Takashi Kawakami, Hiroshi Murakami, and Hiroaki Suga. Preparation of Flexizyme, Microhelix RNA, Suppressor trna Asn-E2 and Initiator

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1 Chemistry & Biology 15 Supplemental Data Messenger RNA-Programmed Incorporation of Multiple N-Methyl-Amino Acids into Linear and Cyclic Peptides Takashi Kawakami, Hiroshi Murakami, and Hiroaki Suga Supplemental Experimental Procedures Preparation of Flexizyme, Microhelix RNA, Suppressor trna Asn-E2 and Initiator trna fmet CAU All oligonucleotides, listed in Supplemental table S1, were purchased from OPERON, Japan. Flexizyme, microhelix RNA and suppressor trna Asn-E2 s were prepared as previously described (Murakami et al., 2006). To prepare the template DNA coding dinitro-flexizyme (dfx) or enhanced flexizyme (efx), O5-1 was annealed with O3-1 or O3-2 respectively and extended by Taq DNA polymerase. The resulting DNA templates were amplified by PCR using the primer pair, O5-2 and O3-3 or O5-2 and O3-4, for dfx or efx respectively. The prepared DNA templates were in vitro transcribed using T7 RNA polymerase according to the standard procedure and the RNA transcripts were purified by 12% denaturing PAGE. A microhelix RNA was prepared by using in vitro transcription in the presence of annealed O5-3 and O3-5 under the same condition as above and purified by 20% denaturing PAGE. For the preparation of DNA template of trna Asn-E2, O5-4 and O3-6 were annealed and extended by Taq DNA polymerase. The resulting dsdna was amplified using O5-2 and O3-7. For the preparation of DNA template of trna fmet CAU, O5-5 and O3-8 were annealed and extended by Taq DNA polymerase. The resulting dsdna was amplified using O5-2 and O3-9. The DNA products were transcribed in the presence of 10 mm GMP under the same condition as above and purified by 8% denaturing PAGE. 1

2 Preparation of DNA Templates Coding Peptides For the DNA template coding fmet-arg- Me aa-arg-flag, O5-6 and O3-10 were annealed and extended by Taq DNA polymerase, and then the resulting dsdna was amplified using O5-2 and O3-11. For the DNA template of m4-i, m4-ii, m4-iii, m4-iv, m5, m1-ii, m2-ii or m3-ii, O5-6 and O3-12, O3-13, O3-14, O3-15, O3-16, O3-17, O3-18 or O3-19 were annealed respectively and extended by Taq DNA polymerase, and then the resulting dsdnas were amplified using P5-2 and P3-20. For the DNA template of m6, m8 or m10, O5-6 and O3-21 were annealed and extended by Taq DNA polymerase. The resulting dsdnas were amplified using O5-2 and O3-22, O3-23 or O3-24 for m6, m8 or m10 respectively, and then these products were further amplified using O5-2 and O3-20. For the DNA template of mc1, mc2 or mc3, O5-6 and O3-25, O3-26 or O3-27 were annealed respectively and extended by Taq DNA polymerase. The resulting dsdnas were amplified using O5-2 and O3-28, O3-29 or O3-30 for mc1, mc2 or mc3 respectively, and then these products were further amplified using O5-2 and O3-20. Prepared DNA templates were purified by the extraction with phenol/chloroform and ethanol precipitation. Aminoacylation Assay with Microhelix RNA by Flexizyme Aminoacylation reactions were performed according to the following procedure: 2.5 μl of 20 μm microhelix RNA in 0.2 M Hepes-KOH [ph 7.5] was heated at 95 C for 1 min and cooled to room temperature over 5 min. 1 μl of 0.1 M MgCl 2 and 0.5 μl of 0.1 mm dfx, or 1 μl of 3 M MgCl 2 and 0.5 μl of 0.1 mm efx were added to the solution. 1 μl of 25 mm N α -methylated amino acid substrate (DBE or CME) in DMSO was then added to the mixture and incubated on ice for 2-24 hours. For Me Thr, Me Tyr, Me Fni and Me Yme, 1 μl of 200 mm substrate was used. The acylation reaction was quenched by addition of 15 μl of the loading buffer (150 mm sodium acetate [ph 5.0], 10 mm EDTA 2

3 and 83% formamide). This sample was analyzed by 20% acid denaturing PAGE, stained with ethidium bromide and quantified by using FLA-5100 (Fuji, JAPAN). Preparation of Ordinary PURE System and wpure System Translation factors, enzymes and ribosome were prepared by the procedure reported before (Josephson et al., 2005; Shimizu et al., 2001). These translation elements were mixed with 50 mm Hepes-KOH [ph 7.6], 20 mm Mg(OAc) 2, 100 mm potassium glutamate, 2 mm spermidine, 1 mm DTT, 30 mm creatine phosphate (Roche), 4 mm ATP, 2 mm GTP, 1 mm CTP, 1 mm UTP, 100 μm 10-formyl-5,6,7,8-tetrahydrofolic acid (Baggott et al., 1995), and 3 mg/ml E. coli total trna (Roche). Translation reaction mixtures contained final concentrations of 0.6 μm MTF, 2.7 μm IF1, 0.8 μm IF2, 1.5 μm IF3, 0.5 μm EF-G, 10 μm EF-Tu, 1 μm EF-Ts, 0.5 μm RF2, 0.5 μm RF3, 1 μm RRF, 0.1 μm T7 RNA polymerase, 0.1 μm nucleotide-diphosphate kinase, 4 μg/ml creatine kinase (Roche), 3 μg/ml myokinase (Sigma), 2 units/ml pyrophosphatase (Sigma), and 2.4 μm ribosome. In the ordinary PURE system for wildtype peptides, the reaction mixtures contained twenty kinds of aarss (0.6 μm AlaRS, 0.2 μm ArgRS, 0.6 μm AsnRS, 0.6 μm AspRS, 0.2 μm CysRS, 0.4 μm GlnRS, 0.6 μm GluRS, 0.2 μm GlyRS, 0.2 μm HisRS, 0.2 μm IleRS, 0.2 μm LeuRS, 0.6 μm LysRS, 0.2 μm MetRS, 0.4 μm PheRS, 0.2 μm ProRS, 0.2 μm SerRS, 0.4 μm ThrRS, 0.6 μm TrpRS, 0.4 μm TyrRS, 0.2 μm ValRS). In the wpure system for linear N-methylated peptides ( Me P4-I, Me P4-II, Me P4-III, Me P4-IV, Me P5, Me P6, Me P8, Me P10, Me P1-II, Me P2-II and Me P3-II), the reaction mixtures contained four kinds of aarss (0.6 μm AspRS, 0.6 μm LysRS, 0.2 μm MetRS, 0.4 μm TyrRS). In the wpure system for cyclic N-methylated peptides ( Me Pc1, Me Pc2 and Me Pc3), the reaction mixtures contained six kinds of aarss (0.6 μm AspRS, 0.2 μm CysRS, 0.6 μm LysRS, 0.2 μm MetRS, 0.2 μm ProRS, 0.4 μm TyrRS). 3

4 Table S1. Primers Used for the Preparation of Flexizyme, Microhelix RNA, trna and DNA Template Coding Peptides XXX indicates anticodon. 4

5 Figure S1. Chemical Structure of N α -methylated Nonproteinogenic Amino Acids Used in This Study Me Nva, N α -methyl-l-norvaline; Me Nle, N α -methyl-l-norleucine; Me Fni, N α -methyl-l-p-nitrophenylalanine; Me Yme, N α -methyl-l-p-methoxyphenylalanine. Figure S2. Flexizyme-Catalyzing Aminoacylation of Microhelix RNA with N α -methylated Amino Acids Lanes 1 and 20, negative controls in the absence of Me aa substrate by using dfx and efx, respectively; Lanes 2-19, dfx-catalyzing aminoacylation with designated Me aa 3,5-dinitrobenzyl ester; lanes 21-25, efx-catalyzing aminoacylation with designated Me aa cyanomethyl ester; Band (a), N α -methyl-aminoacyl-microhelix RNAs; Band (b), uncharged microhelix RNAs; The yield of N α -methyl-aminoacyl-microhelix RNA was calculated based on the fluorescence intensity of bands (a) and (b), fitted to (a)/[(a)+(b)]. Each yield was determined by a mean score of triplicates. Error bars represent standard deviation. 5

6 Figure S3. Expression of Mono-, Di-, Tri-, and Tetra-N-methyl-peptides (A) Sequences of mrna templates (m1-ii, m2-ii, m3-ii, and m4-ii), control wildtype peptides (wt1-ii, wt2-ii, wt3-ii, and wt4-ii), and peptides containing 1, 2, 3 or 4 consecutive N-methyl-peptide bonds ( Me P1-II, Me P2-II, Me P3-II, and Me P4-II). (B) Tricine-SDS-PAGE analysis of control wildtype peptides and N-methyl-peptides expressed from the respective mrna. The wildtype peptides (lanes with odd numbers) were expressed in the ordinary PURE system, while N-methyl-peptides (lanes with even numbers) were expressed in wpure system containing Me Yme-tRNA Asn-E2 GGU for Me P1-II, or Me Yme-tRNA Asn-E2 GGU and Me Phe-tRNA Asn-E2 GAG for Me P2-II, or Me Yme-tRNA Asn-E2 GGU, Me Phe-tRNA Asn-E2 GAG and Me Ser-tRNA Asn-E2 GAA for Me P3-II and Me P4-II, respectively. Absolute expression level of each peptide based on its observed 6

7 radioisotope counts is shown in the upper graph, and relative expression level of each N-methyl-peptide against the corresponding wildtype peptide is shown in the lower graph. Each expression level was determined by a mean score of triplicates. Supplemental References Baggott, J.E., Johanning, G.L., Branham, K.E., Prince, C.W., Morgan, S.L., Eto, I., and Vaughn, W.H. (1995). Cofactor role for 10-formyldihydrofolic acid. Biochem. J. 308 ( Pt 3), Josephson, K., Hartman, M.C., and Szostak, J.W. (2005). Ribosomal synthesis of unnatural peptides. J. Am. Chem. Soc. 127, Murakami, H., Ohta, A., Ashigai, H., and Suga, H. (2006). A highly flexible trna acylation method for non-natural polypeptide synthesis. Nat. Methods 3, Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19,