Consecutive Elongation of D-Amino Acids in Translation

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1 rticle onsecutive Elongation of -mino cids in Translation raphical bstract uthors Takayuki Katoh, Kenya Tajima, Hiroaki Suga orrespondence (T.K.), (H.S.) In Brief Katoh et al. devised a new ribosomal translation system compatible with consecutive incorporation of -amino acids. p to ten consecutive -Ser were successfully introduced into peptides. Macrocyclic peptides with four or five consecutive -amino acids closed by a disulfide or thioether bond were also expressed. Highlights d new ribosomal translation system was devised for -amino acid incorporation d d d Elongator trn sequence and concentrations of IF2, EF-Tu, and EF- were optimized Ten consecutive -Ser residues were ribosomally incorporated into a peptide chain Macrocyclic -peptides closed by a disulfide or thioether bond were also expressed Katoh et al., 2017, ell hemical Biology 24, January 19, 2017 ª 2017 Elsevier td.

2 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), ell hemical Biology rticle onsecutive Elongation of -mino cids in Translation Takayuki Katoh, 1,2, * Kenya Tajima, 1 and Hiroaki Suga 1,3,4, * 1 epartment of hemistry, raduate School of Science, The niversity of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan 2 JST, PRESTO, Hongo, Bunkyo-ku, Tokyo , Japan 3 JST, REST, Hongo, Bunkyo-ku, Tokyo , Japan 4 ead ontact *orrespondence: katoh@chem.s.u-tokyo.ac.jp (T.K.), hsuga@chem.s.u-tokyo.ac.jp (H.S.) SMMRY Recent progress in the field of genetic code reprogramming using a reconstituted cell-free translation system has made it possible to incorporate a wide array of non-proteinogenic amino acids, including N-methyl-amino acids and -amino acids. espite the fact that up to ten N-methyl-amino acid residues can be continuously elongated, the successive incorporation of even two -amino acids into a nascent peptide chain remains a formidable challenge, thus far being nearly impossible. Here we report achievement of continuous -amino acid elongation by the use of engineered trns and optimized concentrations of translation factors, enabling us to incorporate up to ten consecutive -Ser residues into a nascent peptide chain. We have also expressed macrocyclic peptides consisting of four or five consecutive -amino acids consisting of -Phe, -Ser, -la, or -ys closed by either a disulfide bond or a thioether bond. INTROTION In the naturally occurring ribosomal translation system, 20 proteinogenic amino acids are exclusively utilized for synthesizing polypeptides. However, a reconstituted cell-free translation system in combination with an artificially reprogrammed genetic code has successfully been used to elongate various non-proteinogenic amino acids for expression of desired non-standard peptides. To date, not only amino acids with artificial side chains but also N-methyl-a-amino acids, N-alkyl-a-amino acids, b-amino acids, and -amino acids have been successfully introduced into peptide chains by use of genetic code reprogramming (chenbach et al., 2015; Fujino et al., 2013; oto et al., 2008; Kawakami et al., 2008a, 2008b, 2013; Ohta et al., 2007; Subtelny et al., 2008; Tan et al., 2004; Yamagishi et al., 2011). Importantly, the incorporation of certain N-methyl-a-amino acids and N-alkyl-a-amino acids into the nascent peptide chain has been accomplished not only singly but also consecutively, despite the slow rate of peptide bond formation for these secondary amino acids (oerfel et al., 2015; Pavlov et al., 2009). For instance, Kawakami et al. (2008a, 2013) and oto et al. (2011) demonstrated ten successive incorporations of three different N-methyl-a-amino acids and four consecutive incorporations of four different N-alkyl-a-amino acids by means of the flexible in vitro translation (FIT) system, in which these amino acids were pre-charged on orthogonal suppressor trns by means of a flexizyme. In contrast, even two incorporations of -amino acids in a row is extremely inefficient. Fujino et al. (2013) classified 19 -amino acids into three groups based on the efficiency of their single incorporation by the FIT system: group I with a 40% or higher translation yield relative to that of the corresponding -amino acid incorporation (la, Ser, ys, Met, Thr, His, Phe, and Tyr), group II with a 10% 40% yield (sn, ln, Val, and eu), and group III with less than a 10% yield or truncated peptide production (rg, ys, sp, lu, Ile, Trp, and Pro). However, two consecutive incorporations of -amino acids was nearly impossible even with the group I -amino acids (e.g., -Phe and -la). chenbach et al. (2015) also reported that consecutive -amino acid incorporation is difficult in their reconstituted translation system, and generally shows less than a 10% yield compared with the consecutive incorporation of -amino acids. To improve the accommodation rate of the -aminoacyl-trn in the ribosome, chenbach et al. (2015) also engineered a trn ly with a high binding affinity for EF-Tu (chenbach et al., 2015) based on previously reported data that the improved trn-ef-tu binding affinity might influence the incorporation efficiency of nonproteinogenic amino acids (oi et al., 2007; Ieong et al., 2014; ariviere et al., 2001; Schrader et al., 2011; Terasaka et al., 2014). Moreover, EF-Tu mutants that have a higher binding affinity for trns were also constructed for the attempts. However, use of these mutants did not result in an improvement of the efficiency of consecutive incorporations of -amino acids. More recently, edkova et al. (2003, 2006) reported that the incorporation of -Met and -Phe could be improved by introducing point mutations at the peptidyl transferase center of the ribosome. Their result indicates that acceleration of the slow peptidyl transfer reaction could also be effective for improving -amino acid incorporation. However, this study using the mutant ribosomes has yet to be extended to improve the outcomes with either more difficult -amino acids or consecutive -amino acid incorporations. Based on the preceding unsuccessful outcomes, we have revisited attempts at the consecutive incorporation of -amino acids by tuning two critical components, the trn body 46 ell hemical Biology 24, 46 54, January 19, 2017 ª 2017 Elsevier td.

3 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), sequence and the concentrations of translation protein factors. First, we have tuned the trn sequence and native EF-Tu concentration in order to improve the trn-ef-tu interaction and its accommodation rate. We have previously utilized trn sne2 for the incorporation of many non-proteinogenic amino acids in our conventional FIT system, but here substituted it with an engineered trn lue2. Second, the concentrations of IF2 and EF- were tuned to improve the initiation and translocation efficiencies, respectively. onsequently, we were able to achieve more than a 5-fold improvement of the translation yield of a -la--la-containing peptide. sing this optimized FIT system, we have successfully demonstrated the elongation of ten consecutive -Ser residues, as well as five consecutive incorporations of three different -amino acids. Moreover, expression of macrocylic peptides containing -amino acids, cyclized via a disulfide or thioether bond has also been demonstrated. RESTS Optimization of Translation onditions for Incorporation of onsecutive -mino cids To tune the sequence of the trn for the consecutive incorporation of -amino acids, we designed a model peptide based on the earlier work by Fujino et al. (2013), where the peptide contained two -la residues in a row (Figure 1, rp1). In this particular FIT system, Ser, SerRS, and other amino acid/aminoacyltrn synthetase pairs not coded by the mrn (mr1) were removed, and instead -alanyl-trn, prepared using a flexizyme (dfx), was added (Figure S1). -la, assigned to the codon of the Ser, was introduced at two consecutive codons in an mrn, mr1, to produce a model peptide, rp1 (Figure 1) under the reprogrammed genetic code where only -amino acids required to express rp1 were assigned (Figure S1B). RF1 was omitted from the translation system, and thus only was assigned as a stop codon designated by RF2 (note that, also recognized by RF2, could also act as the terminator codon, but was not utilized in our experiments). s a positive control, -la-trn, prepared using a flexizyme, was also prepared for introducing -la at the codons. In the past, we have extensively used an orthogonal trn sne2, derived from E. coli trn sn, for incorporation of various non-proteinogenic amino acids in the FIT system (Figure 1B). lthough trn sne2 was fairly efficient for the single incorporation of -amino acids (Fujino et al., 2013), it was inefficient for two consecutive incorporations, even for the group I -la (Figure 1E, lane 3). Therefore, we firstly investigated another trn aiming at improving the efficiency of consecutive -la incorporations. ale et al. (2004) and ale and hlenbeck (2005) have determined the binding affinity of various aminoacyltrns to EF-Tu, showing that the body sequence of trns contributes to the degree of affinity. For instance, 0 values of -Val-tRN sn, -Val-tRN ly, and -Val-tRN lu are 8.8, 10.7, and 11.7 kcal/mol, respectively, which are the sixth highest, the fourth lowest, and the lowest 0 values among those of the E. coli trns. This translates to the affinity between -Val-tRN lu and EF-Tu being 110-fold higher than between -Val-tRN sn and EF-Tu. Since -aminoacyl-trns intrinsically have weaker affinities for EF-Tu than the ordinary -aminoacyltrns, it is likely better to use a stronger affinity trn body for the consecutive incorporation of -amino acids. Based on the above considerations, we engineered E. coli trn lu to be a new orthogonal trn, referred to as trn lue2 (Figures 1 and 1) (Terasaka et al., 2014). To introduce -la at codon, the naturally occurring anticodon loop sequence of the wild-type trn lu was substituted with that of E. coli trn Ser (Figure 1, trn lue2 ), which was then charged with -la by dfx. The rp1 peptide was translated in the presence of -la-trn lue2 and [ 14 ]sp and subsequently separated by tricine SS-PE (Figure 1E, lane 4), followed by autoradiographic quantification. We also performed translation of rp1 in the presence of the -la counterpart charged by dfx onto trn sne2 and trn lue2 as positive controls (Figure 1E, lanes 1 and 2). The translation yield of rp1 using -la-trn lue2 was significantly improved by comparison with translation using -la-trn sne2 (Figure 1E, lane 4 versus 3; 0.20 and 0.07 mm, respectively). The respective rp1 peptides containing -la and -la (rp1-2 and rp1-2 ) appeared to run as a single mobility band on PE, but their mobilities were unmistakably different. This is consistent with our previous results where the mobility of peptides containing even a single -amino acid differ from those with the -amino acid counterpart (Fujino et al., 2013; oto et al., 2008). The respective rp1 peptides were analyzed by MI-TOF mass spectrometry (Figure 1F), which demonstrated that all peptides had the same molecular mass. Therefore, the observed difference in mobility between rp1-2 in lane 2 (or lane 1) and rp1-2 (lane 4) must originate from the different composition of the residues, i.e., -la--la or -la--la (Figure 1E). This in turn confirms that the -la residues were consecutively incorporated into the nascent chain of rp1-2. For single incorporation of -la or -la, translation of mr2 mrn into rp2 peptide was also conducted (Figure S2). Single incorporation of -la is efficient enough even with trn sne2 as shown in the previous report (Fujino et al., 2013). Thus, no significant improvement in the yield of -la peptide was observed by using trn lue2 instead of trn sne2. When we set the peptide expression under competing conditions in the presence of both -la-trn sne2 /trn lue2 and -latrn sne2 /trn lue2, the observed yields of the -la- and -la-containing peptides were 0.07 and 0.95 mm in trn sne2 and 0.19 and 1.20 mm in trn lue2, respectively. This result indicates that the intrinsic difference in efficiency of -la versus -la incorporation is an approximately 10-fold, but the ratio of -la incorporation was slightly more in trn lue2 compared with trn sne2. Next, concentrations of IF2, EF-Tu, and EF- were optimized in the translation of rp1-2 based on the considerations described below: as IF2 is the initiation factor that supports entry of fmet-trn ini into the ribosomal P site, elongation of -amino acids should not be directly affected by changing IF2 concentration. However, initiation is a critical step in translation that has influence on the overall translation rate. Therefore, we considered that elevating the concentration of IF2 might enhance the yield of full-length peptide via an IF2-mediated stimulation of the initiation event. EF-Tu is a factor that binds aminoacyl-trns and accommodates them into the ribosomal site. Therefore, by elevating the concentration of EF-Tu it ell hemical Biology 24, 46 54, January 19,

4 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), mrn (mr1): wild type peptide (wp1): reprogrammed peptide (rp1): fm K K K S S Y K K (stop) fm K K K /- /- Y K K (stop) flag B trn sne2 trn lu trn lue2 E ane Stereochemistry of la trn Peptide yield ( M) 1 sne lue sne lue rp1-2 rp1-2 F Intensity (a.u.) -la lue2 alc.: Obs. : la lue2 alc.: Obs. : m/z m/z Figure 1. Incorporation of Two onsecutive -la Residues into a Model Peptide using trn lue2 () mrn sequence (mr1) and the corresponding peptide sequences (wp1 and rp1) used in this experiment. The sequence of rp1 is reprogrammed based on the codon table shown in Figure S1B. Bold letters indicate the reprogrammed codons and the corresponding amino acids. (B ) Sequences of transcribed trn sne2 (B), trn lu (), and trn lue2 (). lteration of nucleotides from the wild-type E. coli trn sn (B) and trn lu () are indicated in red. (E) Tricine SS-PE analysis of the rp1 peptide synthesized by the FIT system. trn sne2 or trn lue2 was used for incorporation of - or -la at the codons of mr1. rrows indicate the bands corresponding to the desired products. (F) MI-TOF MS analysis of peptide rp1 synthesized by the FIT system using trn lue2 for introduction of - or -la. rrows indicate the peaks corresponding to the desired products. alc. and Obs. indicate calculated and observed m/z values, respectively. might be possible to modulate the accommodation rate of the -aminoacyl-trn. By contrast, EF- induces translocation of P site deacyl-trn and site peptidyl-trn into the E and P sites, respectively. Moreover, it has also been suggested that EF- is involved in releasing peptidyl-trns from the stalled ribosome (Rao and Varshney, 2001). If the presence of the peptidyl--xaa--xaa-trn (-Xaa represents certain -amino acids) in the P site could cause ribosomal stalling, drop-off of this peptidyl--xaa--xaa-trn would be promoted by EF-, resulting in poor elongation. This hypothesis prompted us to reduce EF- concentration to suppress such undesired peptidyl-trn release from the ribosome. For the initial experiments described above (Figures 1E and 1F) we used our conventional conditions for the FIT system (oto et al., 2011), with concentrations of these factors set at 0.4 mm IF2, 10 mm EF-Tu/Ts and 0.26 mm EF-, respectively. 48 ell hemical Biology 24, 46 54, January 19, 2017

5 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), Yield of peptide ( M) M EF- 10 M EF-Tu/Ts trn lue B Yield of peptide ( M) M EF M IF2 trn lue IF2 ( M) EF-Tu/Ts ( M) Yield of peptide ( M) M IF2 10 M EF-Tu/Ts trn lue EF- ( M) Figure 2. Titration of Translation Factors in Translation of rp1-2 Peptide () Titration of IF2 concentration. Translation was carried out for 30 min with 0.1 mm EF- and 10 mm EF-Tu/Ts using trn lue2 for introduction of -la. Error bars, S (n = 3). (B) Titration of EF-Tu/Ts concentration. Translation was carried out for 30 min with 0.1 mm EF- and 3 mm IF2 using trn lue2 for introduction of -la. Error bars, S (n = 3). () Titration of EF- concentration. Translation was carried out for 30 min with 0.4 mm IF2 and 10 mm EF-Tu/Ts using trn lue2 for introduction of -la. Error bars, S (n = 3). Note that EF-Tu was co-purified with EF-Ts as a complex and added to the translation system. Thus, the concentration of EF-Ts should be equal to that of EF-Tu. To optimize the conditions, a series of titration experiments of the respective IF2, EF-Tu/Ts, and EF- concentrations thought to influence the expression yield of rp1-2 were carried out. These revealed that 3 mm IF2, 20 mm EF-Tu/Ts, and 0.1 mm EF- resulted in the highest peptide yield (Figures 2 2); the concentrations of IF2 and EF-Tu/Ts were elevated, whereas that of EF- was reduced from the conventional conditions, as expected. The yield of the rp1-2 peptide was improved more than 5-fold compared with the original translation conditions from 0.07 to 0.38 mm (Figure 1E, lane 3 and Figure 2B, 20 mm EF-Tu/Ts). Thus, we adopted these conditions for our subsequent investigations. Incorporation of Ten onsecutive -Ser Residues into Peptides sing our newly optimized set of conditions for translation with the FIT system, we analyzed how many -amino acids could be continuously introduced into a peptide. - and -Ser were charged on trn lue2 using a flexizyme, and introduced at three to ten consecutive codons of mr3 to produce the corresponding rp3-(/-ser) n peptides (Figure 3) using a reprogrammed codon table shown in Figure S1. Note that we chose /-Ser instead of /-la for the longer consecutive elongation to avoid the risk of the peptide product becoming insoluble in buffer solution. Once again the respective rp3-(- Ser) n and rp3-(-ser) n pairs with the same length (n = 3 10) showed different mobilities on tricine SS-PE, indicating that these peptides differ in physical properties even though both were expressed from the same mrn template (Figure 3B). The translation efficiency of rp3-(-ser) n decreased when the number of consecutive -Ser increased from 1.16 mm for rp3-(-ser) 3 to 0.01 mm for rp3-(-ser) 10 (note this was still above our detection limit). On the other hand, the native rp3-(-ser) n showed only a moderate decrease from 2.46 mm rp3-(-ser) 3 to 0.32 mm rp3-(-ser) 10. In this experiment, orthogonal trn lue2 was pre-charged with -Ser by dfx, and therefore cannot be recycled by SerRS once deacylated. Thus, it is likely that starvation of -Ser-tRN could occur in incorporation of a large number of -Ser. This would be the reason for the observed lower yield of multiple -Ser-containing peptides. ell hemical Biology 24, 46 54, January 19,

6 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), mrn, mr3: peptide, rp3-(/-s) n : () n flag fm K K K (/-S) n flag (stop) B ane Stereochemistry of Ser n Peptide yield ( M) rp3-(/-s) n Intensity (a.u.) rp3-(-s) 5 alc. : Obs. : alc. : rp3-(-s) 6 rp3-(-s) Obs. : alc. : Obs. : m/z m/z m/z Intensity (a.u.) rp3-(-s) alc. : alc. : rp3-(-s) Obs. : rp3-(-s) Obs. : alc. : Obs. : m/z m/z m/z Figure 3. Incorporation of Ten onsecutive -Ser Residues into a Model Peptide () mrn sequence (mr3) and the corresponding peptide sequence (rp3). n indicates the number of consecutive codons in mr3 and - or -Ser in rp3. Sequence of flag is the same as mr1 and rp1. The sequence of rp3 is reprogrammed by using the codon table shown in Figure S1. Bold letters indicate the reprogrammed codons and the corresponding amino acids. (B) Tricine SS-PE analysis of rp3 synthesized by the FIT system. n indicates the number of consecutive - or -Ser. () MI-TOF MS analysis of peptide rp3 containing consecutive -Ser. rrows indicate the peaks corresponding to desired products. alc. and Obs. indicate calculated and observed m/z values, respectively. See also Figure S3 for the result of peptide rp3 containing consecutive -Ser. Fidelity of translation was confirmed by MI-TOF mass spectrometry (Figures 3 and S3). The observed peaks corresponding to rp3-(-ser) n (Figure 3) were consistent with the calculated values, indicating that the Ser residues were all incorporated. The observed mass values of rp3-(-ser) n were also consistent with the calculated values (Figure S3). These results indicate that the mobility difference between rp3-(-ser) n and rp3-(-ser) n in the tricine SS-PE analysis (Figure 3B) originated from the difference in the incorporation of -Ser or -Ser in the respective nascent peptide chain. We thus conclude that rp3-(-ser) n and rp3-(-ser) n were correctly expressed under the reprogrammed genetic code. Most importantly, this demonstration represents ribosomal incorporation of up to ten consecutive -amino acid residues for the first time. Translation of Macrocyclic -Peptides losed by a isulfide Bond or Thioether Bond To demonstrate consecutive elongation of different types of -amino acids, we conducted the translation of a peptide consisting of -ys--ser--la--ser--ys using the FIT system (Figure 4, rp4). rp4 was translated from mr4 using -ystrn lue2, -Ser-tRN lue2, and -la-trn lue2 based on the reprogrammed codon table shown in Figure S1. In addition to the full--rp4 peptide (-ys--ser--la- -Ser--ys), all the possible combinations of - and -amino acids were tested using /-ys-trn lue2, /-Ser-tRN- lue2, and /-la-trn lue2 (Figure 4B). The full--peptide showed a unique mobility in tricine SS-PE compared with other mixed /-rp4 peptides containing -ys, -Ser, 50 ell hemical Biology 24, 46 54, January 19, 2017

7 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), mrn (mr4): flag wild type peptide (wp4): fm K K K H T I T H flag (stop) reprogrammed peptide (rp4): fm K K K /- /-S /- /-S /- flag (stop) S S B ane Stereochemistry ys () Ser () la () Peptide yield ( M) Intensity (a.u.) full--rp4: ys/-ser/-la/-ser/-ys and/or -la. The observed mass value of each peptide by MI-TOF mass spectrometry was consistent with the calculated value (Figures 4 and S4), for which the two ys side chains were closed via a disulfide bond, indicating that the desired disulfide-macrocyclic full--rp4 was expressed. Macrocyclic peptides closed by non-reducible thioether bonds have greater potential for therapeutic uses than those closed by reducible disulfide bonds. We thus examined a -peptide closed by a thioether bond under a reprogrammed genetic code including an initiation codon reassignment to N-chloroacetyl- -phenylalanine (- lc Phe, Figure S1E). - lc Phe charged on initiator trn fmet was introduced at the N terminus of a peptide, rp5 (Figures 5 and 5B), followed by elongation of -Ser--Ser--ys dictated by the reprogrammed codon table, where the sulfhydryl group of the -ys spontaneously attacks the a carbon of the N-terminal lc group to form the thioether bond. Translation of the full--rp5 as well as all the possible combinations of - and -amino acids were tested using /- lc Phe-tRN fmet, /-Ser-tRN lue2, and /-ystrn lue2 (Figure 5). gain, the full--rp5 peptide showed a unique mobility compared with the other mixed /-rp5 peptides containing -Phe, -Ser, or -ys. These peptides were also analyzed by MI-TOF mass spectrometry (Figures 5 and S5), showing that the m/z value of the main peak is matched with that of the thioether macrocyclic peptide. Taken together, this demonstrates that we were able to express full--peptides closed by a thioether bond via initiation and elongation code reprogramming. ISSSION rp4 m/z alc. : (cyclic) (linear) Obs. : full--rp4: -ys/-ser/-la/-ser/-ys alc. : (cyclic) (linear) Obs. : We devised a new FIT system compatible with consecutive incorporation of -amino acids by optimizing an elongator m/z Figure 4. Ribosomal Synthesis of -Form Macrocyclic Peptide losed by a isulfide Bond () mrn sequence (mr4) and the corresponding peptide sequences (wp4 and rp4). Sequence of flag is the same as mr1 and rp1. The sequence of rp4 is reprogrammed based on the codon table shown in Figure S1. Bold letters indicate the reprogrammed codons and the corresponding amino acids. Two cysteine residues included in the rp4 form a disulfide bond to give a macrocyclic structure. (B) Tricine SS-PE analysis of rp4 synthesized by the FIT system. ombinations of stereochemistry in ys, Ser, and la are indicated. () MI-TOF MS analysis of peptide rp4 with -ys/-ser/-la and -ys/-ser/-la. rrows indicate the peaks of desired products. alc. and Obs. indicate calculated and observed m/z values, respectively. See also Figure S4 for the result of peptide rp4 with other combinations of /-ys, /-Ser, and /-la. trn sequence and concentrations of translation factors. The translation yield of a -la-containing peptide (rp1) was improved by more than 5-fold by using pre-charged -alanyl-trn lue2 in combination with optimized concentration of IF2, EF-Tu/Ts, and EF- (Figures 1 and 2). The concentrations of IF2 and EF- Tu/Ts were increased from 0.4 to 3 mm and 10 to 20 mm, respectively, and that of EF- was reduced from 0.26 to 0.1 mm in comparison with our conventional FIT system. The increase of EF-Tu/Ts concentration and change of trn from trn sne2 to trn lue2, which has higher binding affinity to EF-Tu, presumably leads to an acceleration of the accommodation rate of the -aminoacyl-trn in the ribosomal site. The decrease of the EF- concentration would contribute to suppression of the EF- -mediated drop-off event of peptidyl--aminoacyl-trn from the ribosomal P site. IF2 would not directly accelerate -amino acid incorporation, but would contribute to an acceleration of the initiation event, resulting in an improvement of the overall yield of -peptide expression. Based on the optimized translation conditions, we could successfully perform incorporation of up to ten consecutive -Ser (Figure 3). s reported previously, -Ser is one of the efficient -amino acid substrates classified in group I, and therefore consecutive incorporation of -Ser would be relatively easier than that of groups II and III -amino acids whose incorporation efficiencies are much lower (Fujino et al., 2013). lthough the reason for the difference of incorporation efficiency among -amino acids is yet unclear, it should be attributed to differences in the conformation, orientation, and position of each -amino acid at the peptidyl transferase center. Therefore, to further improve the efficiency of consecutive -amino acid incorporation for other -amino acids in groups II and III, it will likely be necessary to optimize other critical factors associated with the peptidyl transfer event. lthough we have thus far been unsuccessful in improving the incorporation efficiencies of -amino acids using ribosome mutants (H. Kimura and.s. eiermann, ell hemical Biology 24, 46 54, January 19,

8 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), mrn (mr5): flag wild type peptide (wp5): fm H K K K flag (stop) reprogrammed peptide (rp5): /- lc F /-S /-S /- K K K flag (stop) S Thioether bond formation ane lc Phe () Stereochemistry Ser () ys () Peptide yield ( M) Intensity (a.u.) rp5 - lc Phe/-Ser/-Ser/-ys m/z alc. : Obs. : B - lc Phe/-Ser/-Ser/-ys alc. : Obs. : m/z O -F S -S - -S K-K-K-flag Figure 5. Ribosomal Synthesis of -Form Macrocyclic Peptide losed by a Thioether Bond () mrn sequence (mr5) and the corresponding peptide sequences (wp5 and rp5). Sequence of flag is the same as mr1 and rp1. The sequence of rp5 is reprogrammed by using the codon table shown in Figure S1E. Bold letters indicate the reprogrammed codons and the corresponding amino acids. lc F indicates N-chloroacetyl phenylalanine. (B) Structure of macrocyclic peptide rp5. The sulfhydryl group of the ys in rp5 attacks the a carbon of the N-terminal chloroacetyl group to form a thioether bond and give a macrocyclic structure. () Tricine SS-PE analysis of rp5 synthesized by the FIT system. ombinations of stereochemistry in lc Phe, Ser, and ys are indicated. () MI-TOF MS analysis of peptide rp5 with - lc Phe/-Ser/-ys, and - lc Phe/-Ser/ -ys. rrows indicate the peaks corresponding to desired products. alc. and Obs. indicate calculated and observed m/z values, respectively. See also Figure S5 for the result of peptide rp5 with other combinations of /- lc Phe, /-Ser, and /-ys. unpublished data), further engineering of the translation apparatus may make it possible to achieve this goal. In addition, we successfully demonstrated the ribosomal synthesis of macrocyclic -peptides closed by both disulfide and thioether bonds (Figures 4 and 5). Both of the peptides (rp4 and rp5) consist of three different types of -amino acids (-ys/-ser/-la or -Phe/-Ser/-ys, respectively). In particular, the thioether macrocyclic peptides containing -amino acids should provide a good drug-discovery platform due to their high serum stability derived from their non-reducibility, structural rigidity, and protease resistance. Previously, -amino acids could not be reprogrammed into the elongation table because consecutive incorporation would result in termination of translation. However, we now have a new way to incorporate -amino acids belonging to group I (e.g., -la, Ser, ys, Met, Thr, His, Phe, and Tyr) in combination with a selection of desired -amino acids. This advancement now allows the integration of a random macrocyclic peptide library containing appropriate -amino acids and other non-proteinogenic amino acids such as N-methyl--amino acids with RaPI or other related systems providing us a new opportunity to explore a tremendously expanded chemical space for the discovery of peptide ligands against therapeutic targets (Passioura et al., 2014). SINIFINE Here, we have reported a translation system where the body sequence of -aminoacyl-trn and the concentrations of three translation protein factors, IF2, EF-Tu, and EF-, have been tuned for the expression of peptides containing consecutive -amino acids. s an example of the scope of this improved translation system, we have shown expression of peptides containing up to ten consecutive -Ser residues for the first time ever reported in the literature. Moreover, macrocyclic peptides consisting of four or five consecutive -amino acids were also synthesized. The integration of a random macrocyclic peptide library containing the -amino acids and other proteinogenic and N-methyl- -amino acids with the RaPI system opens a new door to expand the chemical space for the discovery of peptide ligands against therapeutic targets. EXPERIMENT PROERES Preparation of Flexizyme and trn Flexizymes (dfx and efx) and trns used for incorporation of -amino acids were prepared by in vitro transcription using T7 RN polymerase. Template Ns for the transcription reaction were prepared by extension of forward and reverse extension primer pairs (Table S1), followed by PR using forward and reverse PR primers (Table S1). These primers were purchased from Eurofins Scientific. The PR products were purified by phenol/chloroform extraction and ethanol precipitation, and then transcribed at 37 for 16 hr in a 250 m reaction mixture containing 40 mm Tris-Hl (ph 8.0), 22.5 mm Mgl 2, 1 mm TT, 1 mm spermidine, 0.01% Triton X-100, 120 nm T7 RN polymerase, 0.04 /m RNasin RNase Inhibitor (Promega), and 3.75 mm nucleoside triphosphate mix. For trn transcription, 5 mm guanosine monophosphate or cytidine monophosphate was added to the above solution to introduce 5 end or with monophosphate, respectively. The resulting RN transcripts were treated with RQ1 Nase (Promega) for 30 min at 37, and then purified by 8% or 12% denaturing PE containing 6 M urea. minoacylation of trn ctivated - and -amino acids (alanine 3,5-dinitrobenzyl ester, serine 3,5-dinitrobenzyl ester, cysteine 3,5-dinitrobenzyl ester, and chloroacetyl phenylalanine cyanomethyl ester) were synthesized by previously reported methods (Murakami et al., 2006; Saito et al., 2001). minoacylation was carried out at 52 ell hemical Biology 24, 46 54, January 19, 2017

9 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), 0 in reaction mixtures containing 50 mm HEPES-KOH (ph 7.5), 600 mm Mgl 2, 20% MSO, 25 mm dfx or efx, 25 mm trn, and 5 mm activated amino acid. dfx was used for dinitrobenzyl ester-activated amino acids and efx for cyanomethyl ester-activated ones. Reaction was carried out for 2 hr for la and lc Phe, and 6 hr for Ser and ys. The aminoacyl-trn was precipitated by ethanol, and then the pellet was washed twice with 70% ethanol containing 0.1 M sodium acetate (ph 5.2), once with 70% ethanol, and dissolved in 1 mm sodium acetate (ph 5.2). Translation of Model Peptides ll translation reactions were carried out using the FIT system (oto et al., 2011). s shown in Figure S1, the reaction mixture consisted of the following components: 50 mm HEPES-KOH (ph 7.6), 100 mm potassium acetate, 12.3 mm magnesium acetate, 2 mm TP, 2 mm TP, 1 mm TP, 1 mm TP, 20 mm creatine phosphate, 0.1 mm 10-formyl-5,6,7,8-tetrahydrofolic acid, 2 mm spermidine, 1 mm TT, 1.5 mg/m E. coli total trn, 1.2 mm E. coli ribosome, 0.6 mm methionyl-trn formyltransferase, 2.7 mm IF1, 3 mm IF2, 1.5 mm IF3, 0.1 mm EF-, 20 mm EF-Tu/Ts, 0.25 mm RF2, 0.17 mm RF3, 0.5 mm RRF, 4 mg/m creatine kinase, 3 mg/m myokinase, 0.1 mm inorganic pyrophosphatase, 0.1 mm nucleotide diphosphate kinase, 0.1 mm T7 RN polymerase, 0.13 mm sprs, 0.11 mm ysrs, 0.03 mm MetRS, 0.02 mm TyrRS, 0.05 mm [ 14 ]aspartic acid, 0.5 mm lysine, 0.5 mm methionine, 0.5 mm tyrosine, 25 mm each pre-charged aminoacyl-trn, and 0.5 mm N template. In the case of initiation suppression, 10-formyl- 5,6,7,8-tetrahydrofolic acid, methionyl-trn formyltransferase, MetRS, and methionine were omitted from the above solution. ifferent concentrations of EF-, IF2, and EF-Tu/Ts were also tested in the titration experiments (Figure 2). Translation reactions were carried out for 30 min at 37 in a 2.5 m solution. The reaction was stopped by addition of an equal volume of stop solution (0.9 M Tris-Hl [ph 8.45], 8% SS, 30% glycerol, and 0.001% xylene cyanol) and incubated at 95 for 2 min, prior to analysis by 15% tricine SS-PE, and autoradiography using a Typhoon F 7000 (E Healthcare). Peptide yield was normalized by intensity of [ 14 ]sp band. For MI-TOF mass-spectrometric analysis, peptides were translated in a non-radioactive reaction mix that contained 0.5 mm cold sp instead of [ 14 ]sp. The reaction was carried out at 37 for 30 min, and then diluted with an equal volume of 23 HEPES-buffered saline (HBS) buffer (100 mm HEPES-KOH [ph 7.6], 300 mm Nal). Then, the peptide was incubated with 5 m slurry of NTI-F M2 ffinity el (Sigma) for 30 min at 25. The gel beads were washed with 25 m of 13 HBS buffer (50 mm HEPES-KOH [ph 7.6], 150 mm Nal), and the peptide eluted from the beads with 15 m of 0.2% trifluoroacetic acid. The peptide was then desalted with SPE -TIP (Nikkyo Technos) and eluted with 1.2 m of 80% acetonitrile and 0.5% acetic acid solution with 50% saturated (R)-cyano-4-hydroxycinnamic acid. MI-TOF mass-spectrometric analysis of the peptide was performed in reflector/positive mode using an ultraflextreme (Bruker altonics). Peptide alibration Standard II (Bruker altonics) was used for external mass calibration. SPPEMENT INFORMTION Supplemental Information includes five figures and one table and can be found with this article online at THOR ONTRIBTIONS ll authors contributed to the writing of this work. T.K. carried out the experiments in all sections. H.S. directed the program. KNOWEMENTS We thank ouise J. Walport for proofreading of the manuscript. This work is supported by Japan Science and Technology gency (JST) PRESTO of Molecular Technology and reation of New Functions; JST REST Rising Star ward of Molecular Technology; JSPS rant-in-id for hallenging Exploratory Research ( ) to T.K.; JST REST of Molecular Technologies; the Japan Society for the Promotion of Science (JSPS) rant-in-id for Scientific Research (S) ( ) to H.S. Received: September 3, 2016 Revised: November 1, 2016 ccepted: November 21, 2016 Published: ecember 29, 2016 REFERENES chenbach, J., Jahnz, M., Bethge,., Paal, K., Jung, M., Schuster, M., lbrecht, R., Jarosch, F., Nierhaus, K.H., and Klussmann, S. (2015). Outwitting EF-Tu and the ribosome: translation with d-amino acids. Nucleic cids Res. 43, ale, T., and hlenbeck, O.. (2005). mino acid specificity in translation. Trends Biochem. Sci. 30, ale, T., Sanderson,.E., and hlenbeck, O.. (2004). The affinity of elongation factor Tu for an aminoacyl-trn is modulated by the esterified amino acid. Biochemistry 43, edkova,.m., Fahmi, N.E., olovine, S.Y., and Hecht, S.M. (2003). Enhanced d-amino acid incorporation into protein by modified ribosomes. J. m. hem. Soc. 125, edkova,.m., Fahmi, N.E., olovine, S.Y., and Hecht, S.M. (2006). onstruction of modified ribosomes for incorporation of d-amino acids into proteins. Biochemistry 45, oerfel,.k., Wohlgemuth, I., Kubyshkin, V., Starosta,.., Wilson,.N., Budisa, N., and Rodnina, M.V. (2015). Entropic contribution of elongation factor P to proline positioning at the catalytic center of the ribosome. J. m. hem. Soc. 137, oi, Y., Ohtsuki, T., Shimizu, Y., eda, T., and Sisido, M. (2007). Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system. J. m. hem. Soc. 129, Fujino, T., oto, Y., Suga, H., and Murakami, H. (2013). Reevaluation of the d-amino acid compatibility with the elongation event in translation. J. m. hem. Soc. 135, oto, Y., Murakami, H., and Suga, H. (2008). Initiating translation with d-amino acids. RN 14, oto, Y., Katoh, T., and Suga, H. (2011). Flexizymes for genetic code reprogramming. Nat. Protoc. 6, Ieong, K.W., Pavlov, M.Y., Kwiatkowski, M., Ehrenberg, M., and Forster,.. (2014). trn body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids. RN 20, Kawakami, T., Murakami, H., and Suga, H. (2008a). Messenger RN-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. hem. Biol. 15, Kawakami, T., Murakami, H., and Suga, H. (2008b). Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids. J. m. hem. Soc. 130, Kawakami, T., Ishizawa, T., and Murakami, H. (2013). Extensive reprogramming of the genetic code for genetically encoded synthesis of highly N-alkylated polycyclic peptidomimetics. J. m. hem. Soc. 135, ariviere, F.J., Wolfson,.., and hlenbeck, O.. (2001). niform binding of aminoacyl-trns to elongation factor Tu by thermodynamic compensation. Science 294, Murakami, H., Ohta,., shigai, H., and Suga, H. (2006). highly flexible trn acylation method for non-natural polypeptide synthesis. Nat. Methods 3, Ohta,., Murakami, H., Higashimura, E., and Suga, H. (2007). Synthesis of polyester by means of genetic code reprogramming. hem. Biol. 14, Passioura, T., Katoh, T., oto, Y., and Suga, H. (2014). Selection-based discovery of druglike macrocyclic peptides. nnu. Rev. Biochem. 83, Pavlov, M.Y., Watts, R.E., Tan, Z., ornish, V.W., Ehrenberg, M., and Forster,.. (2009). Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proc. Natl. cad. Sci. 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10 Please cite this article as: Katoh et al., onsecutive Elongation of -mino cids in Translation, ell hemical Biology (2017), mediates peptidyl-trn release and ribosome recycling in Escherichia coli. EMBO J. 20, Saito, H., Kourouklis,., and Suga, H. (2001). n in vitro evolved precursor trn with aminoacylation activity. EMBO J. 20, Schrader, J.M., hapman, S.J., and hlenbeck, O.. (2011). Tuning the affinity of aminoacyl-trn to elongation factor Tu for optimal decoding. Proc. Natl. cad. Sci. S 108, Subtelny,.O., Hartman, M.., and Szostak, J.W. (2008). Ribosomal synthesis of N-methyl peptides. J. m. hem. Soc. 130, Tan, Z., Forster,.., Blacklow, S.., and ornish, V.W. (2004). mino acid backbone specificity of the Escherichia coli translation machinery. J. m. hem. Soc. 126, Terasaka, N., Hayashi,., Katoh, T., and Suga, H. (2014). n orthogonal ribosome-trn pair via engineering of the peptidyl transferase center. Nat. hem. Biol. 10, Yamagishi, Y., Shoji, I., Miyagawa, S., Kawakami, T., Katoh, T., oto, Y., and Suga, H. (2011). Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. hem. Biol. 18, ell hemical Biology 24, 46 54, January 19, 2017

11 ell hemical Biology, Volume 24 Supplemental Information onsecutive Elongation of -mino cids in Translation Takayuki Katoh, Kenya Tajima, and Hiroaki Suga

12 /-a.a. IF1 IF2 IF3 [ 14 ]sp Met ys Tyr EF-Tu EF-Ts EF- sprs MetRS ysrs TyrRS pre-charged aminoacyl-trn RF2 RF3 RRF translation factors 4 amino acid/rs pairs template N T7 RN polymerase native trn mix ribosome other enzymes B 1st 2nd fmet /-la Tyr stop ys sp 3rd 1st 2nd fmet /-Ser Tyr stop ys sp 3rd 1st 2nd /-la /-Ser fmet Tyr stop /-ys ys sp 3rd E 1st 2nd /- lc Phe /-Ser Tyr stop /-ys ys sp 3rd Figure S1. Related to Figure 1, 3, 4 and 5. enetic code reprogramming by means of the FIT system () Main components of the FIT system. In vitro transcribed trn sne2 or trn lue2 was pre-charged with - or -amino acids by means of the flexizyme technology. The other amino acids were charged on the corresponding natural E. coli trns de novo by the aminoacyl trn synthetases (RSs) present in the FIT system. [ 14 ]sp was used for radioisotopic labeling of peptides. "Other enzymes" include methionyltrn formyltransferase, creatine kinase, myokinase, inorganic pyrophosphatase, and nucleotide diphosphate kinase. See methods section for details. (B-E) Reprogrammed codon tables used for translation of rp1, rp2(b), rp3(), rp4() and rp5(e). Empty codons are left unassigned.

13 mrn (mr2): wild type peptide (wp2): reprogrammed peptide (rp2): flag fm K K K fm K K K S /- flag (stop) flag (stop) B ane Stereochemistry of la trn Peptide yield (µm) 1 sne lue sne lue sne2 0.07() 0.95() 6 + lue2 0.19() 1.20() rp2- rp2- Figure S2. Related to Figure 1. Incorporation of single -la into a model peptide. () mrn sequence (mr2) and the corresponding peptide sequences (wp2 and rp2) used in this experiment. The sequence of rp2 is reprogrammed based on the codon table shown in Figure S1B. Sequence of flag is the same as mr1 and rp1. (B) Tricine SS-PE analysis of the rp2 peptide synthesized by the FIT system. trn sne2 or trn lue2 was used for incorporation of - or -la at the codons of mr2. For lanes 5 and 6, 25 µm each -la-trn and -la-trn were cointroduced. rrows indicate the bands corresponding to the desired products.

14 Intensity (a.u.) Intensity (a.u.) rp3-(-s) 5 rp3-(-s) 6 rp3-(-s) 7 alc. : alc. : Obs. : Obs. : m/z m/z m/z rp3-(-s) 8 alc. : rp3-(-s) 9 rp3-(-s) 10 alc. : Obs. : Obs. : alc. : Obs. : alc. : Obs. : m/z m/z m/z Figure S3. Related to Figure 3. MI-TOF MS spectra of peptide rp3 with consecutive -Ser rrows indicate the peaks corresponding to the desired products. alc. and Obs. indicate calculated and observed m/z values, respectively. See also Figure 3 for spectra of rp3 with -Ser.

15 Intensity (a.u.) Intensity (a.u.) -ys/-ser/-la/-ser/-ys -ys/-ser/-la/-ser/-ys -ys/-ser/-la/-ser/-ys alc. : (cyclic) Obs. : alc. : (cyclic) Obs. : m/z m/z m/z alc. : (cyclic) Obs. : ys/-ser/-la/-ser/-ys -ys/-ser/-la/-ser/-ys -ys/-ser/-la/-ser/-ys alc. : (cyclic) Obs. : alc. : (cyclic) Obs. : alc. : (cyclic) Obs. : m/z m/z m/z Figure S4. Related to Figure 4. MI-TOF MS spectra of peptide rp4 with various combinations of - or -ys, - or -Ser and - or - la rrows indicate the peaks corresponding to the desired products. alc. and Obs. indicate calculated and observed m/z values, respectively. See also Figure 4 for spectra of full- and full- peptides.

16 Intensity (a.u.) - lc Phe/-Ser/-Ser/-ys - lc Phe/-Ser/-Ser/-ys - lc Phe/-Ser/-Ser/-ys alc. : Obs. : alc. : Obs. : alc. : Obs. : m/z m/z m/z - lc Phe/-Ser/-Ser/-ys - lc Phe/-Ser/-Ser/-ys - lc Phe/-Ser/-Ser/-ys Intensity (a.u.) alc. : Obs. : alc. : Obs. : alc. : Obs. : m/z m/z m/z Figure S5. Related to Figure 5. MI-TOF MS spectra of peptide rp5 with various combinations of - or - lc Phe, - or -Ser and - or -ys rrows indicate the peaks corresponding to the desired products. alc. and Obs. indicate calculated and observed m/z values, respectively. See also Figure 5 for spectra of full- and full- peptides.