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• W3136643486 abstract "•Improving the yield of aaRS-free translation using solely flexizyme-charged tRNAs•Translating multiple protein enzymes of distinct functions in the absence of aaRS•Producing an active aaRS (TrpRS) under aaRS-free conditions•Realizing mirror-image tRNA charging with D-amino acids using L-flexizyme The requirement of dozens of large aminoacyl-tRNA synthetases (aaRSs) for tRNA charging presents a major hurdle in building synthetic ribosome translation systems including the mirror-image translation apparatus. The discovery of tRNA-charging ribozymes, such as the flexizyme, opens the possibility of circumventing the laborious synthesis of the aaRS proteins. Here, through improving the translation yield by concentrating cation-depleted tRNAs, we show that a highly simplified translation system using solely flexizyme-charged tRNAs is capable of producing protein enzymes of up to 334 aa with all 20 proteinogenic amino acids. Furthermore, we demonstrate the mirror-image tRNA charging with D-amino acids using a synthetic L-flexizyme, suggesting the feasibility of realizing mirror-image translation in a similar aaRS-free translation system. Protein translation from the ribosome seemingly requires dozens of sophisticated aminoacyl-tRNA synthetases (aaRSs). Despite the discovery of tRNA-aminoacylating ribozymes, such as the flexizyme, synthesizing protein enzymes from highly simplified translation systems in the absence of aaRS remains undemonstrated. Here, we show the translation of multiple protein enzymes of distinct functions using solely flexizyme-charged tRNAs, through improving the translation yield by concentrating cation-depleted tRNAs. Notably, we used the aaRS-free translation system to produce an active aaRS (TrpRS), which in turn catalyzed the charging of more tRNAs. Moreover, toward realizing mirror-image translation, we performed the mirror-image tRNA charging with D-amino acids by a synthetic L-flexizyme. Our work demonstrates the feasibility of translating protein enzymes from a highly simplified translation apparatus without aaRS and drastically reduces the requirement to chemically synthesize dozens of large aaRS proteins for realizing mirror-image translation. Protein translation from the ribosome seemingly requires dozens of sophisticated aminoacyl-tRNA synthetases (aaRSs). Despite the discovery of tRNA-aminoacylating ribozymes, such as the flexizyme, synthesizing protein enzymes from highly simplified translation systems in the absence of aaRS remains undemonstrated. Here, we show the translation of multiple protein enzymes of distinct functions using solely flexizyme-charged tRNAs, through improving the translation yield by concentrating cation-depleted tRNAs. Notably, we used the aaRS-free translation system to produce an active aaRS (TrpRS), which in turn catalyzed the charging of more tRNAs. Moreover, toward realizing mirror-image translation, we performed the mirror-image tRNA charging with D-amino acids by a synthetic L-flexizyme. Our work demonstrates the feasibility of translating protein enzymes from a highly simplified translation apparatus without aaRS and drastically reduces the requirement to chemically synthesize dozens of large aaRS proteins for realizing mirror-image translation. The emergence of protein enzymes is key to the transition from RNA-based life to modern biology.1Lohse P.A. Szostak J.W. Ribozyme-catalysed amino-acid transfer reactions.Nature. 1996; 381: 442-444Crossref PubMed Scopus (181) Google Scholar, 2Zhang B. Cech T.R. Peptide bond formation by in vitro selected ribozymes.Nature. 1997; 390: 96-100Crossref PubMed Scopus (220) Google Scholar, 3Noller H.F. Evolution of protein synthesis from an RNA world.Cold Spring Harb. Perspect. Biol. 2012; 4: a003681Crossref PubMed Scopus (70) Google Scholar The discovery of tRNA-aminoacylation ribozymes suggests the possibility of synthesizing protein enzymes from highly simplified translation systems with tRNAs charged by ribozymes.4Illangasekare M. Sanchez G. Nickles T. Yarus M. Aminoacyl-RNA synthesis catalyzed by an RNA.Science. 1995; 267: 643-647Crossref PubMed Scopus (278) Google Scholar, 5Lee N. Bessho Y. Wei K. Szostak J.W. Suga H. Ribozyme-catalyzed tRNA aminoacylation.Nat. Struct. Biol. 2000; 7: 28-33Crossref PubMed Scopus (144) Google Scholar, 6Saito H. Kourouklis D. Suga H. An in vitro evolved precursor tRNA with aminoacylation activity.EMBO J. 2001; 20: 1797-1806Crossref PubMed Scopus (99) Google Scholar, 7Murakami H. Saito H. Suga H. A versatile tRNA aminoacylation catalyst based on.RNA. Chem. Biol. 2003; 10: 655-662Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 8Murakami H. Ohta A. Ashigai H. Suga H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis.Nat. Methods. 2006; 3: 357-359Crossref PubMed Scopus (263) Google Scholar Additionally, other systems using pre-charged tRNAs prepared by aaRS, urzyme, and chemical acylation have also been reported.9Ganoza M.C. Cunningham C. Green R.M. Isolation and point of action of a factor from Escherichia coli required to reconstruct translation.Proc. Natl. Acad. Sci. USA. 1985; 82: 1648-1652Crossref PubMed Scopus (22) Google Scholar, 10Li L. Francklyn C. Carter C.W. Aminoacylating urzymes challenge the RNA world hypothesis.J. Biol. Chem. 2013; 288: 26856-26863Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Robertson S.A. Ellman J.A. Schultz P.G. A general and efficient route for chemical aminoacylation of transfer RNAs.J. Am. Chem. Soc. 1991; 113: 2722-2729Crossref Scopus (192) Google Scholar, 12Forster A.C. Tan Z. Nalam M.N.L. Lin H. Qu H. Cornish V.W. Blacklow S.C. Programming peptidomimetic syntheses by translating genetic codes designed de novo.Proc. Natl. Acad. Sci. USA. 2003; 100: 6353-6357Crossref PubMed Scopus (154) Google Scholar, 13Forster A.C. Cornish V.W. Blacklow S.C. Pure translation display.Anal. Biochem. 2004; 333: 358-364Crossref PubMed Scopus (35) Google Scholar Among them, the highly robust and versatile tRNA-aminoacylating ribozyme, flexizyme, discovered through in vitro selection has been shown capable of charging a wide variety of amino acids to tRNAs.8Murakami H. Ohta A. Ashigai H. Suga H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis.Nat. Methods. 2006; 3: 357-359Crossref PubMed Scopus (263) Google Scholar Using tRNAs charged by flexizyme and aaRS, incorporation of multiple unnatural amino acids into translated peptides was achieved, enabling the practical selection of peptide drugs.14Sakai K. Passioura T. Sato H. Ito K. Furuhashi H. Umitsu M. Takagi J. Kato Y. Mukai H. Warashina S. et al.Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor.Nat. Chem. Biol. 2019; 15: 598-606Crossref PubMed Scopus (34) Google Scholar, 15Nawatha M. Rogers J.M. Bonn S.M. Livneh I. Lemma B. Mali S.M. Vamisetti G.B. Sun H. Bercovich B. Huang Y. et al.De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains.Nat. Chem. 2019; 11: 644-652Crossref PubMed Scopus (39) Google Scholar, 16Katoh T. Sengoku T. Hirata K. Ogata K. Suga H. Ribosomal synthesis and de novo discovery of bioactive foldamer peptides containing cyclic β-amino acids.Nat. Chem. 2020; 12: 1081-1088Crossref PubMed Scopus (29) Google Scholar However, in part due to the low translation yield, when using solely flexizyme-charged tRNAs in the absence of aaRS (hereinafter referred to as “aaRS-free”), only short peptides were translated,17Terasaka N. Hayashi G. Katoh T. Suga H. An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center.Nat. Chem. Biol. 2014; 10: 555-557Crossref PubMed Scopus (42) Google Scholar whereas the ribosomal production of full-length, functional protein enzymes with all 20 proteinogenic amino acids under aaRS-free conditions has remained undemonstrated thus far. One of the main reasons for the low yield of aaRS-free translation is that, compared with tRNA aminoacylation by aaRS,18Walker S.E. Fredrick K. Preparation and evaluation of acylated tRNAs.Methods. 2008; 44: 81-86Crossref PubMed Scopus (62) Google Scholar,19Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Cell-free translation reconstituted with purified components.Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1224) Google Scholar the flexizyme charging of tRNAs lacks recycling.8Murakami H. Ohta A. Ashigai H. Suga H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis.Nat. Methods. 2006; 3: 357-359Crossref PubMed Scopus (263) Google Scholar In addition, the use of in vitro transcribed, unmodified tRNAs for aaRS-free charging also contributes to the low translation yield.20Cui Z. Stein V. Tnimov Z. Mureev S. Alexandrov K. Semisynthetic tRNA complement mediates in vitro protein synthesis.J. Am. Chem. Soc. 2015; 137: 4404-4413Crossref PubMed Scopus (18) Google Scholar,21Hibi K. Amikura K. Sugiura N. Masuda K. Ohno S. Yokogawa T. Ueda T. Shimizu Y. Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs.Commun. Biol. 2020; 3: 350Crossref PubMed Scopus (15) Google Scholar Here, we set out to test the ability of aaRS-free systems to translate protein enzymes with all 20 proteinogenic amino acids using tRNAs charged by flexizyme. Our results show that through improving the translation yield by concentrating the flexizyme-charged, cation-depleted tRNAs, multiple protein enzymes of distinct functions, such as the lysozyme, luciferase, and even aaRS itself can be translated (Figure 1). Moreover, we carried out the charging of mirror-image (L-) tRNAs with mirror-image (D-) amino acids by a synthetic mirror-image (L-) flexizyme, which will enable the future realization of a highly simplified mirror-image translation apparatus. We first analyzed the aaRS-free translation system to address the apparent low yield issue.17Terasaka N. Hayashi G. Katoh T. Suga H. An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center.Nat. Chem. Biol. 2014; 10: 555-557Crossref PubMed Scopus (42) Google Scholar Our rationale was that the yield of aaRS-free translation might be improved by increasing the concentrations of flexizyme-charged tRNAs to compensate for the lack of tRNA recycling. Earlier studies showed that adding excessive tRNAs in Escherichia coli (E. coli) translation systems with aaRS inhibited the translation,22Rojiani M.V. Jakubowski H. Goldman E. Relationship between protein synthesis and concentrations of charged and uncharged tRNATrp in Escherichia coli.Proc. Natl. Acad. Sci. USA. 1990; 87: 1511-1515Crossref PubMed Scopus (26) Google Scholar,23Anderson W.F. The effect of tRNA concentration on the rate of protein synthesis.Proc. Natl. Acad. Sci. USA. 1969; 62: 566-573Crossref PubMed Scopus (78) Google Scholar which was attributed to uncharged tRNAs competing out the charged tRNAs by occupying the ribosomal A-site22Rojiani M.V. Jakubowski H. Goldman E. Relationship between protein synthesis and concentrations of charged and uncharged tRNATrp in Escherichia coli.Proc. Natl. Acad. Sci. USA. 1990; 87: 1511-1515Crossref PubMed Scopus (26) Google Scholar or cation imbalance from the addition of large amounts of tRNAs.23Anderson W.F. The effect of tRNA concentration on the rate of protein synthesis.Proc. Natl. Acad. Sci. USA. 1969; 62: 566-573Crossref PubMed Scopus (78) Google Scholar However, all of these experiments were performed in the presence of aaRS, and thus the exact charging yields were not determined, making it difficult to differentiate between the influence of inefficient charging and altered cation (e.g., Mg2+) concentrations. To evaluate the effect of tRNA concentrations and charging yields on aaRS-free translation, we performed the aaRS-free translation of a short peptide using fluorescein (FAM)-labeled phenylalanine (Fph-tRNAfMet) to facilitate the quantification of translation yields17Terasaka N. Hayashi G. Katoh T. Suga H. An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center.Nat. Chem. Biol. 2014; 10: 555-557Crossref PubMed Scopus (42) Google Scholar (Figures 2A, 2B, S1, and S2). The mRNA template was decoded by tRNAfMet, tRNALys, tRNATyr, and tRNAAsp, among which tRNAfMet was charged with Fph-tRNAfMet by the 45-nt enhanced flexizyme,17Terasaka N. Hayashi G. Katoh T. Suga H. An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center.Nat. Chem. Biol. 2014; 10: 555-557Crossref PubMed Scopus (42) Google Scholar and the others were charged with their cognate amino acids by the 46-nt dinitro-flexizyme.8Murakami H. Ohta A. Ashigai H. Suga H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis.Nat. Methods. 2006; 3: 357-359Crossref PubMed Scopus (263) Google Scholar Unmodified tRNAs transcribed in vitro by the T7 RNA polymerase were used, as they have been shown to operate in ribosomal peptide synthesis assays.24Iwane Y. Hitomi A. Murakami H. Katoh T. Goto Y. Suga H. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes.Nat. Chem. 2016; 8: 317-325Crossref PubMed Scopus (70) Google Scholar The individual charging yield for each tRNA was determined by polyacrylamide gel electrophoresis under acidic conditions (acid PAGE), which was used to deduce the weighted average (overall) charging yield of the translation system (Table S1). We first performed tRNA titration by adding charged total tRNAs with an overall charging yield of ∼44% (mixed Fph-tRNAfMet: Lys-tRNALys: Tyr-tRNATyr: Asp-tRNAAsp at 1:2:2:2 molar ratio) and total tRNA concentrations from 20–644 μM in the final translation system, and discovered that the translation yield reached the highest level when the total tRNA concentration was at ∼160 μM, and without plateauing, the translation yield decreased upon further increases of tRNA concentrations (Figure S1A). We attributed the observed inhibition not to the flexizyme buildup in the translation system, because in a control experiment, the addition of purified dinitro-flexizyme to a fixed amount of total tRNAs did not inhibit the translation (Figure S1B). Next, we added 90–470 μM of uncharged tRNAs to the aaRS-free translation system in the presence of 70 μM charged tRNAs, with the overall charging yield decreased from 44% to 13%, but the overall translation yield remained largely unaffected (Figure 2B). We reasoned that another factor that could be responsible for the observed translation inhibition was the increased cation concentration due to Mg2+ carryover from the flexizyme-charged tRNAs. To test this theory, we added exogenous flexizyme and tRNAs with MgCl2 to the aaRS-free translation system and discovered that the translation was indeed inhibited by increased MgCl2 carryover (Figures S1C and S1D). Enlighted by this result, we used the high-performance liquid chromatography (HPLC) equipped with a C18 column to purify the flexizyme-charged tRNAs to reduce the Mg2+ concentrations (except Fph-tRNAfMet, which was treated by ultrafiltration instead to minimize fluorescence quenching). This process also removed most of the flexizyme and modestly improved the overall charging yield from 44% to 56% (Figure S3). We then added the purified, flexizyme-charged tRNAs to the aaRS-free translation system and discovered that the translation yield was significantly improved by 5-fold as a result of concentrating the flexizyme-charged tRNAs. An additional 2-fold improvement was observed upon reducing the Mg2+ contamination by HPLC, resulting in a ∼10-fold overall improvement of translation yield (Figures 2B and S1A), with the optimal total tRNA concentration shifted from 160 to 500 μM. However, when tRNA concentrations further increased from 500 to 1,000 μM, the overall translation yield reduced by about 50%, potentially resulting from the Mg2+ associated with Fph-tRNAfMet. Moreover, similar titration assays with high concentrations of uncharged tRNAs in the presence of aaRS did not lead to improvement of translation yield (Figure S4), suggesting that the improvement of aaRS-free translation yield likely resulted from the increased concentrations of flexizyme-charged tRNAs per se. Having discovered that concentrating the flexizyme-charged, cation-depleted tRNAs improved the yield of aaRS-free translation, we sought to test the aaRS-free translation on multiple short peptides and determine the translation fidelity under high flexizyme-charged tRNA concentrations. We obtained a minimal set of 21 E. coli tRNAs through in vitro transcription by the T7 RNA polymerase, including one tRNA (tRNAfMet) for translation initiation and 20 others for translation elongation (Table S2). Each tRNA was separately charged by flexizyme with charging yields ranging from 20%-60% (Figure S5A). The flexizyme-charged tRNAs were mixed at a molar ratio according to the abundance of their cognate codons on the mRNA before being added to the aaRS-free translation system to a final concentration ranging from 170–520 μM (Table S1). We designed and in vitro transcribed five distinct mRNA sequences that allowed Watson-Crick base pairing to the anticodon of flexizyme-charged tRNAs, and the aaRS-free translated short peptides were evaluated by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to examine the translation fidelity (Figures 2C–2G). The MALDI-TOF MS results showed that all 21 flexizyme-charged tRNAs accurately decoded the mRNAs with up to ∼200-fold molar excess over the ribosome (e.g., 414 μM tRNAs: 2 μM ribosome, with mRNA #5). In the control experiments with uncharged tRNAs and free amino acids (Figures 2C–2G), no peptide products were detected, thus minimizing contamination concerns of aaRS and charged tRNAs from ribosome preparation. Notably, we encoded a short message “MITRNACHARGINGSYSTEM” into mRNA #6 (Figure 2G) and successfully translated the full-length information-carrying peptide. However, when we increased the total tRNA concentration to 520 μM, an additional +12 Da peak was detected (Figure S6), potentially due to mRNA misdecoding resulting from the high tRNA concentration and use of unmodified tRNAs for translation. Encouraged by the successful translation of short peptides, we carried out the aaRS-free translation of protein enzymes solely with in vitro transcribed, unmodified tRNAs charged by flexizyme. We first chose two small enzymes, the 130-aa chicken lysozyme and the 169-aa Gaussia luciferase, as models. Neither of the enzymes is native to E. coli and thus minimizes contamination concerns from ribosome preparation. A subset (underlined amino acids in Figures S7A and S7B) of the 21 flexizyme-charged tRNAs were purified by HPLC to reduce Mg2+ carryover, and the individual charging yields after HPLC purification were determined by acid PAGE (Figure S5B), resulting in an overall charging yield of ∼40%. The total tRNA concentrations of ∼330 μM for chicken lysozyme and ∼430 μM for Gaussia luciferase (Table S1) were approximately ∼10- to 20-fold higher than those used in other in vitro translation systems.17Terasaka N. Hayashi G. Katoh T. Suga H. An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center.Nat. Chem. Biol. 2014; 10: 555-557Crossref PubMed Scopus (42) Google Scholar,20Cui Z. Stein V. Tnimov Z. Mureev S. Alexandrov K. Semisynthetic tRNA complement mediates in vitro protein synthesis.J. Am. Chem. Soc. 2015; 137: 4404-4413Crossref PubMed Scopus (18) Google Scholar,24Iwane Y. Hitomi A. Murakami H. Katoh T. Goto Y. Suga H. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes.Nat. Chem. 2016; 8: 317-325Crossref PubMed Scopus (70) Google Scholar We first tested the aaRS-free translation of the full-length proteins using the FAM-labeled Fph-tRNAfMet reporter. Analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the fluorescently labeled product bands were consistent with the molecular weight of the chicken lysozyme and Gaussia luciferase (14.8 and 18.7 kDa, respectively), and the mobility of the product bands was also similar to that of commercial chicken lysozyme and recombinant Gaussia luciferase, respectively (Figures 3A , 3C, and S8A–S8D). In comparison, the product bands were absent in the control experiments lacking DNA templates, or with uncharged tRNAs and free amino acids, respectively. These results suggested that aaRS-free translation was sufficiently processive to accomplish the synthesis of full-length proteins before the flexizyme-charged tRNAs were hydrolyzed. Attempts to characterize the aaRS-free translated proteins from the excised product bands using liquid chromatography-tandem mass spectrometry (LC-MS/MS) were unsuccessful due to ribosomal protein contamination (however, this was addressed by translating a larger protein with a molecular weight more different from those of the ribosomal proteins, as described below). Next, we replaced the FAM-labeled Fph-tRNAfMet with unlabeled Met-tRNAfMet for translation initiation and performed enzymatic assays to test whether the translated proteins can fold correctly in vitro and possess their corresponding catalytic activities. The results showed that after incubation for up to 24 h in the folding buffers (see Experimental procedures), the aaRS-free translated enzymes carried out the catalysis of their corresponding substrates: the chicken lysozyme released FAM-labeled cell debris and the Gaussia luciferase emitted bioluminescence, respectively (Figures 3B and 3D), whereas the control experiments lacking DNA templates, or with uncharged tRNAs and free amino acids, did not generate detectable signals, thus minimizing contamination concerns of auto-fluorescence or contaminating luminescence from the aaRS-free translation system. Comparing the emitted bioluminescence of the aaRS-free translated Gaussia luciferase with known standards of recombinant luciferase suggested a translation yield of ∼25 nM (Figure S9), which was ∼80-fold lower than the maximal yield of the aaRS-free translation of a 7-aa peptide (Figure 2B), likely as a result of the lower availability of flexizyme-charged tRNAs for each translated codon, as well as the limited folding efficiency of the Gaussia luciferase with multiple disulfide bonds.25Yu T. Laird J.R. Prescher J.A. Thorpe C. Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding.Protein Sci. 2018; 27: 1509-1517Crossref PubMed Scopus (4) Google Scholar Next, we sought to explore the possibility for the aaRS-free translation system to produce functional aaRS itself, an important step in establishing a proposed self-reproducing translation apparatus.26Awai T. Ichihashi N. Yomo T. Activities of 20 aminoacyl-tRNA synthetases expressed in a reconstituted translation system in Escherichia coli.Biochem. Biophys. Rep. 2015; 3: 140-143PubMed Google Scholar,27Libicher K. Hornberger R. Heymann M. Mutschler H. In vitro self-replication and multicistronic expression of large synthetic genomes.Nat. Commun. 2020; 11: 904Crossref PubMed Scopus (26) Google Scholar Here, we used the 334-aa E. coli TrpRS as a model. A large portion (14 out of 21 in total) of the in vitro transcribed flexizyme-charged tRNAs were purified by HPLC to reduce Mg2+ carryover (underlined amino acids in Figure S7C), resulting in an overall charging yield of ∼42% and total tRNA concentration of ∼170 μM (Table S1). Again, we used the FAM-labeled Fph-tRNAfMet reporter to test the translation of the full-length protein, and a product band indicative of the 334-aa E. coli TrpRS (37.8 kDa) was observed by SDS-PAGE (the mobility of the fluorescently labeled protein band was similar to that of recombinant TrpRS, Figures S8E and S8F), whereas this band was absent in the control experiments lacking DNA templates, or with uncharged tRNAs and free amino acids (Figure 4A). We also observed several faster migrating bands, which likely correspond to the truncated TrpRS translation products and unused Fph-tRNAfMet (Figure S8G). To further confirm the aaRS-free translation of TrpRS, we analyzed the protein content from the excised product band using LC-MS/MS and identified four non-overlapping peptide segments from E. coli TrpRS, resulting in a sequence coverage of ∼16% (Table S3). In comparison, no peptides corresponding to E. coli TrpRS were detected in the control experiment with uncharged tRNAs and free amino acids, suggesting that the detected TrpRS was not from endogenous aaRS contamination. To further examine the tRNA-aminoacylating activity of the aaRS-free translated TrpRS, we designed and synthesized an internally Cy5-labeled tRNA substrate (Cy5-tRNATrp, Figure 4B), allowing the in situ detection of the charged Cy5-tRNATrp without interference from other charged tRNA species. Using Met-tRNAfMet for translation initiation, and after the TrpRS was translated, we added Cy5-tRNATrp along with tryptophan and adenosine triphosphate (ATP) to the aaRS-free translation system. The aaRS-free translated TrpRS successfully charged tryptophan onto Cy5-tRNATrp, whereas in the control experiments lacking DNA templates, or with uncharged tRNAs and free amino acids, no Cy5-tRNATrp charging was observed (Figure 4C), suggesting that the observed Cy5-tRNATrp charging activity was unlikely due to endogenous aaRS contamination from ribosome preparation or flexizyme charging. After the realization of mirror-image genetic replication, transcription, and reverse transcription,28Wang Z. Xu W. Liu L. Zhu T.F. A synthetic molecular system capable of mirror-image genetic replication and transcription.Nat. Chem. 2016; 8: 698-704Crossref PubMed Scopus (123) Google Scholar,29Wang M. Jiang W. Liu X. Wang J. Zhang B. Fan C. Liu L. Pena-Alcantara G. Ling J.-J. Chen J. Zhu T.F. Mirror-image gene transcription and reverse transcription.Chem. 2019; 5: 848-857Abstract Full Text Full Text PDF Scopus (13) Google Scholar the next major step in establishing the mirror-image central dogma of molecular biology is to build a mirror-image ribosome translation machine.28Wang Z. Xu W. Liu L. Zhu T.F. A synthetic molecular system capable of mirror-image genetic replication and transcription.Nat. Chem. 2016; 8: 698-704Crossref PubMed Scopus (123) Google Scholar, 29Wang M. Jiang W. Liu X. Wang J. Zhang B. Fan C. Liu L. Pena-Alcantara G. Ling J.-J. Chen J. Zhu T.F. Mirror-image gene transcription and reverse transcription.Chem. 2019; 5: 848-857Abstract Full Text Full Text PDF Scopus (13) Google Scholar, 30Peplow M. Mirror-image enzyme copies looking-glass DNA.Nature. 2016; 533: 303-304Crossref PubMed Scopus (7) Google Scholar, 31Peplow M. A conversation with Ting Zhu.ACS Cent. Sci. 2018; 4: 783-784Crossref PubMed Scopus (5) Google Scholar, 32Ling J.J. Fan C. Qin H. Wang M. Chen J. Wittung-Stafshede P. Zhu T.F. Mirror-image 5S ribonucleoprotein complexes.Angew. Chem. Int. Ed. Engl. 2020; 59: 3724-3731Crossref PubMed Scopus (6) Google Scholar However, the total chemical synthesis of the dozens of mirror-image aaRS proteins, which typically range from 300–1,000 aa in size, poses a significant challenge. We reasoned that a mirror-image version of the highly simplified aaRS-free translation apparatus capable of producing protein enzymes could circumvent the laborious synthesis of dozens of the mirror-image aaRS proteins, despite the challenges to chemically synthesize the mirror-image ribosomal proteins32Ling J.J. Fan C. Qin H. Wang M. Chen J. Wittung-Stafshede P. Zhu T.F. Mirror-image 5S ribonucleoprotein complexes.Angew. Chem. Int. Ed. Engl. 2020; 59: 3724-3731Crossref PubMed Scopus (6) Google Scholar and translation factors. In a proof of concept experiment to test the charging of mirror-image (L-) tRNAs with mirror-image (D-) amino acids by a synthetic mirror-image (L-) flexizyme (Figure 5A), we applied our previously established mirror-image gene transcription system based on the mirror-image version of a designed mutant of the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)29Wang M. Jiang W. Liu X. Wang J. Zhang B. Fan C. Liu L. Pena-Alcantara G. Ling J.-J. Chen J. Zhu T.F. Mirror-image gene transcription and reverse transcription.Chem. 2019; 5: 848-857Abstract Full Text Full Text PDF Scopus (13) Google Scholar,33Xu W. Jiang W. Wang J. Yu L. Chen J. Liu X. Liu L. Zhu T.F. Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction.Cell Discov. 2017; 3: 17008Crossref PubMed Scopus (30) Google Scholar,34Jiang W. Zhang B. Fan C. Wang M. Wang J. Deng Q. Liu X. Chen J. Zheng J. Liu L. et al.Mirror-image polymerase chain reaction.Cell Discov. 2017; 3: 17037Crossref PubMed Scopus (28) Google Scholar to transcribe the mirror-image tRNAs (Figure S10A). To avoid the high cost of synthesizing 21 different L-RNA primers, we applied a universal primer for the transcription of mirror-image tRNAs (Figure S10B). The universal primer was modified near the 3′ end by phosphorothioate so that the fully extended primers were efficiently cleaved by I2 via a previously reported mechanism,35Suydam I.T. Strobel S.A. Nucleotide analog interference mapping.Methods Enzymol. 2009; 468: 3-30Crossref PubMed Google Scholar,36Gish G. Eckstein F. DNA and RNA sequence determination based on phosphorothioate chemistry.Science. 1988; 240: 1520-1522Crossref PubMed Scopus (194) Google Scholar generating full-length mirror-image tRNAs. The I2-mediated cleavage generates RNA with hydroxyl-terminated 5′-end, as verified by MALDI-TOF MS (Figures S11A–S11C). The transcribed mirror-image tRNAs were, as expected, resistant to natural RNase A digestion and unable to be charged by natural aaRS (Figures S12A and S12B). We also showed that the enzymatically transcribed 76-nt mirror-image tRNA was of much higher purity than that prepared by chemical synthesis (Figure S10C) an" @default.
• W3136643486 created "2021-03-29" @default.
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• W3136643486 date "2021-03-01" @default.
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• W3136643486 title "Translating protein enzymes without aminoacyl-tRNA synthetases" @default.
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