASFV pol X. After incubation at 37 oC for up to 12 h, the extension products were analyzed by 20% PAGE in 8 M urea, stained with SYBR Gold. c, ESI-MS spectrum of the fully extended L-DNA primer. The observed M.W. of 5516.9 corresponds to the expected full-length product (calculated M.W. 5517.6), with the presence of the template (observed M.W. 5481.9, calculated M.W. 5481.6) as impurity. d, Sequence validation of the D-DNAzyme polymerized by synthetic L-ASFV pol X. The 44-nt DNAzyme with an additional 12-nt primer sequence was polymerized by L-ASFV pol X, purified by PAGE, and ligated to a pEASY T3 vector for Sanger sequencing. e, Experimental design of the mirror-image ASFV pol X repeated cycles of polymerization. The FAM-labeled L-DNA primer can only be efficiently extended to full length when annealed to the enzymatically polymerized L-DNA from the first cycle, and the repeated cycles of polymerization proceed to produce more copies of L-DNA molecules of the same sequence.



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Supplementary Figure S9 | The observed rates of reaction of the natural and the mirror-image polymerase systems. a, The observed rates of reaction were defined from the rates of disappearance of primers. Time points were taken as fractions of unextended primer remaining versus time. Both reactions were carried out in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl, containing the same quantity of 0.7 μg L- and D-ASFV pol X, 2.5 μM FAM-primer12, 2.5 μM template18, 0.2 mM dNTPs (each) of the corresponding chirality. The products were analyzed 20% PAGE in 8 M urea and quantified using the ImageQuant software. Dotted lines are linear fits, with the slop being kobs (observed rate of reaction) (h-1). b, Time course of the natural and the mirror-image RNA polymerization systems. Both reactions were carried out in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl, containing the same quantity of 0.7 μg L- and D-ASFV pol X, 2.5 μM FAM-primer12, 2.5 μM template18, 0.2 mM NTPs (each) of the corresponding chirality. The products were analyzed 20% PAGE in 8 M urea and quantified using the ImageQuant software. Dotted lines are linear fits, with the slop being kobs (h-1). c, Time course of the chirally mixed experiment. The reactions were carried out in the same buffer condition of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl, containing the same quantity of 0.7 μg L- and D-ASFV pol X. The natural system used 2.5 μM D-Cy5-primer20, 2.5 μM D-template26, and 0.2 mM D-dNTPs (each), while the mirror-image system used 2.5 μM L-FAM-primer12, 2.5 μM L-template18, and 0.2 mM L-dNTPs (each). The longer (20-nt) D-primer appeared to have affected the efficiency of the natural system. The chirally mixed system contained the same concentration of polymerases, primers, templates, and dNTPs. Dotted lines are linear fits, with the slop being kobs (h-1). d, kobs in the natural and the mirror-image DNA polymerization systems. Data are presented as mean±SEM, n=3.



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Supplementary Table S1 | Peptide sequencing of synthetic L-ASFV pol X by mass spectrometry

Segment sequence XCorr Charge MH+ [Da] ΔM [ppm] RT [min] EEKmLNDVDLLIIVPEK 4.86360168 2 2014.09123 6.67686844 43.7154 HVLPNIRIKGLSFSVKVcGER

2.49292898 3 2409.36637 4.78211214 44.34

KcVLFIEWEK 3.71132851 2 1351.71477 5.0557844 38.6446 KKTYQLDLFTALAE 3.88172317 2 1640.89983 6.31761428 43.7574 KLLKHVLPNIR 3.55191755 2 1330.87224 3.02709865 31.6344 KTYQLDLFTALAEEKPYAIFHFTGPVSYLIR

6.0060935 3 3631.95664 10.0763472 54.9092

LNQYGLFKNQTLVPLKITTEKELIKELGFTYRIPKKRL

2.74559188 5 4533.6656 7.29527526 54.3708

mLTLIQGKKIVNHLRSRLAFEYNGQLIK

2.66363716 3 3299.90256 7.61680303 49.6173

NYKLNQYGLFK 3.87206316 2 1387.74187 3.58682542 33.5826 SRLAFEYNGQLIKILSKNIVAVGSLRREEK

2.5462563 5 3431.98255 9.8659898 54.0116



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Supplementary Table S2 | DNA oligonucleotide sequences Oligo name Sequence

D-/L-primer12 5'-ACTACGAACGCG

D-/L-FAM-primer12 5'-FAM-ACTACGAACGCG

D-/L-template18 5'-CTCAGTCGCGTTCGTAGT

D-/L-DNAzymeTemplate 5'-TGTACAGCCACTTCAACTAATTGCTCAACTATGGCTGTAGCACCCGCGTTCGTAGTATGCAATGCA

Cy5-primer20 5'-Cy5-AGTGCGATACTACGAACGCG

template26 5'-CTCAGTCGCGTTCGTAGTATCGCACT

template18A 5'-CTCAGACGCGTTCGTAGT

template18C 5'-CTCAGCCGCGTTCGTAGT

template18G 5'-CTCATGCGCGTTCGTAGT

L-primer15 5'-GATCACAGTGAGTAC

L-template21 5'-CTATTGTACTCACTGTGATC

DNAzyme marker 5'-FAM-ACTACGAACGCGGGTGCTACAGCCATAGTTGAGCAATTAGTTGAAGTGGCTGTACA

L-template27 5'-CGCGCTGTTATAGGGATACGGCAAAAA

L-primer11 5'-CGCGCTGTTAT

L-FAM-primer11 5'-FAM-CGCGCTGTTAT

L-reverse11 5'-GCCGTATCCCT

Oligonucleotides without D-/L- prefix are D-DNA.



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Supplementary References 1 Raibaut, L., Ollivier, N. & Melnyk, O. Sequential native peptide ligation strategies for

total chemical protein synthesis. Chem Soc Rev 41, 7001-7015, (2012).

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SUEMENTARY INRMATIN - Nature · SUEMENTARY INRMATIN.10/.2517 1 ... MS spectra were obtained on an Agilent 1200 Series HPLC system with LC-MS ... or Shimadzu LCMS-IT/ TOF