Protein translation system

Abstract

The present disclosure provides a cell-free and aaRS-free protein translation system and its use in producing proteins and active enzymes.

Description

[Technical Field]

[0001] In some embodiments, the present invention relates to a cell-free protein translation system, and more specifically, but not limited to, a method for synthesizing proteins and their enantiomers without aminoacyl-tRNA synthetase, as well as the use thereof. [Background technology]

[0002] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 150,641, filed on 18 February 2021, the contents of which are incorporated in their entirety by reference.

[0003] Statement regarding sequence listings An ASCII file titled 90912 Sequence Listing.txt, created on 16 February 2022, containing 36,864 bytes, and filed concurrently with the filing of this application, is incorporated by reference into this disclosure.

[0004] Cell-free protein synthesis is a crucial tool for molecular biologists in both basic and applied sciences. It is increasingly used in high-throughput functional genomics and proteomics, offering significant advantages over protein expression in living cells. Cell-free protein synthesis is essential for the construction of protein arrays, such as nucleic acid programmable protein arrays (NAPPA), and for enzyme engineering using display technologies. The cell-free approach provides the fastest way to correlate phenotype (the function of expressed proteins) with genotype. Protein synthesis can be performed in hours using mRNA templates in translation systems or DNA templates (plasmid DNA or PCR fragments) in coupled transcription and translation systems. Furthermore, cell-free protein expression systems are essential for the expression of toxic proteins, membrane proteins, viral proteins, and proteins that undergo rapid proteolysis by intracellular proteases.

[0005] Most cell-free protein expression is based on lysates, which are produced from cells engaged in high-rate protein synthesis. The most frequently used cell-free expression systems require macromolecular components for translation, such as ribosomes, tRNA, aminoacyl-tRNA synthetase, initiation factors, elongation factors, and termination factors. To ensure efficient translation, commercially available extracts must be supplemented with amino acids, energy sources (ATP, GTP), energy regeneration systems, and salts (Mg2+, K+, etc.). For eukaryotic systems, creatine phosphate and creatine phosphokinase act as energy regeneration systems, while for prokaryotic systems, phosphoenolpyruvate and pyruvate kinase are supplemented. For linked transcription and translation systems, phage-derived RNA polymerase is supplemented to enable the expression of cloned genes downstream of the polymerase promoter.

[0006] The emergence of protein enzymes is key to the transition from RNA-based life to modern biology. The discovery of tRNA-aminoacylated ribozymes suggested the possibility of synthesizing protein enzymes from a highly simplified translation system using tRNA charged by ribozymes. Other systems have also been reported, using aaRS, urzymes, and pre-loaded tRNA prepared by chemical acylation. Among these, a highly robust and versatile tRNA-aminoacylated ribozyme system called flexizyme, discovered through in vitro selection, has been shown to be able to load a wide variety of amino acids into tRNA. Using tRNA loaded by flexizyme and aaRS, the incorporation of multiple non-natural amino acids into translated peptides has been achieved, enabling the selection of peptide drugs in practical applications. However, due to partially low translation yields, when using only tRNA loaded by flexizyme without the presence of aaRS (hereinafter referred to as "aaRS-free"), only short peptides (less than 7 amino acid residues in length) are translated. On the other hand, ribosome-mediated production of full-length functional protein enzymes containing all 20 protein constituent amino acids under aaRS-free conditions has not yet been demonstrated.

[0007] Terasaka, N. et al. [Terasaka, N., Hayashi, G., Katoh, T., and Suga, H. (2014). An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Nat. Chem. Biol. 10, 555-557] reported a modified system using rRNA-tRNA pairs with compensatory mutations, which specifically utilize a genetic code programmed differently from the natural genetic code and thus synthesize peptides orthogonally from their wild-type counterparts. These translation mechanisms allow a single mRNA to produce two distinct peptides according to an artificially programmed genetic code. [Overview of the project]

[0008] Aspects of the present invention relate to cell-free, aaRS-free protein translation / expression / synthesis systems and methods, as well as their use. This disclosure relates to the use of only tRNA charged by flexizyme, and Mg 2+ This invention provides successful translation of multiple proteins, including active enzymes with different functions, by improving translation yield through lowering the concentration and increasing the tRNA concentration. An aaRS-free translation system for producing active aaRS (TrpRS) is demonstrated, which further catalyzes the loading of more tRNA. Enantiomer tRNAs loaded with D-amino acids by synthetic L-flexizymes are also demonstrated. This disclosure demonstrates the feasibility of translating protein enzymes without aaRS from a highly simplified translation apparatus, alleviating the need to chemically synthesize dozens of large aaRS proteins to achieve enantiomer translation. Cation-depleted and flexizyme-loaded tRNAs are useful for the translation of complete or partial non-natural peptides, either in combination with or without other aaRS proteins.

[0009] Therefore, according to certain embodiments of the present invention, mRNA molecules that code for proteins; Multiple loaded tRNA molecules; and Cell-free translation mix, Includes Mg in the system +2 The concentration is less than 100 mM. A system for producing proteins is provided.

[0010] According to some embodiments, the system essentially lacks aminoacyl-tRNA synthetase. According to some embodiments, the concentration of the loaded tRNA molecules is greater than 60 μM. According to some embodiments, the concentration of the loaded tRNA molecule exceeds 160 μM, and Mg +2 The concentration is less than 100 mM.

[0011] According to some embodiments, at least one of the multiple loaded tRNA molecules is loaded by the flexizyme. According to some embodiments, non-natural amino acid residues are loaded into the tRNA molecule. According to some embodiments, the non-natural amino acid residue is a D-amino acid residue.

[0012] According to some embodiments, the tRNA molecule contains an L-ribonucleic acid residue (L-tRNA). According to some embodiments, L-tRNA is prepared using D-polymerase. According to some embodiments, the D-polymerase is a mirror image protein of Dpo4 (D-Dpo4).

[0013] According to some embodiments, the D-Dpo4 is D-Dpo4-5m-Y12S (Sequence ID 126). According to some embodiments, the flexizyme comprises an L-ribonucleic acid residue (L-flexizyme). According to some embodiments, the protein is selected from the group consisting of active L-protein enzymes and active D-protein enzymes. According to some other embodiments of the present invention, Mg at concentrations below the specified level +2 To provide multiple loaded tRNA molecules having different concentrations; and The loaded tRNA molecule is brought into contact with a protein-coding mRNA molecule in a cell-free translation mix, thereby obtaining the protein. A method for producing a protein using the system provided in this disclosure is also provided.

[0014] According to some embodiments, the system used in the method essentially lacks aminoacyl-tRNA synthetase. According to some embodiments, providing multiple loaded tRNA molecules involves Mg before the contact step. +2 This includes adjusting (reducing or depleting) the concentration of [the substance]. According to some embodiments, Mg +2 Adjusting the concentration of the substance may involve using techniques such as chromatography, alcohol precipitation and pellet washing, ultrafiltration, and dialysis. According to some embodiments, providing multiple loaded tRNA molecules further includes adjusting the concentration of the loaded tRNA molecules to more than twice the concentration of loaded tRNA in other protein translation systems, including the aaRS enzyme. According to some embodiments, the concentration of loaded tRNA molecules is greater than 160 μM.

[0015] According to some other aspects of several embodiments of the present invention, a method is provided for loading D-amino acids into L-tRNA, and the method is Prepare the L-tRNA molecule using D-polymerase; Prepare activated D-amino acids; To prepare L-aminoacylated ribozymes; and The L-tRNA, the L-aminoacylated ribozyme, and the activated D-amino acid are brought into contact to obtain a D-amino acid-loaded L-tRNA molecule. It is executed by [the specified method / system].

[0016] According to some embodiments, the L-aminoacylated ribozyme is an L-flexizyme. According to some embodiments, the method can be analyzed by PAGE analysis of the reaction mixture of D-amino acid-loaded L-tRNA molecules, where the PAGE gel is characterized by clear peaks for loaded tRNA species and clear peaks for unloaded tRNA species.

[0017] According to some other embodiments of the present invention, an L-flexizyme comprising an L-ribonucleotide residue is provided. In some embodiments, the L-flexizyme contains 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of L-ribonucleotide residues. In some embodiments, the L-flexizyme consists of an L-ribonucleotide residue.

[0018] In some embodiments, the L-flexizyme has a sequence that exhibits more than 80% identity with 5'-ggaucgaaagauuuccgcauccccgaaaggguacauggcguuaggu-3' (SEQ ID NO: 82). According to some other aspects of several embodiments of the present invention, a protein prepared by the method provided in this disclosure is provided. In some embodiments, the protein is selected from the group consisting of a protein containing at least one non-canonical amino acid residue, a protein containing at least one D-amino acid residue, an L-protein, and a D-protein.

[0019] In some embodiments, the protein is selected from the group consisting of chicken lysozyme, Gaussia luciferase, and Escherichia coli TrpRS. In some embodiments, the protein has a sequence that can be decoded into text information and / or numerical information, and includes natural amino acids and / or non-natural amino acids. In some embodiments, the protein is encoded by mRNA#6. According to some other aspects of several embodiments of the present invention, a library of randomized or partially randomized peptides obtained by the method described is provided, at least of the peptides comprising at least one non-natural amino acid.

[0020] Unless otherwise specified, all technical and / or scientific terms used in this disclosure have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing embodiments of the invention, but exemplary methods and / or materials are described below. In case of any conflict, the patent specification shall prevail, including definitions. Furthermore, the materials, methods and examples are illustrative and not necessarily intended to limit the scope. [Brief explanation of the drawing]

[0021] Some embodiments of the present invention are described in this disclosure with reference to the accompanying drawings, merely as examples. While the drawings are referred to here specifically and in detail, it should be emphasized that the illustrated matters are illustrative and for illustrative purposes only, for the purpose of illustrating embodiments of the present invention. In this regard, the description made with reference to the drawings will make it clear to those skilled in the art how embodiments of the present invention may be carried out. In the drawings,

[0022] [Figure 1] Figure 1 shows a schematic diagram of several aspects of the present invention, in particular of aaRS-free translation of a protein using Flexizyme-loaded tRNA (10), where tRNA 11 is loaded by the Flexizyme system 12 to produce a population of loaded tRNA 13 representing protein constituent amino acids for translation of the protein enzyme, and the aaRS-free translation further comprises step 14a, in which the loaded tRNA is purified by HPLC to reduce Mg2+14b contamination, and step 15, in which the loaded tRNA 13 is enriched in ribosome 17 for aaRS-free translation of mRNA 16, and the translated polypeptide 18 can be folded into an active protein enzyme 19a containing aaRS 19b, which can be used to load tRNA, thereby completing the cycle. [Figure 2]Figure 2 shows the acidic PAGE analysis of tRNA loading yield before and after HPLC purification. Here, "U" represents unloaded tRNA, "C" represents crude loaded tRNA, and "P" represents purified loaded tRNA. The tRNA loading yield was determined using the integrated peak area of ​​loaded tRNA relative to total tRNA using the software package IMAGEJ. [Figure 3A] Figures 3A-E illustrate the concept and results of flexizyme loading onto tRNA in the process of aaRS-free loading onto enantiomer tRNA according to several embodiments of the present invention. These figures show D-tRNA loading catalyzed by D-flexizyme, and its enantiomer version, i.e., enantiomer tRNA loading catalyzed by L-flexizyme (PDB source: 1EHZ(tRNA), 3CUL(flexizyme) (Figure 3A), loading of D-alanine by L-flexizyme onto enzymatically transcribed enantiomer tRNAAla (with natural chirality counterpart for comparison) (Figure 3B), loading of glycine by L-flexizyme onto enzymatically transcribed enantiomer tRNAGly (with natural chirality counterpart for comparison). Figure 3C shows loading of D-lysine onto enzymatically transcribed enantiomer tRNALys with L-flexizyme (with natural chirality counterpart for comparison) (Figure 3D), and Figure 3E shows loading of D-phenylalanine onto enzymatically transcribed enantiomer tRNAPhe with L-flexizyme (with natural chirality counterpart for comparison). Here, tRNA loading yield was determined using the software package IMAGEJ, with the integrated peak area of ​​loaded tRNA relative to total tRNA. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 4A]Figures 4A-G show the results of aaRS-free translation of several short peptides according to several embodiments of the present invention. Shown are MALDI-TOF-MS analysis of the translated short peptide from mRNA#1 (Figure 4A), aaRS-free translation yield of the short peptide analyzed by trichine-SDS-PAGE, where the unloaded tRNA concentration was in the range of 160–540 μM, but the flexizyme-loaded tRNA concentration remained at 70 μM, resulting in a loading yield in the range of 44–13% (top of Figure 4B), where the total tRNA concentration was in the range of 16–1003 μM, and the loading yield remained at 56% (bottom of Figure 4B) (error bars represent the standard deviation from three independent experiments), and MALDI-TOF-MS analysis of the translated short peptide from mRNA#2 (Figure 4C), mRNA#3 (Figure 4D), mRNA#4 (Figure 4E), mRNA#5 (Figure 4F), and mRNA#6 (Figure 4G). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 5A] Figures 5A–D show the results of aaRS-free translation of mRNA #1 under various conditions. It is shown that the cargo yield remained at 44% when the total tRNA concentration ranged from 20–644 μM (Figure 5A), the total tRNA concentration remained at 160 μM when the total flexizyme concentration ranged from 240–525 μM (Figure 5B), and that flexizyme and 0–380 μM of unloaded tRNA were mixed in 10 mM MgCl2 (in Figure 5C) and 100 mM MgCl2 (in Figure 5D), desalted by ethanol precipitation, and added to the aaRS-free translation mix, where the concentration of flexizyme-loaded tRNA remained at 70 μM (error bars represent the standard deviation from three independent experiments). [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 6A] Figures 6A-E show the tricine-SDS-PAGE gel analysis for calculating the aaRS-free translation yield, with Figures 6A-E corresponding to Figures 4B, 5A, 5B, 5C, and 5D, respectively, for calculating the aaRS-free translation yield. "M" is the synthetic peptide standard (Fph-KYDKYD (SEQ ID NO: 125)). [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 7A] Figures 7A and 7B show the results of in vitro translation experiments in the presence of LysRS, TyrRS, and AspRS, and show tricine-SDS-PAGE analysis of translation products and Fph-tRNAfMet preloaded with enhanced flexizyme at unloaded, unmodified total tRNA concentrations ranging from 22 to 680 μM in the presence of LysRS, TyrRS, and AspRS (Figure 7A), as well as calculated translation yields (Figure 7B) (error bars represent the standard deviation from three independent experiments). [Figure 7B] Same as above. [Figure 8A] Figures 8A and 8B show the flexizyme loading yields of 21 tRNAs that have their own corresponding (cognate) protein constituent amino acids. Figure 8A shows the loading yield determined after ethanol precipitation, and Figure 8B shows the loading yield determined after HPLC purification of 14 flexizyme-loaded tRNAs, where N / A indicates that purification of flexizyme-loaded tRNAs was not performed. [Figure 8B] Same as above. [Figure 9]Figure 9 shows the MALDI-TOF MS analysis of aaRS-free translated mRNA #6. It indicates that when the total tRNA concentration increased (520 μM) in the aaRS-free translation system, a mistranslated product with a molecular weight of 2,252.7 Da was observed, while the correctly translated product had a molecular weight of 2,240.7 Da. Here, au represents an arbitrary unit, and C and O represent the calculated and observed m / z values, respectively. [Figure 10A] Figures 10A–C show the amino acid sequences of aaRS-free translated protein enzymes, specifically chicken lysozyme (Figure 10A), Gaussia luciferase (Figure 10B), and E. coli TrpRS (Figure 10C). The positions translated by flexizyme-loaded tRNA were purified by either ethanol precipitation or HPLC (underlined). [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 11A]Figures 11A-G show the SDS-PAGE analysis of aaRS-free translated protein enzymes, with Figure 12A showing the overall gel image (Figure 11A), a 400 ng sample of commercially available chicken lysozyme purified from chicken egg white analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue (Figure 11B), a whole gel image shown in Figure 12C (Figure 11C), a 400 ng sample of recombinant Gaussia luciferase expressed and purified from E. coli BL21 strain analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue (Figure 11D), a whole gel image shown in Figure 14A (Figure 11E), and 15% SDS-PAGE. Figure 11F shows a 300 ng sample of recombinant E. coli TrpRS expressed and purified from E. coli strain BL21, analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue, as well as 5 μM Fph-CME, 1 μM Fph-tRNAfMet, and 5 μM Fph-tRNAfMet, analyzed by 15% SDS-PAGE with or without heating at 98°C for 3 minutes and scanned by Typhoon FLA 9500 under Cy2 mode, where M is the benchmark fluorescent protein standard. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 11G] Same as above. [Figure 12A]Figures 12A-D show the results of experimental demonstrations of the concept of aaRS-free translation of protein enzymes according to several embodiments of the present invention, aaRS-free translation of N-terminal FAM-labeled chicken lysozyme (M represents a benchmark fluorescent protein standard) (Figure 12A), analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode (Figure 12B), enzyme assay of crude aaRS-free translated chicken lysozyme using fluorescently labeled bacterial (Micrococcus lysodeikticus) cell wall material as a substrate, analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode Figure 12C shows aaRS-free translation of N-terminal FAM-labeled Gaussia luciferase scanned by 9500, and Figure 12D shows an enzyme assay of crude aaRS-free translated Gaussia luciferase using coelenterazine as a substrate (RFU represents relative fluorescence units, RLU represents relative luminescence units). [Figure 12B] Same as above [Figure 12C] Same as above [Figure 12D] Same as above [Figure 13] Figure 13 shows yield estimates for aaRS-free translated Gaussia luciferase, where standard curves are plotted using recombinant Gaussia luciferases of 0, 25 nM, 50 nM, 100 nM, and 250 nM (indicated by squares), and the yield of translated Gaussia luciferase was estimated to be approximately 25 nM (indicated by triangles). [Figure 14A]Figures 14A-C show aaRS-free translation of TrpRS, analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode, aaRS-free translation of N-terminal FAM-labeled E. coli TrpRS (M represents a benchmark fluorescent protein standard) (Figure 14A), sequence and secondary structure of internal Cy5-labeled tRNATrp (Figure 14B), and an enzymatic assay of crude aaRS-free translated TrpRS using Cy5-tRNATrp as a substrate, analyzed by 8% acidic PAGE and scanned by Typhoon FLA 9500 under Cy5 mode (Figure 14C). [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 15A] Figures 15A and 15B show the results of transcription of enantiomer tRNALys by D-Dpo4-5m-Y12S, showing the extension of a 5'-FAM-labeled L-universal primer on an L-ssDNA template, where polymerization was carried out by synthetic D-Dpo4-5m-Y12S polymerase. Reaction aliquots were terminated at different time points and analyzed by a 12% denatured PAGE gel with 7M urea (Figure 15A), as well as enantiomer transcription and I2-mediated cleavage of tRNALys transcripts, which were analyzed by a 10% denatured PAGE gel with 7M urea and stained with SYBR-Green II (Thermo Fisher Scientific, MA, USA) (Figure 15B). [Figure 15B] Same as above. [Figure 16A] Figures 16A and 16B show the results of biochemical characterization of enzymatically transcribed native tRNA and enantiomer tRNA, illustrating RNase A digestion of enzymatically transcribed D- and L-tRNAAla (Figure 16A) and AaRS-catalyzed aminoacylation of enzymatically transcribed D- and L-tRNAAla (Figure 16B). [Figure 16B] Same as above. [Figure 17A]Figures 17A-C show MALDI-TOF MS analysis of I2-mediated cleavage. Figure 17A shows the MALDI-TOF MS spectrum of a synthetic DNA-RNA chimeric oligo cleaved by I2 at a phosphorothioate modification site, Figure 17B shows the MALDI-TOF MS spectrum of the uncleaved oligo under negative linear mode, and Figure 17C shows the MALDI-TOF MS spectrum of the I2-cleaved oligo under negative linear mode (m / z > 4000) and negative reflectron mode (m / z < 4000). Uppercase letters indicate DNA nucleotides, lowercase letters indicate RNA nucleotides, and "*" indicates phosphorothioate modification. 'au' represents an arbitrary unit, and C and O represent the calculated and observed m / z values, respectively. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 18A] Figures 18A and 18B show the translation of complete or partial non-natural peptides using cation-depleted flexizyme-loaded tRNA, illustrating the translation of peptide drugs and non-natural proteins using cation-depleted flexizyme-loaded tRNA in an in vitro translation system (Figure 18A), and the translation, data storage, and ribosome / mRNA display of complete or partial non-natural proteins using cation-depleted flexizyme-loaded tRNA in an in vitro translation system (Figure 18B). [Figure 18B] Same as above. [Figure 19A] Figures 19A and 19B show 8% acidic PAGE photographs and analyses of the experimental proof of the concept of loading pre-activated amino acids onto fully functional L-tRNA molecules enzymatically transcribed by the enantiomer enzyme (D-Dpo4-5m-Y12S). Figure 19A shows the result of loading onto enzymatically transcribed L-tRNA, and Figure 19B shows the result of loading onto synthetically produced L-tRNA. [Figure 19B] Same as above. [Figure 20A]Figures 20A-C show the results of in vitro translation of short peptides containing two consecutive D-phenylalanine molecules. Figure 20A shows the MALDI-TOF-MS analysis of the short peptide translated from mRNA #7, Figure 20B shows the MALDI-TOF-MS analysis of the short peptide translated from mRNA #8, and Figure 20C shows the tricine-SDS-PAGE analysis of the translation products of mRNA #7 or mRNA #8 scanned by Typhoon FLA 9500 under Cy2 mode, using unloaded tRNAPhe only (mRNA #7), 20 μM LPhe-tRNAPhe (mRNA #7), 20 μM DPhe-tRNAGluE2CUA (mRNA #8), or 200 μM DPhe-tRNAGluE2CUA (mRNA #8). [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 21A] Figures 21A and 21B show the results of in vitro translation of short peptides containing three consecutive D-phenylalanine units, where Figure 21A shows the MALDI-TOF-MS analysis of the translated short peptide from mRNA#9, and Figure 21B shows the tricine-SDS-PAGE analysis of the translation product of mRNA#9 using unloaded tRNAPhe only, 30 μM LPhe-tRNAPhe, 30 μM DPhe-tRNAGluE2CUA, or 300 μM DPhe-tRNAGluE2CUA, scanned by Typhoon FLA 9500 in Cy2 mode. [Figure 21B] Same as above. [Figure 22] Figure 22 shows the in vitro translation results of a short peptide containing three consecutive β-Gln molecules, and shows tricine-SDS-PAGE analysis of the translation product of mRNA #10 using only unloaded tRNA, 30 μM βGln-tRNAGluE2CUA, or 300 μM βGln-tRNAGluE2CUA, scanned with a Typhoon FLA 9500 in Cy2 mode. [Modes for carrying out the invention]

[0023] In some embodiments, the present invention relates to cell-free protein translation systems, more specifically, methods for synthesizing proteins and their enantiomers, without the use of aminoacyl-tRNA synthesizers, but not limited to these, and the use thereof.

[0024] The principles and operation of the present invention can be better understood by referring to the drawings and accompanying description. Before describing in detail at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited in its application to the details described below or illustrated by the examples. Other embodiments of the present invention are possible, or can be carried out or performed in various ways.

[0025] As mentioned above, despite the discovery of tRNA-aminoacylated ribozymes such as flexizymes, the synthesis of protein enzymes from a highly simplified translation system in the absence of aaRS remains unproven. One of the main reasons for the low yield of aaRS-free translation is that, compared to tRNA aminoacylation by aaRS, there is no reuse of the tRNA cargo by flexizymes. Furthermore, using in vitro transcribed unmodified tRNA as the aaRS-free cargo may also contribute to the low translation yield.

[0026] While developing the present invention, the inventors undertook to test the ability of an aaRS-free system to translate protein enzymes containing all 20 protein constituent amino acids using tRNA loaded solely by flexizyme. Preliminary test results showed that by increasing the concentration of flexizyme-loaded tRNA and decreasing the concentration of cation Mg2+ through purification, multiple protein enzymes with different functions, such as lysozyme, luciferase, and even aaRS itself, could be synthesized. It has also been demonstrated that enantiomer D-amino acids can be loaded onto enantiomer L-tRNA using synthetic enantiomer L-flexizyme, which ultimately enables the realization of an enantiomer translation apparatus.

[0027] Figure 1 shows a schematic diagram of several aspects of the present invention, in particular aaRS-free translation of proteins using loaded tRNA by Flexizyme (10), where tRNA 11 is loaded by the Flexizyme system 12, producing a population of loaded tRNA 13 representing protein constituent amino acids for protein enzyme translation, and the aaRS-free translation is further achieved when the loaded tRNA is purified by HPLC and Mg 2+ The process includes step 14 to reduce contamination, step 15 in which loaded tRNA 13 is enriched for aaRS-free translation of mRNA 16 in ribosome 17, and the translated polypeptide 18 can be folded into an active protein enzyme 19a containing aaRS 19b, which can be used to load onto tRNA and complete the cycle.

[0028] This disclosure demonstrates aaRS-free translation of protein enzymes using an exclusive set of ribozyme-charged tRNAs. This shows that progressive and reliable ribosome translation does not require aaRS-catalyzed tRNA charging or tRNA reuse, revealing that protein enzymes with more structural motifs and therefore more catalytic activity than short peptides can be translated from a highly simplified aaRS-free translation system. In particular, the average size of modern natural proteins is approximately 270–470 amino acid residues. AaRS-free translation of proteins as large as TrpRS suggests the potential to further improve translation efficiency and fidelity by producing other important protein enzymes, such as tRNA modifying enzymes. Furthermore, this disclosure also shows the finding that high concentrations of ribozyme-loaded tRNA significantly improve the yield of aaRS-free translation. This may elucidate possible conditions for the emergence of protein enzymes on pre-biological Earth, where a rich feedstock of ribozyme-loaded tRNA would have been crucial for the efficient operation of primitive translation systems.

[0029] One limitation of current aaRS-free translation systems is that tRNA loading must be decoupled from translation, as tRNA is pre-loaded before being added to the translation system. This is because flexizymes are nonspecific catalysts that load various amino acids onto tRNA. A methodology using high concentrations of flexizyme-loaded tRNA and removing Mg2+ contamination by purification has been shown to significantly improve the yield of aaRS-free translation and can be applied to other in vitro translation systems (with or without aaRS) that use pre-loaded tRNA for producing peptides or proteins from amino acid groups that are all or partly non-natural, enabling immediate application in many areas of synthetic biology and drug discovery.

[0030] Achieving aaRS-free translation of protein enzymes paves the way for aaRS-free translation machinery, providing a more viable model for achieving enantiomer translation. This is because all aaRS proteins, in total, account for 29% (approximately 1.4 MDa) of the molecular weight of the E. coli translation machinery (approximately 4.9 MDa total molecular weight), including ribosomes, translation factors, aaRS, and tRNA. Furthermore, the translation of the small 169-amino acid residue Gaussia lucifilase demonstrated in this disclosure provides a highly sensitive and chiral-specific assay for testing enantiomer translation.

[0031] aaRS-free cell-free translation system: As discussed above in this disclosure, cell-free protein synthesis provides a simple and rapid method for synthesizing, monitoring, analyzing, and purifying proteins from DNA templates, while also paving the way for gene code extension methods that enable site-specific incorporation of non-natural amino acids (UAAs; also known as non-standard amino acids) into proteins via ribosome translation. Known systems are based on the exogenous addition of an orthogonal translation system (OTS) containing orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase (aaRS) to a cell-free reaction mixture, but the protein translation system provided in this disclosure further extends this concept by enabling the efficient production of proteins without the presence of aminoacyl-tRNA synthetase (aaRS), and an aaRS-free translation system is provided in this disclosure.

[0032] In the context of this disclosure, as used herein, the term “aaRS-free” means a ribosome translation system and / or method and / or platform for preparing proteins from transcriptional templates (e.g., ribonucleic acid molecules) that is essentially devoid of aminoacyl-tRNA synthetase (aaRS). Essentially devoid of aminoacyl-tRNA synthetase means that no step in protein production involves the use or presence of aaRS. According to some embodiments of the present invention, the only exception to the definition of aaRS-free translation system / method / platform is an embodiment in which the aminoacyl-tRNA synthetase is a protein product produced by the system / method / platform. Essentially devoid of tRNA synthetase enzymes means that the system does not include means for loading amino acid residues onto tRNA, and that no aaRS enzyme is introduced into the system at any stage of translation, and that the entire supply of amino acid residues comes from a pre-loaded tRNA molecule.

[0033] Therefore, according to certain embodiments of the present invention, a system for producing a protein is provided, the system is: mRNA molecules that code for proteins; Multiple loaded tRNA molecules; and Cell-free translation mix, The system includes, and the system essentially lacks aminoacyl-tRNA synthetase, and Mg in the system +2 The concentration is less than 100 mM.

[0034] As used in this disclosure, the term “system” refers to the reaction mixture (i.e., solvent, solute, reactants, and optionally present (optional) detection markers) and reaction conditions (concentration, temperature, and mixing) that are essential for carrying out complex chemical reactions such as protein synthesis.

[0035] In the context of some embodiments of the present invention, the term “cell-free translation mix” refers to an in vitro protein translation mixture that does not involve the use of intact / viable cells and includes ribosomes and ribosomal translation factors essential for cell-free in vitro protein translation reactions, as these terms are known in the art. In the context of some embodiments of the present invention, the term “aaRS-free translation mix” refers to a cell-free translation mix as known in the art, except that a cell-free (in vitro) translation mix essentially lacks aaRS proteins, unless otherwise specified.

[0036] A protein translation system includes a messenger RNA molecule that codes for the amino acid sequence of a desired protein to be produced by the system. Alternatively, the system may include means for transcribing a DNA template into an mRNA molecule, i.e., a DNA template and transcription factors (e.g., RNA nucleotides, RNA polymerase, and general transcription factors) for achieving DNA-RNA transcription.

[0037] The protein translation system includes a plurality of charged tRNA molecules, which are also referred to as pre-charged tRNA transcripts in the context of some embodiments of the present disclosure. In some embodiments, the tRNA molecules are synthetically prepared polynucleotides, and in other embodiments, the tRNA molecules are enzymatically prepared transcripts, and the important differences between these two categories are explained below.

[0038] According to some embodiments of the present invention, this plurality of charged tRNA molecules includes at least tRNA molecules loaded with amino acid residues that are encoded by the mRNA and will be present in the protein sequence as encoded by the mRNA molecule. The plurality of charged tRNA molecules also includes tRNA molecules loaded with non-natural amino acid residues such as residues of D-amino acids and other non-standard amino acid residues, as shown in Tables A and B below. Preferably, the frequency and amount of each individual charged tRNA molecule match the frequency of each amino acid in the protein sequence. For example, if the frequency of serine residues in the protein sequence is 8% and the frequency of methionine is 1%, the plurality of charged tRNA molecules in the system will reflect that frequency, with about 8 times more tRNA Met than tRNA Ser included. In some embodiments, the tRNA molecules are made from L-nucleotides, giving a tRNA molecular mirror image of natural tRNA molecules. In some embodiments, the tRNA molecules consist of L-nucleotides and, further, residues of D-amino acids are loaded.

[0039] As used in this disclosure, the terms "residue" and / or "portion" refer to a part of a molecule, typically its main part, or a group of atoms related to a particular function. For example, the term "amino acid residue" refers to an amino acid in the context of a compound to which the amino acid is attached, a peptide is a chain of amino acid residues linked to each other, and a tRNA molecule loaded with ribonucleic acid residues is a ribonucleic acid attached to the tRNA molecule.

[0040] Tables A and B show some optional amino acid residues relevant in the context of several embodiments of the present invention. These are examples and should not be considered limiting.

[0041] [Table 1-1] Table A

[0042] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0043] Cation depletion system: As disclosed above in this disclosure, the cell-free aaRS-free system for protein production according to embodiments of the present invention is effective at low cation concentrations, more specifically, low magnesium ion concentrations. Magnesium is present in relatively high concentrations in most cell-free protein translation mixtures, including commercially available mixtures. Magnesium is also present in most loaded tRNA preparations, particularly Flexizyme loaded tRNA preparations. As shown in the following Examples section, the inventors have surprisingly found that reducing the magnesium concentration in the cell-free aaRS-free protein translation system to a practically minimum level significantly improves the efficiency and fidelity of protein production. Therefore, in order to achieve the improved performance of the system disclosed in this disclosure, the intrinsic presence of magnesium ions carried over from various components in known protein translation systems had to be deliberately reduced by the inventors.

[0044] Therefore, according to some embodiments of the present invention, a system for producing proteins has a lower Mg compared to any known cell-free protein translation system. +2 It is characterized by its concentration. More specifically, the magnesium concentration in the system according to the present invention is the Mg in the loaded tRNA preparation. +2 It is lower than the concentration. In absolute terms, the amount of Mg in the system. +2 The concentrations are less than 100 mM, less than 90 mM, less than 80 mM, less than 70 mM, less than 60 mM, less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, or less than 10 mM.

[0045] In some embodiments, the concentration of magnesium ions in the system is the minimum concentration that can actually be obtained by ion depletion methods, such as, but not limited to, chromatography (HPLC), precipitation and pellet washing in alcohol, ultrafiltration, and dialysis.

[0046] Loaded tRNA concentration: The tRNA molecules in the system of this disclosure may be pre-loaded by any method known in the art, but in some preferred embodiments, the tRNA is loaded by a flexizyme. The concentrations of pre-loaded tRNA molecules present in the system are also subject to modification compared to their concentrations in known cell-free protein translation systems.

[0047] According to some embodiments, the concentration of loaded tRNA is at least twice as high as the concentration in other known cell-free protein translation systems. According to some embodiments, the concentration of loaded tRNA is greater than 50 μM, greater than 60 μM, greater than 80 μM, greater than 90 μM, greater than 100 μM, greater than 110 μM, greater than 120 μM, greater than 130 μM, greater than 140 μM, greater than 150 μM, greater than 160 μM, greater than 170 μM, greater than 180 μM, greater than 190 μM, or greater than 200 μM.

[0048] Inverted chirality element: Because the system provided in this disclosure does not require the aaRS enzyme, it is particularly suitable for translating proteins containing non-natural / non-standard amino acid residues, especially D-amino acid residues. The system can be used to insert D-amino acid residues into any polypeptide chain, including translation into a chain in which all mRNA is D-amino acid. The system was used to translate a complete enantiomer protein, as demonstrated below.

[0049] As described later in this disclosure, the system has been successfully used with tRNA molecules containing or consisting of L-ribonucleic acid residues (L-tRNA). Therefore, according to some embodiments of the present invention, the system includes L-tRNA molecules. L-tRNA is prepared using a D-polymerase such as D-Dpo4-5m-Y12S, but other methods for producing L-tRNA molecules are also conceivable within the scope of the present invention.

[0050] In some embodiments of the present invention, the system includes an L-tRNA molecule preloaded with D-amino acid residues by an L-flexizyme for translation of a D-protein (enantiomer).

[0051] L-aminoacylated ribozymes: According to some embodiments of the present invention, the system comprises L-tRNA preloaded with a ribozyme having aminoacyl-tRNA synthetase (aaRS) activity, i.e., an aminoacylated ribozyme. In some embodiments, the aminoacylated ribozyme is a flexizyme. In some preferred embodiments, the L-tRNA is loaded with an L-flexizyme, which is a ribozyme composed entirely or substantially of L-ribonucleotides.

[0052] Accordingly, according to certain aspects of several embodiments of the present invention, a polyribonucleic acid molecule (RNA) (a mirror image to a corresponding (comparable) natural RNA molecule) containing L-ribonucleotides is provided, which has catalytic activity (tRNA loading activity) to aminoacylate RNA by using activated amino acids (ribozyme). In other words, the present disclosure provides an L-flexizyme.

[0053] As demonstrated in the examples section below, loading D-amino acid residues into L-tRNA molecules becomes more efficient and consistent when using L-flexizymes. The L-flexizymes provided in this disclosure have substantially the same sequence as their enantiomers (D-aaRS ribozymes; D-flexizymes) or exhibit 80% or more sequence identity with D-flexizymes known in the art. For example, according to some embodiments, the L-flexizyme has a sequence that exhibits 80% or more identity with 5'-ggaucgaaagauuuccgcauccccgaaaggguacauggcguuaggu-3' (SEQ ID NO: 82).

[0054] The embodiment of the L-flexizyme results in the use of the L-flexizyme to load pre-activated D-amino acid residues into an L-tRNA molecule. Accordingly, according to some other embodiments of several embodiments of the present invention, a method is provided for loading D-amino acids into L-tRNA, the method being Prepare activated D-amino acids; Prepare the L-tRNA molecule; To prepare L-flexizyme; and The L-tRNA, the L-flexizyme, and the activated D-amino acid are reacted to obtain an L-tRNA molecule into which the D-amino acid has been loaded. It is done by.

[0055] According to some embodiments of the present invention, L-tRNA molecules are prepared using D-polymerase rather than as a product of a synthetic apparatus. As demonstrated in the Examples section below, the reaction of L-flexizyme with L-tRNA showed a significant improvement in the efficiency and fidelity of the aaRS activity (amino acid loading) reaction when the L-tRNA source was enzymatic rather than synthetic (see the description of Figures 3A-3E and 19A-19B below). This advantage can be shown and identified by using PAGE analysis of the reaction mixture of D-amino acid loaded L-tRNA molecules, where distinct peaks for loaded tRNA species and unloaded tRNA species are characteristic. On the other hand, in reactions using mechanically synthesized L-tRNA molecules, the reaction mixture shows a series of large peaks, which indicate multiple intermediates, mismatches, and other side reactions resulting from the use of low-quality L-tRNA as a starting material.

[0056] How to use the aforementioned system: The use of the system provided in this disclosure differs from the use of other known cell-free protein translation systems, and even from previously known aaRS-free protein translation systems, because, at least, the concentration of pre-loaded tRNA molecules is higher than that used in known systems, and Mg+2 Mg used in systems where the concentration is known +2 That's correct in the sense that it's lower than the concentration.

[0057] Therefore, according to some other aspects of several embodiments of the present invention, a method is provided for producing a protein using the cell-free aaRS-free protein translation system provided in this disclosure, the method is The Mg described above in this disclosure +2 Mg below the concentration +2 Preparing multiple pre-loaded tRNA molecules at concentrations (less than half the concentrations of other known cell-free protein translation systems, or less than 100 mM); and These multiple loaded tRNA molecules are brought into contact with mRNA molecules encoding the desired protein in a cell-free translation mix, thereby obtaining the target protein. It is done by.

[0058] In some embodiments, the method involves Mg before contacting the pre-loaded tRNA preparation with the cell-free translation mix. +2 This further includes adjusting the concentration to a desired low concentration. Depletion of ions, particularly cations, from systems containing macromolecules, especially sensitive biomacromolecules, can be achieved by any known procedure in the art. For example, Mg +2 The concentration can be reduced by chromatography (e.g., HPLC), alcohol precipitation and subsequent washing of the precipitated pellet, ultrafiltration, and dialysis, but is not limited to these. Other procedures are also conceivable within the scope of the present invention.

[0059] In some embodiments, the method further includes adjusting the concentration of the preloaded tRNA molecules to a desired high concentration, as this feature has been discussed above in this disclosure, before contacting the preloaded tRNA preparation with the cell-free translation mix. Thus, in some embodiments, the method further includes enriching the preloaded tRNA molecules to a concentration at least twice higher than that in the system comprising aaRS. In some embodiments, the concentration of the preloaded tRNA molecules is 160 μM or higher.

[0060] The following examples section provides a detailed presentation of several embodiments of the system disclosed herein and a method for producing proteins using the system in the cell-free and aaRS-free protein translation system disclosed herein.

[0061] Products of the disclosed systems and methods: As demonstrated by the experimental proof of the concepts presented below, the systems and methods provided in this disclosure can be used to produce proteins characterized by exhibiting the structure and function of comparable proteins produced in any in vitro translation system, cellular system, or any native system. Proteins produced according to the provisions of the present invention may also be enantiomers produced from chirality inversion elements, such as fully active enzymes that catalyze reactions from enantiomer starting materials to produce enantiomer products.

[0062] Accordingly, according to certain aspects of several embodiments of the present invention, a protein produced by a system and / or method provided in this disclosure is provided. In some embodiments, the protein is a mirror image protein (a D-protein consisting substantially of D-amino acid residues).

[0063] Exemplary proteins for which the use of the system provided in this disclosure has been demonstrated include chicken lysozyme, Gaussia luciferase, and Escherichia coli TrpRS.

[0064] Library: According to some embodiments, the systems and methods provided in this disclosure can be used to prepare a library of randomized or partially randomized peptides in which at least one non-natural amino acid is present in the peptides.

[0065] One advantage of the aaRS-free system provided in this disclosure is that it requires 21 tRNAs to function efficiently. There are more than 20 other natural tRNA transcripts available for assigning non-natural amino acids (genetic code reprogramming). In other protein translation systems, these tRNAs are unusable because aaRS loads them with natural amino acids. Therefore, the present invention can provide a means for translating randomized peptides containing multiple non-natural amino acids without encountering the problem of misloaded tRNA molecules.

[0066] The protein translation system provided in this disclosure can be applied to orthogonal ribosome-tRNA pairs with compensatory mutations [Terasaka, N., Hayashi, G., Katoh, T., and Suga, H. "An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center," Nat. Chem. Biol., 2014, 10, 555-557]. In such orthogonal systems, the orthogonal tRNA is loaded by a flexizyme and cannot be loaded by any aaRS protein, resulting in inefficiency problems, which are resolved by the provisions of the present invention. For example, known aaRS-free systems have low yields for translating heptapeptides, e.g., (Fph)-Lys-Tyr-Asp-Lys-Tyr-Asp (SEQ ID NO: 125), with a yield of approximately 0.15 μM and low processability (7 amino acid residues). Under the improved conditions provided by the system according to the embodiments of the present invention, the yield is approximately 2 μM for the same heptapeptide. Furthermore, translation of up to 334 amino acid residues, 48 ​​times longer than previously demonstrated, has been demonstrated using the provisions of the present invention. Thus, the improved cell-free / aaRS-free system according to some embodiments of the present invention is better suited to peptide drug discovery due to better yields (higher peptide pool concentration) and longer products (higher sequence diversity).

[0067] Non-biological use: In the search for ultra-high density, high-fidelity information storage devices, the inventors envisioned the use of the systems and methods disclosed herein in the production of proteinaceous macromolecules having sequences that can be coded and decoded using known procedures but cannot be degraded by natural biochemical elements. Protection from biodegradation is provided by using non-natural amino acid residues in the protein.

[0068] Furthermore, the provisions of the present invention can be used to maximize data density by incorporating unnatural amino acids, which are essentially letters acting as character modifiers when analogous to text. Accordingly, in some embodiments of the present invention, the protein that is the product of using the system provided herein is characterized in that it can be decoded into textual and / or numerical information and has an amino acid sequence containing at least some non-natural amino acid residues. Realization of this concept requires translation of a peptide of any sequence. The inventors of the present invention demonstrated this using 20 protein constituent amino acids in Figures 4C to 4G.

[0069] This concept was realized in the demonstration proof-of-concept experiment presented in the Examples section below, in which the inventors encoded a short message "MITRNACHARGINGSYSTEM" (SEQ ID NO: 123) into mRNA #6 (see Figure 4G) and successfully translated the full-length information-carrying peptide.

[0070] As used in this disclosure, the term "approximately" means ±10%. The terms "comprise," "comprising," "include," "including," and "have," as well as their conjugations, all mean "to include, but not limited to."

[0071] The term "~consists of" means "to include and be limited to ~". The term "essentially consisting of" means that the composition, method, or structure may include additional components, steps, and / or parts, provided that such additional components, steps, and / or parts do not substantially alter the basic and novel features of the claimed composition, method, or structure.

[0072] Where used in this disclosure, the phrases “substantially lacking” and / or “essentially lacking” in the context of a particular substance mean a composition that is completely lacking in the substance or that contains the substance in amounts less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% of the total weight or total volume of the composition. Alternatively, the phrases “substantially lacking” and / or “essentially lacking” in the context of a process, method, property, or feature mean a process, composition, structure, or article that completely lacks a particular process / method step or a particular property or particular feature, or a process / method in which a particular process / method step is achieved in amounts less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% compared to a given standard process / method, or a property or feature characterized by properties or features in amounts less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% compared to a given standard.

[0073] Where applied to the original, desired, or given properties of an object or composition, the term “substantially maintained” as used in this disclosure means that the properties of the treated object or composition have not changed by more than 20%, more than 10%, or more than 5%.

[0074] The term “exemplary” is used in this disclosure to mean “acting as an example, case, or illustration.” Any embodiment described as “exemplary” should not necessarily be construed as being preferable or advantageous to other embodiments, and / or as excluding the incorporation of features from other embodiments.

[0075] The terms “optionally” or “alternatively” are used in this disclosure to mean “given in some embodiments but not in other embodiments.” Any particular embodiment of the present invention may include several “optional” features, provided that such features do not conflict with each other.

[0076] As used in this disclosure, the singular forms "a," "an," and "the" also include multiple subjects unless the context clearly indicates otherwise. For example, the terms "compound" or "at least one compound" encompass multiple compounds, including mixtures thereof.

[0077] Throughout this application, various embodiments of the present invention may be presented in the form of ranges. It should be understood that descriptions in the form of ranges are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of the present invention. Therefore, a description of a range should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0078] Whenever a numerical range is indicated in this disclosure, it means that any cited digit (fractional or integer) within the indicated range is included. The terms "ranging" and "range" between the first and second indicators, and between the first and second indicators are used interchangeably in this disclosure and mean that the range includes the first and second indicators and all fractional numerals and integers between them.

[0079] As used in this disclosure, the terms “process” and “method” mean ways, means, techniques, and procedures for accomplishing a given task, including but not limited to ways, means, techniques, and procedures that are known to practitioners of chemistry, materials, mechanics, computation, and digital technologies or that can be readily developed from known ways, means, techniques, and procedures.

[0080] During the term of the patent granted by this application, it is anticipated that many related methods for aaRS-free protein translation systems will be developed, and the scope of the phrase “aaRS-free protein translation system” is intended to a priori include all such new technologies.

[0081] It is understood that certain features of the Invention described in the context of separate embodiments for clarity may be given in combination in a single embodiment. Conversely, various features of the Invention described in the context of a single embodiment for brevity may also be given separately, in any suitable subcombination, or as appropriate in any other embodiment of the Invention described. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments are inoperable without those elements.

[0082] The following examples provide experimental support for the various embodiments and aspects of the present invention outlined above and claimed in the claims section below. [Examples]

[0083] The following examples are shown below. These, along with the above description, illustrate some non-limiting embodiments of the present invention.

[0084] Example 1 Experimental Procedure material: The amino acid substrates for flexizyme loading were prepared as 3,5-dinitrobenzyl ester (DBE), however, Asn and fluorescein (FAM)-labeled Phe (Fph) were synthesized as 4-chlorobenzylthioester (CBT) and cyanomethyl ester (CME), respectively. The amino acid DBEs were obtained by order from Nantong Pptide Biotech Ltd (Jiangsu, China) or synthesized in-house according to a previously reported method [Murakami, H., Ohta, A., Ashigai, H., and Suga, H. (2006). A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat. Methods 3,357]. All amino acid DBE substrates were prepared as follows: 1 Verification was performed by 1H-NMR or high-resolution mass spectrometry. Fph-CME was synthesized and verified by high-resolution mass spectrometry as described [Terasaka, N., Hayashi, G., Katoh, T., and Suga, H. (2014). An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Nat. Chem. Biol. 10, 555-557].

[0085] Asn-CBT was synthesized as follows: A mixture of 0.5 mmol of Boc-Asn(Trt)-OH, 0.45 mmol of N,N-bis(2-oxo-3-oxazolidiyl)phosphodiamide chloride, 1.5 mmol of triethylamine, and 0.5 mmol of 4-chlorobenzyl mercaptan in 5 ml of dichloromethane was stirred at room temperature for 4 hours. The solution was washed with 0.5 M HCl, 0.5 N NaOH, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated by rotary evaporation. To remove the Boc protecting group and trityl protecting group, 2 ml of a solution containing 19:1 (v / v) trifluoroacetic acid (TFA) / ddH2O was added and stirred at room temperature for 4 hours. The solution was neutralized with saturated NaHCO3, extracted with dichloromethane, and concentrated by rotary evaporation. The crude product was dissolved in methanol and purified using a C18 HPLC column (Inertsil ODS-3, 5 μm, 10 × 150 mm, GL Sciences, Japan) with a gradient of 30–80% acetonitrile in 0.1% TFA. The fractions containing the product were pooled, lyophilized, and validated. 1 ¹H NMR (400 MHz, DMSO-d6) δ 8.40 (s, 3H), 7.74 (d, J=9.3Hz, 1H), 7.44-7.35 (m, 4H), 7.33 (d, J=9.5Hz, 1H), 4.54-4.46 (m, 1H), 4.25 (d, J=9.4Hz, 2H), 2.78 (dh, J=14.3, 7.5, 6.4Hz, 2H). D-DNA oligo was ordered and obtained from Genewiz (Jiangsu, China).

[0086] RNA oligos and DNA-RNA chimeric oligos were ordered and obtained from Tsingke (Beijing, China). L-DNA oligos and L-flexizymes were synthesized using L-deoxynucleoside and L-2'-t-butyldimethylsilyl (TBDMS) phosphoramidite (Chemgenes, MA, USA) in an H-8 DNA synthesizer (K&A Laborgeraete, Germany). Phosphothioate modifications were introduced using Sulfur 42 reagent (Sigma-Aldrich, MO, USA). The synthesized L-oligos were cleaved from CPG with concentrated ammonium hydroxide at 65°C for 2 hours. For the synthesis of L-flexizymes, the 2'-TBDMS protecting group was removed by treatment with 1:1 (v / v) triethylamine trihydrofluoride / DMSO at 65°C for 2.5 hours.

[0087] L-NTPs for mirror image transfer were prepared from unprotected L-nucleosides (Chemgenes, MA, USA) according to a previously reported method [Caton-Williams, J., Hoxhaj, R., Fiaz, B., and Huang, Z. (2013). Use of a 5'-regioselective phosphitylating reagent for one-pot synthesis of nucleoside 5'-triphosphates from unprotected nucleosides. Curr. Protoc. Nucleic Acid Chem. 52, 1.30.1-1.30.21]. L-DNA oligonucleotides and L-NTPs were purified by denaturation PAGE and HPLC, respectively. L-flexizyme was precipitated with ethanol and purified by HPLC.

[0088] The genes for AlaRS, AspRS, LysRS, TrpRS, and TyrRS were amplified and cloned from E. coli K12 MG1655 genomic DNA. The Gaussia luciferase gene was synthesized by Genewiz (Jiangsu, China). Recombinant aaRS protein and Gaussia luciferase with an N-terminal TEV-cleavable His tag were expressed and purified from E. coli BL21 strain as described in the literature [Shimizu, Y., and Ueda, T. (2010). PURE technology. In Cell-Free Protein Production: Methods and Protocols, Y. Endo, K. Takai and T. Ueda, eds. (Totowa, NJ: Humana Press), pp. 11-21]. After purification, the His tag was cleaved with a TEV protease. The refined chicken egg white lysozyme was purchased from Sigma-Aldrich (MO, USA).

[0089] In vitro transcription: Double-stranded DNA (dsDNA) templates for in vitro transcription were prepared by cross-extensioning two partially overlapping primers (1F and 2R) (2 μM forward primer 1F, 3 μM reverse primer 2R, 0.2 mM dNTPs, and 5 U of EasyTaq (TransGen Biotech, Beijing, China) per 100 μl of reaction solution) in 5 cycles of PCR, or by 25 cycles of assembly PCR using four primers (1F, 2R, 3F, and 4R) (2 μM primers 1F and 4R, 0.05 μM primers 2R and 3F, 0.2 mM dNTPs, and 5 U of EasyTaq per 100 μl of reaction solution).

[0090] The PCR products were purified with phenol-chloroform and then ultrafiltration. In tRNA sequences starting at 5' nucleotides other than guanosine, the self-cleaving hammerhead motif is positioned upstream of the tRNA sequence [Cui, Z., Stein, V., Tnimov, Z., Mureev, S., and Alexandrov, K. (2015). Semisynthetic tRNA complement mediates in vitro protein synthesis. J.Am. Chem. Soc. 137, 4404-4413]. All primer DNA oligo sequences for constructing the dsDNA template for in vitro transcription are listed in Table 1 below.

[0091] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0092] Each 1 ml transcription reaction system contained 20 μg of purified dsDNA template, 2 mM NTP, 0.1 mg / ml T7 RNA polymerase, and 400 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, MA, USA) in a 1× transcription buffer containing 25 mM MgCl2, 40 mM Tris-HCl pH 8.0, 2 mM DTT, and 1 mM spermidine. The transcription reaction product was incubated at 37°C for 2 hours, treated with 10 μl of DNase I (New England Biolabs, MA, USA), incubated for a further 0.5 hours, and then quenched by adding 60 μl of 0.5 M EDTA, followed by ethanol precipitation. The transcribed RNA was analyzed by the "disruption and immersion" method. 7 The samples were gel-purified, desalted, and concentrated by ultrafiltration. Using this method, both 5'-triphosphate (tRNA sequences starting with G) and 5'-hydroxyl-terminated tRNA (tRNA sequences starting with A / U / C) were prepared, and these were used to determine the tRNA His With the exception of [specific example], it has been previously shown that it is functionally equivalent to physiological 5'-monophosphate-terminal tRNA [Cui, Z., Stein, V., Tnimov, Z., Mureev, S., and Alexandrov, K. (2015). Semisynthetic tRNA complement mediates in vitro protein synthesis. J.Am.Chem.Soc.137, 4404-4413]. Therefore, the two versions of tRNA His Prepared: 5'-triphosphate-terminal tRNA His This was used in all flexizyme loading assays; 5'-monophosphate-terminal tRNA with an additional G at position-1 His This was prepared using a previously reported method and used as a control requiring aaRS activity.

[0093] Flexizyme-catalyzed tRNA-aminoacylation: The procedure for flexizyme-catalyzed tRNA loading was adapted from a previously reported method [Goto, Y., Katoh, T., and Suga, H. (2011). Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779-790]: 20 μM tRNA was mixed with 30 μM dinitroflexizyme in the presence of 1× folding buffer at pH 7.5 containing 50 mM HEPES-KOH and 100 mM KCl. The mixture was heated at 95°C for 2 minutes, cooled to 25°C for 10 minutes, and then 100 mM MgCl2 was added. The mixture was incubated at room temperature for 10 minutes and on a refrigerated metal block for 3 minutes. 5 mM DBE substrate was added to the system on the refrigerated metal block to initiate the loading reaction. The reaction products were incubated at 4°C for 6 hours, quenched with 2 × volume of 0.6 M NaOAc (pH 5.2), and precipitated with ethanol. For the following amino acids, adjustments were made to the general procedure: for Ile-DBE and Val-DBE, the refolding buffer was changed to 50 mM bicine-KOH at pH 9.0; for Met-DBE and Cys-DBE, 5 mM DTT was added to the substrate; for Pro-DBE, the substrate concentration was increased from 5 mM to 40 mM; for Fph-CME, 30 μM of augmented flexizyme was used to decrease the Fph-CME substrate concentration from 5 mM to 1 mM and increase the MgCl2 concentration from 100 mM to 400 mM; for Asn-CBT, the substrate concentration was increased from 5 mM to 25 mM; and for Trp-DBE, 20% DMSO was further added. The cargo yield was determined by acidic PAGE analysis: The precipitated cargo reaction product was dissolved in a loading buffer containing 93% formamide, 100 mM NaOAc at pH 5.2, 10 mM EDTA, and a trace amount of bromophenol blue. Acidic gels were prepared with 8% acrylamide, 100 mM NaOAc at pH 5.2, and 7 M urea. The gels were run for 16 hours in a refrigerator at 4°C with an aluminum cooling plate using 100 mM NaOAc as the electrophoresis buffer at pH 5.2.The gel was stained with SYBR-Green II (Thermo Fisher Scientific, MA, USA), scanned with a Typhoon FLA 9500 operating in Cy2 mode, and analyzed with the ImageJ software package [Schneider, CA; Rasband, WS & Eliceiri, KW (2012), "NIH Image to ImageJ: 25 years of image analysis", Nature methods 9(7):671-675, PMID 22930834]. tRNA. Asp Excluding the above, the peak area was integrated to calculate the yield of loaded tRNA. Asp-tRNA Asp Peak height was used to estimate the yield of Asp-tRNA. Asp This is unloaded tRNA Asp It moves very close to (see Figure 2), and Asp-tRNA Asp The estimated cargo yield (approximately 50%) was consistent with previously reported results [Murakami, H., Ohta, A., Ashigai, H., and Suga, H. (2006). A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat. Methods 3,357].

[0094] Figure 2 shows the acidic PAGE analysis of tRNA loading yield before and after HPLC purification. In the figure, "U" represents unloaded tRNA, "C" represents crude loaded tRNA, and "P" represents purified loaded tRNA. The tRNA loading yield was determined using the ImageJ software package, using the integrated peak area of ​​loaded tRNA relative to total tRNA.

[0095] aaRS-free translation of multiple short peptides: dsDNA templates for aaRS-free peptide translation were prepared by 25-cycle assembly PCR using the primers listed in Table 2.

[0096] [Table 2-1] [Table 2-2]

[0097] All DNA templates were purified by 10% denaturing PAGE. To translate mRNA#1, the reaction mixture was prepared so that each codon was decoded by the corresponding (cognate) flexizyme-loaded tRNA in the range of 1.25–80 μM. The tRNA concentrations corresponded to a total tRNA of 16–1000 μM. HPLC purification of the flexizyme-loaded tRNA was performed as described in the reference [Zhang, J., and Ferre-D'Amare, Adrian R. (2014). Direct evaluation of tRNA aminoacylation status by the T-Box riboswitch using tRNA-mRNA stacking and steric readout. Mol. Cell 55, 148–155]. The loaded tRNA was stored and determined by acidic PAGE analysis to be stable as a dry pellet for up to 3 days at -80°C. The loaded tRNA pellet was dissolved in 1 mM NaOAc at pH 5.2 at a volume of 0.5 × translation volume. Serial dilutions were performed to prepare 2-fold concentrated tRNAs at the concentrations shown in Table 3. Table 3 shows the total tRNA concentrations used for aaRS-free translation.

[0098] [Table 3]

[0099] Translation was initiated by mixing equal volumes of 2×aaRS-free translation mix, pre-incubated at 37°C for 5 minutes, with tRNA. All translation reactions were incubated at 37°C for 2 hours. Translation was terminated by placing the reaction mixture at -20°C before analysis by 17% or 20% tricine-SDS-PAGE to determine the translation yield of the Fph-labeled peptide. 0.125–4 μM peptide standards (Fph-KYDKYD (SEQ ID NO: 125)) custom synthesized by Genscript (Jiangsu, China) were also loaded for calibration. Titration of unloaded tRNA was performed. fMet :tRNA Lys :tRNA Tyr :tRNA Asp The flexizyme-loaded tRNA was mixed with flexizyme-loaded tRNA in a molar ratio of 1:2.5:2.5:2.5, incubated briefly at room temperature, and then added to the aaRS-free translation system. The final concentration of unloaded tRNA was 90–470 μM. Before initiating the translation reaction by mixing with an equal volume of 2× aaRS-free mix, flexizyme titration was performed by mixing dinitro-flexizyme with flexizyme-loaded tRNA in 1 mM NaOAc. The final concentration of flexizyme was 240–520 μM. Titration of the folded flexizyme-tRNA complex was performed with 50 μM dinitro-flexizyme and 6 μM tRNA fMet , each containing 15 μM tRNA Lys tRNA Tyr , and tRNA Asp The mixture was run in a system containing the following components and heated to 95°C for 2 minutes in the presence of 50 mM HEPES-KOH and 100 mM KCl at pH 7.5. After slowly cooling the mix to 25°C, either 100 mM MgCl2 or 10 mM MgCl2 was added and incubated at room temperature for 10 minutes. The mix was then precipitated with ethanol, washed twice with 70% ethanol, and air-dried. The pellet was dissolved in a small amount of ddH2O and serially diluted to reach ×2 concentrations of the flexizyme-tRNA complex, which were 750, 375, and 188 μM, respectively, and then added to an equal volume of aaRS-free translation system along with a control containing only ddH2O.

[0100] MALDI-TOF MS: AaRS-free translation of mRNA #2–#6 was analyzed using MALDI-TOF MS. To reduce peptide dropout, the scale of each loading reaction was adjusted according to the codon abundance in each gene, so that each codon was matched with flexizyme-loaded tRNA at an equal concentration (10 μM per codon for mRNA #2–#5, and 5 μM for mRNA #6). A control containing unloaded tRNA was used, with 30 μM of tRNA (each). Asn tRNA Glu tRNA Lys tRNA Ile The mixture contained 5 μM of other tRNAs and 100 μM of each amino acid. The loading reaction was quenched, precipitated, and washed once with 70% ethanol. The washed pellets of various flexizyme-loaded tRNAs were dissolved in 0.3 M NaOAc, mixed, precipitated again with ethanol, stored at -80°C, and washed once with 70% ethanol before use. AaRS-free translation was performed by mixing flexizyme-loaded tRNAs with an equal volume of 2 × aaRS-free translation mix. After translation at 37°C for 2 hours, TFA was added to the translation system to lower the pH to less than 4, the sample was briefly centrifuged, and the supernatant was desalted using a C18 spin column (Thermo Fisher Scientific, MA, USA). After elution, the sample volume was reduced to approximately 2-3 μl using a centrifugal vacuum concentrator (Eppendorf, Germany), and 0.5 μl of this was used for MALDI-TOF analysis under positive reflectron mode (Applied Biosystems 4800 plus MALDI TOF / TOF analyzer, CA, USA). Control experiments were conducted using unloaded tRNA and free amino acids (100 μM for each amino acid species), desalted, and analyzed in parallel by MALDI-TOF MS. The concentration of unloaded tRNA used in the control experiment was tRNA Asn tRNA Glu tRNA Lys tRNA IleThe concentration for each of the tRNAs was 30 μM, and the concentration for the other tRNAs was 5 μM each.

[0101] Protein identification by LC-MS / MS: Crude aaRS-free, translated N-terminal FAM-labeled E. coli TrpRS from 20 μl was isolated by 15% SDS-PAGE and silver-stained with the ProteoSilver silver staining kit (Sigma-Aldrich, MO, USA). The protein band between EF-Tu (43 kDa) and MTF (34 kDa) was excised from the gel, reduced with 5 mM dithiothreitol, and alkylated with 11 mM iodoacetamide. Intragel digestion was performed overnight at 37°C using sequencing-grade trypsin in 50 mM ammonium bicarbonate. The peptide was extracted twice with 0.1% TFA in 50% acetonitrile aqueous solution for 30 minutes. The extract was then concentrated using a centrifugal vacuum concentrator. The peptide produced by trypsin digestion was dissolved in 20 μl of 0.1% TFA and analyzed by LC-MS / MS. Control experiments with unloaded tRNA and free amino acids using the above concentrations were performed in parallel and analyzed.

[0102] Enzymatic transcription of enantiomer tRNA: The synthesis and folding of D-polymerase D-Dpo4-5m-Y12S for mirror-image transfer has been previously reported in [Jiang, W., Zhang, B., Fan, C., Wang, M., Wang, J., Deng, Q., Liu, X., Chen, J., Zheng, J., Liu, L., et al. (2017). Mirror-image polymerase chain reaction. Cell Discov. 3, 17037; Xu, W., Jiang, W., Wang, J., Yu, L., Chen, J., Liu, X., Liu, L., and Zhu, TF (2017). Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction. Cell]. Discov.3,17008;Wang,M.,Jiang,W.,Liu,X.,Wang,J.,Zhang,B.,Fan,C.,Liu,L.,Pena-Alcantara,G.,Ling,J.-J.,Chen,J.,et al.(2019).Mirror-image gene transcription and reverse transcription.Chem 5,848-857]. All L-DNA primers, template sequences, and L-nucleotide oligo sequences are listed in Table 4. In the table, "*" indicates phosphorothioate modification, uppercase letters indicate L-DNA nucleotides, and lowercase letters indicate L-RNA nucleotides.

[0103] [Table 4]

[0104] Mirror image transcription was performed using a tethered 24nt primer binding site attached to the 3' end of a mirror image single-stranded DNA (L-ssDNA) template, facilitating RNA purification by PAGE due to the resulting difference in product length (the L-RNA transcript being 23 nucleotides shorter than the 99-nucleotide L-ssDNA template, which can be separated by 12% denatured PAGE with 7M urea). The L-primer was designed to contain a phosphorothioate-modified L-RNA nucleotide at its 3' end. Mirror image tRNA Ala tRNA Gly tRNA Lys and tRNA Phe Enzymatic transcription was performed. After enantiomer transcription, the L-primer was efficiently cleaved at the phosphorothioate site with 100 μM I2 in ethanol at 70°C for 10 minutes, yielding a mature enantiomer tRNA transcript. For RNase A digestion, 0.4 μM D- or L-tRNA was added. Ala The mixture was mixed with 4 μM RNase A, incubated at 37°C for 15 minutes, and analyzed by 10% denatured PAGE with 7 M urea. 5 μM D- or L-tRNA was added for aaRS-catalyzed aminoacylation. Ala The mixture was mixed with 1 μM AlaRS in the presence of 10 mM ATP and 100 μM L- or D-alanine, incubated at 37°C for 1 hour, and analyzed by 8% acidic PAGE.

[0105] Enantiomer-tRNA cargo: Enantiomer tRNA loading was performed using the same aminoacylation method as above, except that the L-tRNA concentration and L-flexizyme concentration were scaled down to 2 μM and 10 μM, respectively. Enantiomer tRNA was transcribed using synthetic D-Dpo4-5m-Y12S polymerase, and native tRNA was transcribed using the recombinant Y12S mutant of Dpo4 (L-Dpo4-5m-Y12S) (SEQ ID NO: 126) (tRNA Ala tRNA Gly and tRNA Lys ) or T7 RNA polymerase (tRNA PheThe tRNA was synthesized using one of the following methods. The tRNA loading yield was determined using the ImageJ software package, using the integrated peak area of ​​loaded tRNA relative to total tRNA.

[0106] Example 2 Flexizyme-catalyzed tRNA aminoacylation: Twenty-one tRNAs were individually loaded with the corresponding (cognate) amino acids using either a 46-nucleotide dinitro-flexizyme or a 45-nucleotide augmented flexizyme. The loading reaction was quenched with 0.3 M NaOAc and precipitated. The pellet was purified by washing with 70% ethanol or by a Shimadzu Prominence HPLC system (Japan) to the appropriate degree (see Figures 8A and 8B).

[0107] Figures 3A to 3E illustrate the concept and results of flexizyme-catalyzed loading onto tRNA, which is on the way to aaRS-free loading onto enantiomer tRNA, according to several embodiments of the present invention, including D-tRNA loading catalyzed by D-flexizyme and its enantiomer version, i.e., enantiomer tRNA loading catalyzed by L-flexizyme (PDB source: 1EHZ(tRNA), 3CUL(flexizyme)) (Figure 3A), loading of D-alanine onto enzymatically transcribed enantiomer tRNAAla by L-flexizyme, and natural chirality Figure 3B shows the loading of glycine onto enzymatically transcribed enantiomer tRNAGly by L-flexizyme, with the natural chirality equivalent also shown for comparison (Figure 3C), the loading of D-lysine onto enzymatically transcribed enantiomer tRNALys by L-flexizyme, with the natural chirality equivalent also shown for comparison (Figure 3D), and the loading of D-phenylalanine onto enzymatically transcribed enantiomer tRNAPhe by L-flexizyme, with the natural chirality equivalent also shown for comparison (Figure 3E). The tRNA loading yield was determined using the integrated peak area of ​​loaded tRNA relative to total tRNA using the ImageJ software package.

[0108] Symmetry Shield RP18 columns (3.5 μm, 4.6 × 150 mm and 3.5 μm, 4.6 × 100 mm) (Waters Corp, MA, USA) were used for HPLC purification, and the elution conditions were adopted from the literature [Zhang, J., and Ferre-D'Amare, Adrian R. (2014). Direct evaluation of tRNA aminoacylation status by the T-Box riboswitch using tRNA-mRNA stacking and steric readout. Mol. Cell 55, 148-155]. The fraction containing flexizyme-loaded tRNA was precipitated and dissolved in 10 mM NaOAc at pH 5.2, and the concentration was measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, MA, USA). The desired amount of tRNA was then mixed in and precipitated again with ethanol. The pellet was air-dried and stored at -80°C until use.

[0109] Example 3 Cell-free in vitro translation Cell-free in vitro translation mixes were prepared according to a previously reported method [Terasaka, N., Hayashi, G., Katoh, T., and Suga, H. (2014). An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Nat. Chem. Biol. 10, 555-557] with the following modifications: Recombinant IF1, IF2, IF3, EF-Ts, EF-Tu, EF-G, RF-2, RF-3, RRF, and MTF proteins were expressed in E. coli BL21 strain with N-terminal TEV-cleavable His tags. The mixtures were purified by Ni-NTA Superflow resin (Senhui Microsphere Tech., Suzhou, China), cleaved by TEV protease (Sigma-Aldrich, MO, USA), further purified by ion-exchange chromatography, and refracted at 50 mM at pH 7.6. The samples were replaced with a buffer containing HEPES, 100 mM potassium glutamate, 10 mM magnesium acetate, 7 mM β-mercaptoethanol, and 30% glycerol. The buffer and small molecule components were prepared as described in the literature [Goto, Y., Katoh, T., and Suga, H. (2011). Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779-790]. aaRS-free E. coli ribosomes were purchased from New England Biolabs (MA, USA).

[0110] Example 4 Protein enzyme aaRS free translation 20-codon DNA templates for chicken lysozyme, Gaussia luciferase, and E. coli TrpRS were synthesized and assembled by Genewiz (Jiangsu, China) and cloned into the pUC-57 vector. Table 5 shows the DNA template sequences for aaRS-free translation of chicken lysozyme, Gaussia luciferase, and E. coli TrpRS.

[0111] [Table 5]

[0112] DNA plasmids were double-digested and purified by 1% agarose gel before use. After recovery from -80°C, the dried flexizyme-loaded tRNA pellet was washed twice with 70% ethanol and dissolved in 10-20 μl of 1 mM NaOAc at pH 5.2. The lysed tRNA mix was then added to an aaRS-free translation mix pre-incubated at 37°C for 5 minutes, where the final DNA template concentration was approximately 10 ng / μl. For lysozyme and luciferase translation, approximately 1 μM of flexizyme-loaded tRNA was used for each codon to be translated. For TrpRS translation, approximately 1 μM of flexizyme-loaded FAM-labeled Fph-tRNA was used. fMet Approximately 0.4 μM of flexizyme-loaded tRNA was used for each of the Cys and Pro codons, and approximately 0.2 μM of flexizyme-loaded tRNA was used for each of the remaining codons (total tRNA concentrations are shown in Table 3 above). Control experiments without a DNA template were performed using the same flexizyme-loaded tRNA concentrations, but control experiments using unloaded tRNA were performed using tRNA Asn tRNA Glu tRNA Lys tRNA Ile30 μM was used for each of the following: 5 μM for the other tRNAs, and 100 μM for the free amino acids. Translation reactions were incubated at 37°C for 2 hours for lysozyme and luciferase, and for 4 hours for TrpRS. For analysis by 15% SDS-PAGE, 10 μl aliquots were sampled from the translation reaction products, mixed with 2 μl of 6× protein loading dye, and heated at 98°C for 3 minutes for loading. Alexa Fluor 488-labeled Benchmark fluorescent protein standards were purchased from Thermo Fisher Scientific (MA, USA). The gels were scanned using a Typhoon FLA 9500 (GE Healthcare, UK) operating in Cy2 mode.

[0113] Example 5 Biochemical characterization of aaRS-free translated protein enzymes aaRS-free translation of chicken lysozyme, Gaussia luciferase, and E. coli TrpRS using Met-tRNA fMetThe analysis was performed using the following method. The chicken lysozyme translation mix was diluted with an equal volume of 2× folding buffer (pH 7.5) containing 0.1M sodium phosphate and 0.1M NaCl, incubated at room temperature for 24 hours, and assayed using the EnzChek Lysozyme Assay Kit (Thermo Fisher Scientific, MA, USA). The translation mix for Gaussia luciferase has been previously shown to promote disulfide bond formation in recombinant Gaussia luciferase [Yu, T., Laird, JR, Presser, JA, and Thorpe, C. (2018). Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding. Protein Science 27, 1509-1517]. The mixture was diluted in equivolute 2× folding buffer (pH 7.3) containing 6 mM reduced glutathione and 4 mM oxidized glutathione, incubated at room temperature for 16 hours, and assayed using the Pierce Gaussia Luciferase Glow Assay Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. Cy5-tRNA was used for translation of E. coli TrpRS. Trp The tRNA was prepared by enzymatic ligation of two synthetic oligonucleotides and purified by 10% denatured PAGE as described in the literature [Suddala, KC, Cabello-Villegas, J., Michnicka, M., Marshall, C., Nikonowicz, EP, and Walter, NG (2018). Hierarchical mechanism of amino acid sensing by the T-box riboswitch. Nat.Commun. 9, 1896]. Table 6 shows the internally Cy5-labeled tRNA. Trp This shows the RNA oligo sequence for enzymatic ligation.

[0114] [Table 6]

[0115] 2 μM Cy5-tRNA Trp A mixture of 250 μM tryptophan and 1 mM ATP was added to the reaction mixture after translation completion, incubated at 37°C for 1 hour, quenched with 0.3 M NaOAc, and extracted with phenol-chloroform. The loaded samples were analyzed by 8% acidic PAGE (Supplementary Information) and scanned with a Typhoon FLA 9500 operating in Cy5 mode. 2 μM unloaded Cy5-tRNA Trp , and 2 μM Cy5-tRNA loaded by 100 nM recombinant E. coli TrpRS Trp The sample was used as the standard. All control experiments, using DNA-lacking or unloaded tRNA and free amino acids, were assayed under the same conditions.

[0116] Example 6 Maximizing the yield of aaRS-free translation To address the apparent low yield problem reported in the literature [Terasaka, N., Hayashi, G., Katoh, T., and Suga, H. (2014). An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Nat. Chem. Biol. 10, 555-557], we studied an aaRS-free translation system. The rationale was that increasing the concentration of flexizyme-loaded tRNA to compensate for the lack of tRNA reuse might improve the yield of aaRS-free translation. Previous research showed that the addition of excessive tRNA inhibits translation in an E. coli (E. coli) translation system using aaRS [Rojiani, MV, Jakubowski, H., and Goldman, E. (1990). Relationship between protein synthesis and concentrations of charged and uncharged tRNA]. Trp[in Escherichia coli. Proc. Natl Acad. Sci. USA 87, 1511; Anderson, WF (1969). The effect of tRNA concentration on the rate of protein synthesis. Proc. Natl Acad. Sci. USA 62, 566]. This was attributed to unloaded tRNA overcoming loaded tRNA by occupying the ribosome A site, or to cation imbalance caused by the addition of large amounts of tRNA. However, since all these experiments were performed in the presence of aaRS, the exact loading yield could not be determined, and inefficient loading and altered cations (e.g., Mg) could be considered. 2+ It was difficult to identify the effect of the concentration.

[0117] To evaluate the effects of tRNA concentration and cargo yield on aaRS-free translation, fluorescein (FAM)-labeled phenylalanine (Fph-tRNA) was used. fMet We performed aaRS-free translation of short peptides using (see Figures 4A-4B, 5A-5D, and 6A-6E) to facilitate the quantification of translation yield.

[0118] Figures 4A to 4G show the results of aaRS-free translation of several short peptides according to several embodiments of the present invention. Shown are MALDI-TOF-MS analysis of short peptides translated from mRNA #1 (Figure 4A), aaRS-free translation yield of short peptides analyzed by trichine-SDS-PAGE, where the unloaded tRNA concentration was in the range of 160-540 μM and the flexizyme-loaded tRNA concentration was 70 μM, resulting in a loading yield in the range of 44-13% (top of Figure 4B), and the total tRNA concentration was in the range of 16-1003 μM, with the loading yield remaining at 56% (Figure 4 (Bottom of B) (Error bars represent the standard deviation from three independent experiments), MALDI-TOF-MS analysis of translated short peptides from mRNA#2 (Figure 4C), mRNA#3 (Figure 4D), mRNA#4 (Figure 4E), mRNA#5 (Figure 4F), and mRNA#6 (Figure 4G), where 55–135 μM of unloaded tRNA and 100 μM of each amino acid were used as translational controls (for mRNA#1, FAM-labeled Fph-tRNA loaded with 5 μM of flexizyme). fMet (This was added to both the control experiment and the aaRS-free translation experiment), and aaRS-free translation was also performed using 170-414 μM total flexizyme-loaded tRNA (see Table 3). au represents an arbitrary unit, and C and O represent the calculated and observed m / z values ​​(Figures 4C to 4G, respectively).

[0119] mRNA template to tRNA fMet tRNA Lys tRNA Tyr and tRNA Asp Decoded by, and among them the tRNA fMet It is enhanced by a 45-nucleotide flexizyme that enhances Fph-tRNA fMetOne tRNA was loaded, while others were loaded with their corresponding (cognate) amino acids via a 46-nucleotide dinitro-flexizyme [Murakami, H., Ohta, A., Ashigai, H., and Suga, H. (2006). A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat. Methods 3, 357]. Unmodified tRNAs transcribed in vitro by T7 RNA polymerase were used because they have been shown to function in ribosomal peptide synthesis assays. Individual loading yields for each tRNA were determined by polyacrylamide gel electrophoresis under acidic conditions (acidic PAGE), and this was used to estimate the weighted average (overall) loading yield of the translation system (see Table 3). Titration of the tRNAs was performed, and the total loading yield was approximately 44% (Fph-tRNAs mixed in a molar ratio of 1:2:2:2). fMet :Lys-tRNA Lys :Tyr-tRNA Tyr :Asp-tRNA Asp Initially, when we added loaded total tRNA with a total tRNA concentration of 20–644 μM in the final translation system, we found that the translation yield reached its highest level at a total tRNA concentration of approximately 160 μM and then decreased as the tRNA concentration increased further without plateauing (see Figure 5A).

[0120] Figures 5A to 5D show the results of aaRS-free translation of mRNA #1 under various conditions. It is shown that the cargo yield remained at 44% when the total tRNA concentration was in the range of 20–644 μM (Figure 5A), the total tRNA concentration remained at 160 μM when the total flexizyme concentration was in the range of 240–525 μM (Figure 5B), and that flexizyme and 0–380 μM of unloaded tRNA were mixed in 10 mM MgCl2 (in Figure 5C) and 100 mM MgCl2 (in Figure 5D), desalted by ethanol precipitation, and added to the aaRS-free translation mix, while the concentration of flexizyme-loaded tRNA remained at 70 μM (error bars represent the standard deviation from three independent experiments).

[0121] Figures 6A to 6 E The figure shows the tricine-SDS-PAGE gel analysis for calculating the aaRS-free translation yield, and the corresponding gel images (Figures 6A-6E, respectively) for calculating the aaRS-free translation yield are shown in Figures 4A, 4B, 5A, 5B, 5C, and 5D. "M" represents the synthetic peptide standard (Fph-KYDKYD).

[0122] The observed inhibition was not due to the accumulation of flexizymes in the translation system, because in the control experiment, adding purified dinitro-flexizymes to a fixed amount of total tRNA did not inhibit translation (see Figure 5B).

[0123] Next, when 90–470 μM of unloaded tRNA was added to the aaRS-free translation system in the presence of 70 μM of loaded tRNA, the overall loading yield decreased from 44% to 13%, but the overall translation yield was hardly affected (see Figure 4B).

[0124] Another factor that may cause observed translation inhibition is Mg from flexizyme-loaded tRNA. 2+It was hypothesized that this was due to an increase in cation concentration caused by carryover. To test this theory, exogenous MgCl2 was added to flexizyme-loaded tRNA before adding it to the aaRS-free translation system, and it was found that translation was indeed inhibited by an increase in MgCl2 carryover (see Figures 5C and 5D).

[0125] Based on the above observations, flexizyme-loaded tRNA was purified using high-performance liquid chromatography (HPLC) equipped with a C18 column and then Mg 2+ The concentration of Fph-tRNA was reduced (treated by ultrafiltration instead to minimize fluorescence quenching). fMet (Excluding [specific components]). This process also removed most of the flexizymes and slightly improved the overall cargo yield from 44% to 56% (see Figure 2).

[0126] Subsequently, the purified flexizyme-loaded tRNA was added to the aaRS-free translation system, and it was observed that concentrating only the flexizyme-loaded tRNA significantly improved the translation yield by 5 times. HPLC revealed Mg 2+ Reducing the contamination level resulted in a further twofold improvement, leading to an overall improvement of approximately 10 times in translation yield (see Figures 4B and 5A), and the optimal total tRNA concentration shifted from 160 μM to 500 μM.

[0127] However, when the tRNA concentration was further increased from 500 μM to 1000 μM, the overall translation yield decreased by approximately 50%, and this is due to Fph-tRNA fMet Mg associated with 2+ This may be due to the following. Furthermore, similar titration assays using high concentrations of unloaded tRNA in the presence of aaRS did not result in improved translation yield (see Figures 7A-7B), suggesting that the improvement in aaRS-free translation yield is likely due to the increased concentration of flexizyme-loaded tRNA itself.

[0128] Figures 7A - 7B show the results of in vitro translation experiments in the presence of LysRS, TyrRS, and AspRS, showing translation products when using uncharged, unmodified total tRNA concentrations ranging from 22 - 680 μM in the presence of LysRS, TyrRS, and AspRS, and tricine - SDS - PAGE analysis (Figure 7A) and calculated translation yields (Figure 7B) of Fph - tRNAfMet pre - charged by an enhanced flexizyme (error bars represent standard deviation from three independent experiments).

[0129] Example 7

[0130] aaRS - free translation of multiple short peptides Since it was discovered that increasing the tRNA concentration improves the yield of aaRS - free translation, the inventors of the present application tested aaRS - free translation for multiple short peptides and attempted to determine the translation fidelity at high flexizyme - loaded tRNA concentrations. A minimum set of 21 E. coli tRNAs, including one tRNA (tRNA fMet ) for translation initiation and 20 other tRNAs for translation elongation, was obtained by in vitro transcription with T7 RNA polymerase. Table 7 shows the relevant tRNA sequences.

[0131]

Table 7 - 1

Table 7 - 2

[0132] Each tRNA was separately charged by the flexizyme, and the charging yields were in the range of 20 - 60% (see Figure 8A). Figures 8A-8B show the charging yields of cognate protein-constituting amino acids to 21 types of tRNAs by their corresponding flexizymes, the charging yields determined after ethanol precipitation (Figure 8A), and the charging yields determined after HPLC purification of 14 types of flexizyme-charged tRNAs. N / A indicates those for which the purification of flexizyme-charged tRNAs was not performed (Figure 8B). For promoting purification as reported previously, reversible N-pentenoylation was performed on gly-tRNA Gly with respect to it.

[0133] Flexizyme-charged tRNAs were mixed at a molar ratio according to the abundance of their corresponding codons on mRNA and then added to the aaRS-free translation system to a final concentration in the range of 170-520 μM (Table 3). The inventors of the present application designed five different mRNA sequences capable of forming Watson-Crick base pairs with the anticodons of flexizyme-charged tRNAs, transcriptionally transcribed them in vitro, and evaluated the translation fidelity by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for aaRS-free translated short peptides (see Figures 4C-4G).

[0134] The results of MALDI-TOF MS showed that all 21 types of flexizyme-charged tRNAs accurately decoded mRNA up to about 200-fold molar excess relative to ribosomes (for example, in the case of mRNA#5, 2 μM of ribosomes relative to 414 μM of tRNA). In control experiments using uncharged tRNAs and free amino acids (see Figures 4C-4G), no peptide products were detected, and thus concerns about contamination of aaRS and charged tRNAs from ribosome preparations were minimized.

[0135] In particular, the inventors of the present application encoded the short message "MITRNACHARGINGSYSTEM" (SEQ ID NO: 123) into mRNA#6 (see Figure 4G) and successfully translated the full length of the information-carrying peptide.

[0136] However, when the total tRNA concentration was increased to 520 μM, an additional +12Da peak was detected (see Figure 9). This may be due to mRNA misdecoding resulting from the high tRNA concentration and the use of unmodified tRNA for translation.

[0137] Figure 9 shows the MALDI-TOF MS analysis of aaRS-free translated mRNA #6. It indicates that in the aaRS-free translation system, when the total tRNA concentration increased (520 μM), a mistranslated product with a molecular weight of 2,252.7 Da was observed, while the correctly translated product had a molecular weight of 2,240.7 Da. 'au' represents an arbitrary unit, and C and O represent the calculated and observed m / z values, respectively.

[0138] Example 8 Protein enzyme aaRS free translation Successful translation of short peptides led to aaRS-free translation of protein enzymes using only in vitro transcribed, unmodified tRNA loaded by the Flexizyme system. Two small enzymes, chicken lysozyme (130 amino acid residues) and Gaussia luciferase (169 amino acid residues), were selected as models. Neither enzyme is present in E. coli itself, which minimizes concerns about contamination from ribosome preparations.

[0139] Figures 10A–10C show the amino acid sequences of aaRS-free translated protein enzymes: chicken lysozyme (Figure 10A), Gaussia luciferase (Figure 10B), and E. coli TrpRS (Figure 10C). The position translated by flexizyme-loaded tRNA was purified by either ethanol precipitation or HPLC (underlined).

[0140] A subset consisting of 21 types of flexizyme-loaded tRNAs (underlined amino acids in Figures 10A-10B) was purified by HPLC and then Mg 2+Carryover was reduced, and the yield of individual cargoes after HPLC purification was measured by acidic PAGE (see Figure 8B). The overall cargo yield was approximately 40%. The total tRNA concentrations of approximately 330 μM for chicken lysozyme and approximately 430 μM for Gaussia luciferase (Table 3) were about 10 to 20 times higher than those used in other in vitro translation systems [Terasaka, N., Hayashi, G., Katoh, T., and Suga, H. (2014). An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center. Nat. Chem. Biol. 10, 555-557; Iwane, Y., Hitomi, A., Murakami, H., Katoh, T., Goto, Y., and Suga, H. (2016). Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes. Nat. Chem. 8, 317-325; and Cui, Z., Stein, V., Tnimov, Z., Mureev, S., and Alexandrov, K. (2015). Semisynthetic tRNA complement mediates in vitro protein synthesis. J. Am. Chem. Soc. 137, 4404-4413].

[0141] Full-length protein aaRS-free translation using FAM-labeled Fph-tRNA fMet The tests were performed using reporters. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fluorescently labeled product bands were consistent with the molecular weights of chicken lysozyme and Gaussia luciferase (14.8 kDa and 18.7 kDa, respectively), and the mobility of the product bands was similar to that of commercially available chicken lysozyme and recombinant Gaussia luciferase, respectively (see Figures 11A-11D).

[0142] Figures 11A to 11G show the SDS-PAGE analysis of aaRS-free translated protein enzymes. Figure 12A shows the overall gel image (Figure 11A), a sample of 400 ng of commercially available chicken lysozyme purified from chicken egg white, analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue (Figure 11B), a sample of 400 ng of recombinant Gaussia luciferase expressed and purified from E. coli strain BL21, analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue (Figure 11D), a sample of recombinant E. coli TrpRS expressed and purified from E. coli strain BL21, analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue. Figure 14A shows the overall gel image (Figure 11E), a sample of recombinant E. coli TrpRS expressed and purified from E. coli strain BL21, analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue. 300 ng of sample (Figure 11F), heated to 98°C for 3 minutes or without heating, was analyzed by 15% SDS-PAGE and scanned with a Typhoon FLA 9500 under Cy2 mode, detecting 5 μM Fph-CME and 1 μM Fph-tRNA. fMet , and 5 μM Fph-tRNA fMet The sample (Figure 11G) is shown, where M is the benchmark fluorescent protein standard.

[0143] On the other hand, no product band was observed in control experiments lacking a DNA template, or in control experiments using unloaded tRNA and free amino acids (see Figures 12A and 12C).

[0144] Figures 12A to 12D show the results of experimental demonstrations of the concept of aaRS-free translation of protein enzymes according to several embodiments of the present invention, aaRS-free translation of N-terminal FAM-labeled chicken lysozyme (M represents a benchmark fluorescent protein standard) (Figure 12A), analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode, enzyme assay of crude aaRS-free translated chicken lysozyme using fluorescently labeled bacterial (Micrococcus rhizodiccus) cell wall material as substrate (Figure 12B), analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode Figure 12C shows aaRS-free translation of N-terminal FAM-labeled Gaussia luciferase scanned by 9500, and Figure 12D shows an enzyme assay of crude aaRS-free translated Gaussia luciferase using coelenterazine as a substrate (RFU represents relative fluorescence units, and RLU represents relative emission units).

[0145] These results suggest that aaRS-free translation is sufficiently progressive to achieve full-length protein synthesis before the flexizyme-loaded tRNA is hydrolyzed. Attempts to characterize the aaRS-free translated protein from the cleaved product band using liquid chromatography-tandem mass spectrometry (LC-MS / MS) were unsuccessful due to contamination by ribosomal proteins. However, this was addressed by translating a larger protein, whose molecular weight is even more different from that of the ribosomal protein, as described in this disclosure. Next, FAM-labeled Fph-tRNA was used for translation initiation. fMet Unlabeled Met-tRNA fMetIt was replaced and an enzyme assay was performed to test whether the translated protein could fold correctly in vitro and have its corresponding catalytic activity. The results showed that after incubation for up to 24 hours in folding buffer, the aaRS-free translated enzymes catalyzed their corresponding substrates: chicken lysozyme released FAM-labeled cell debris and Gaussia luciferase emitted bioluminescence (see Figures 12B and 12D), while control experiments without a DNA template or using uncharged tRNA and free amino acids did not produce a detectable signal, thus minimizing concerns about autofluorescence or contaminating luminescence from the aaRS-free translation system.

[0146] Comparing the emitted bioluminescence of aaRS-free translated Gaussia luciferase with the known standard of recombinant luciferase suggested a translation yield of approximately 25 nM (see Figure 13), which was approximately 80-fold lower than the maximum yield of aaRS-free translation of a 7-amino acid residue peptide (see Figure 4B), probably as a result of the low availability of flexizyme-charged tRNA for each translation codon and the limited folding efficiency of Gaussia luciferase with multiple disulfide bonds.

[0147] Figure 13 shows the estimated yields of aaRS-free translated Gaussia luciferase, with a standard curve plotted using recombinant Gaussia luciferase at 0, 25 nM, 50 nM, 100 nM, and 250 nM (shown as squares), and the yield of translated Gaussia luciferase was estimated to be approximately 25 nM (shown as triangles).

[0148] Example 9 aaRS-free translation of aaRS The inventor of the present application attempted to explore the possibility of an aaRS-free translation system for producing a functional aaRS itself. This is an important step in establishing a self-replicating translation apparatus. For this purpose, Escherichia coli TrpRS of 334 amino acid residues was used as a model. Most of the in vitro transcribed flexizyme-loaded tRNAs (14 out of a total of 21 species) were purified by HPLC to reduce the carryover of Mg 2+ (underlined amino acids in Figure 10A), and an overall loading yield of about 42% and a total tRNA concentration of about 170 μM were obtained (see Table 3).

[0149] The inventor of the present application tested the translation of full-length proteins using a FAM-labeled Fph-tRNA fMet reporter, and a product band representing Escherichia coli TrpRS of 334 amino acid residues (37.8 kDa) was observed by SDS-PAGE (the mobility of the fluorescently labeled protein band was similar to that of recombinant TrpRS) (see Figures 11E and 11F), but this band was not present in control experiments lacking a DNA template or using unloaded tRNA and free amino acids (see Figure 14A).

[0150] Figures 14A to 14C show the aaRS-free translation of TrpRS, analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 in Cy2 mode for the aaRS-free translation of N-terminal FAM-labeled Escherichia coli TrpRS (M represents the benchmark fluorescent protein standard) (Figure 14A), the sequence and secondary structure of internally Cy5-labeled tRNA Trp (Figure 14B), and the enzyme assay of the crude aaRS-free translated TrpRS using Cy5-tRNA Trp as a substrate, analyzed by 8% acidic PAGE and scanned by Typhoon FLA 9500 in Cy5 mode (Figure 14C).

[0151] Also, some faster-moving bands were observed, which may correspond to truncated TrpRS translation products and unused Fph-tRNA fMet (see Figure 11G).

[0152] To further confirm aaRS-free translation of TrpRS, protein content from excised product bands was analyzed using LC-MS / MS, identifying four non-overlapping peptide segments from E. coli TrpRS, resulting in approximately 16% sequence coverage. In contrast, control experiments using unloaded tRNA and free amino acids did not detect peptides corresponding to E. coli TrpRS, suggesting that the detected TrpRS did not originate from endogenous aaRS contamination (Table 8).

[0153] Table 8 shows the peptide sequences detected by LC-MS / MS from aaRS-free translated E. coli TrpRS (aaRS-free translation using a DNA template for E. coli TrpRS).

[0154] [Table 8]

[0155] To further test the tRNA-aminoacylation activity of aaRS-free translated TrpRS, an internal Cy5-labeled tRNA substrate (Cy5-tRNA) was used. Trp (See Figure 14B) was designed and synthesized.

[0156] The placement of Cy5 labeling allows for the removal of loaded Cy5-tRNA without interference from other loaded tRNA species. Trp Enables in-situ detection. Met-tRNA for translation initiation. fMet Using this method, after TrpRS is translated, Cy5-tRNA Trp TrpRS was added to the aaRS-free translation system along with tryptophan and adenosine triphosphate (ATP). The aaRS-free translated TrpRS was converted to Cy5-tRNA. Trp In contrast to the successful loading of tryptophan onto the above, control experiments using tRNA lacking a DNA template or unloaded tRNA and free amino acids showed that Cy5-tRNA TrpNo cargo was observed (see Figure 14C). This is because the observed Cy5-tRNA Trp This suggests that the cargo activity is unlikely to be due to endogenous aaRS contamination from ribosome preparations or residual flexizyme activity.

[0157] Example 10 aaRS-free cargo onto enantiomer tRNA As a proof-of-concept experiment to test the loading of mirror image D-amino acids onto mirror image L-tRNAs by a synthetic mirror image L-flexizyme (see Figure 3A), the inventors transcribed mirror image tRNAs by applying a previously established mirror image gene transcription system based on a mirror image version of a designed mutant of Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) (see Figure 15A).

[0158] Figures 15A and 15B show mirror-image tRNAs created by D-Dpo4-5m-Y12S. Lys The transcription results are shown, including the extension of a 5'-FAM-labeled L-universal primer on an L-ssDNA template polymerized by synthetic D-Dpo4-5m-Y12S polymerase, and reaction aliquots terminated at different time points and analyzed by 12% denatured PAGE gel with 7M urea (Figure 15A), and tRNA stained with SYBR-Green II (Thermo Fisher Scientific, MA, US) and analyzed by 10% denatured PAGE gel with 7M urea. Lys The mirror image transfer and I2-mediated cleavage of the transfer material are shown (Figure 15B).

[0159] To avoid the high cost of synthesizing 21 different L-RNA primers, the inventors of this application applied a universal primer for the transcription of enantiomer tRNA (see Figure 15B).

[0160] The universal primer was modified with a phosphorothioate near its 3' end so that the fully extended primer would be efficiently cleaved by I2 via a previously reported mechanism to produce full-length enantiomer tRNA (see Figure 15B). As expected, this full-length enantiomer tRNA was resistant to digestion by native RNase A and could not be loaded by native aaRS (see Figures 16A-16B).

[0161] Figures 16A and 16B show the results of biochemical characterization of enzymatically transcribed native tRNA and enantiomer tRNA, specifically D- and L-tRNA. Ala RNase A digestion (Figure 16A), and enzymatically transcribed D- and L-tRNA Ala AaRS-catalyzed aminoacylation is shown (Figure 16B).

[0162] I2-mediated cleavage produces RNA with hydroxyl-terminal 5'-terminus, as verified by MALDI-TOF MS (see Figures 17A-17C). Figures 17A to 17C show MALDI-TOF MS analysis of I2-mediated cleavage. Figure 17A shows the MALDI-TOF MS spectrum of a synthetic DNA-RNA chimeric oligo cleaved by I2 at the phosphorothioate modification site, Figure 17B shows the MALDI-TOF MS spectrum of the uncleaved oligo under negative linear mode, and Figure 17C shows the MALDI-TOF MS spectrum of the oligo cleaved by I2 under negative linear mode (m / z > 4000) and negative reflectron mode (m / z < 4000). Uppercase letters indicate DNA nucleotides, lowercase letters indicate RNA nucleotides, and "*" indicates phosphorothioate modification. au represents an arbitrary unit, and C and O represent the calculated m / z value and observed m / z value, respectively.

[0163] Next, using a chemically synthesized 46-nucleotide L-flexizyme (dinitro-flexizyme), we successfully loaded four representative D-amino acids (lysine, alanine, glycine, and phenylalanine) belonging to different amino acid categories (polar (Lys), nonpolar (Ala), achiral (Gly), and aromatic (Phe)) onto their corresponding cognate enantiomer tRNAs with efficiency comparable to that of the natural system (Figures 3B-3E).

[0164] Example 11 Translation of complete or partial non-natural peptides using cation-depleted flexizyme-loaded tRNA. It is inferred that the flexizyme system can be used to incorporate multiple non-natural amino acids for peptide translation, and this can be used in combination with or without other aaRS proteins. The provisions of the present invention make it possible to test whether non-natural peptides can be translated using cation-depleted flexizyme-loaded tRNA, or at least using a preparation of flexizyme-loaded tRNA in which the Mg+2 concentration has been essentially reduced to the minimum possible level, and whether purification by means such as HPLC and ultrafiltration, and enrichment of cation-depleted flexizyme-loaded tRNA can increase the translation yield, particularly for difficult-to-translate peptides, such as complete or partial non-natural peptides. In the translation system, aaRS proteins may be added to enhance the loading of specific tRNAs not loaded by flexizyme (see Figures 18A-18B).

[0165] Figures 18A and 18B show flowchart translations of complete or partial non-natural peptides using cation-depleted flexizyme-loaded tRNA, illustrating the translation of peptide drugs and non-natural proteins using cation-depleted flexizyme-loaded tRNA in an in vitro translation system (see Figure 18A), and the translation, data storage, and ribosome / mRNA display of complete or partial non-natural proteins using cation-depleted flexizyme-loaded tRNA in an in vitro translation system (see Figure 18B).

[0166] In this experiment, non-natural amino acids are first loaded onto unmodified tRNA. Non-natural amino acids may include, but are not limited to, D-amino acids and β-amino acids, such as D-Phe, D-His, D-Cys, D-Ala, D-Ser, D-Met, D-Thr, D-Tyr, N-chloroacetyl-D-Tyr, D-Trp, N-chloroacetyl-D-Trp, L-β-homomethionine (β-hMet), L-β-homoglutamine (β-hGln), L-β-homophenylglycine (β-hPhg), 2-aminocyclohexanecarboxylic acid (2-ACHC), or 2-aminocyclopentanecarboxylic acid (2-ACPC). The flexizyme-loaded tRNA is purified by techniques to reduce cation contamination, including but not limited to HPLC. Other purification techniques may include ultrafiltration and dialysis. Then, the flexizyme-loaded tRNA is concentrated to 100-500 μM and used as a substrate for in vitro translation. The translation product is analyzed by MALDI-TOF MS and tricine-SDS-PAGE.

[0167] As a proof of concept, the peptide drug is translated using the aaRS-free translation system. (See Figure 18A) The amino acid sequence of peptide drugs is, AcThe peptide is yFAYDRR(2-ACHC)LSNN(2-ACHC)RNYcG-NH2 (SEQ ID NO: 124), where the first amino acid is acetyl-D-Tyr and the second to last amino acid is D-Cys, which spontaneously forms a cyclic bond with the acetyl-D-Tyr residue. This peptide has been previously shown to inhibit human factor XIIa. The translation product is analyzed by MALDI-TOF MS.

[0168] As another proof of concept, protein enzymes such as Gaussia luciferase, which consists of 169 amino acid residues, are tRNA Asn tRNA Ile and tRNA Lys Translation is performed using cation-depleted flexizyme-loaded tRNAs, including but not limited to these (see Figure 18A). Other tRNAs are loaded by recombinant aaRS proteins. Furthermore, the non-natural amino acid fluorescein-labeled phenylalanine (Fph) is translated by flexizyme into initiator tRNAs. fMet The samples are loaded onto a transceiver. The translation products are analyzed by measuring bioluminescence and SDS-PAGE. Since the Fph residue causes Gaussia luciferase to fluoresce on the SDS-PAGE gel, the purity of the Gaussia luciferase thus translated can be easily determined based on its fluorescence. This application is useful for high-throughput analysis of translation purity without requiring radioisotopes or cumbersome protein purification procedures.

[0169] Translation of complete or partial non-natural peptides using cation-depleted flexizyme-loaded tRNA, either in combination with or without other aaRS proteins, can be applied to peptide drug selection in combination with selection schemes such as ribosome display and mRNA display, as well as to data storage using complete or partial non-natural peptides based on amino acid letters (see Figure 18B).

[0170] Example 12 Loading of enzymatically transcribed L-tRNA using L-flexizyme Figures 19A and 19B show 8% acidic PAGE photographs and analyses of an experimental proof of concept in loading fully functional, enzymatically transcribed L-tRNA molecules with pre-activated amino acids. Figure 19A shows the loading results for enzymatically transcribed L-tRNA, and Figure 19B shows the loading results for synthetically produced L-tRNA.

[0171] As seen in Figures 19A and 19B, band shift is evident when enzymatically transcribed L-tRNA is loaded (Figure 19A). However, when L-tRNA prepared using commercially available synthesis equipment is loaded with pre-activated amino acids using L-flexizyme, no band shift is observed, and loaded L-tRNA molecules cannot be distinguished from unloaded tRNA molecules. This is likely due to the low quality of synthetically prepared tRNA.

[0172] This experiment clearly demonstrates the advantages of obtaining enzymatically transcribed L-tRNA molecules and also clearly shows the advantages of using L-flexizymes and obtaining D-enzymes that can enzymatically transcribe and produce L-RNA molecules.

[0173] Example 13 Translation of peptides containing two or three consecutive D-phenylalanine molecules To investigate the effects of increasing the concentration of cation-depleted tRNA and whether this can improve the translation yield of difficult-to-translate non-natural amino acids such as D-amino acids, the inventors of this invention have developed a short peptide (mRNA#7):Fph-KKK D F D We attempted to translate FDYKDDDDK (SEQ ID NO: 127) by loading its fluorescein-labeled L-phenylalanine (Fph) and L-lysine (K) onto their corresponding (cognate)tRNAs using flexizymes, while loading L-aspartic acid (D) and L-tyrosine (Y) onto their corresponding (cognate)tRNAs using aaRS (AspRS and TryRS, respectively).

[0174] Table 9 below shows the tRNA sequences for in vitro translation of mRNA #7 to mRNA #10.

[0175] [Table 9]

[0176] Following the instructions of Katoh, T. et al. ["Consecutive Elongation of D-Amino Acids in Translation", Cell Chemical Biology, 2017, 24, pp. 46-54], D-phenylalanine ( D F) is a modified tRNA having a sequence optimized for D-amino acid incorporation. GluE2 CUA (See Table 9) Loaded by Flexizyme ( D Phe-tRNA GluE2 CUA This peptide contains two consecutive D-phenylalanine units, which have been previously shown to be more difficult to translate and have a yield of less than 15% compared to peptides with the same sequence but containing two consecutive L-phenylalanine units [Achenbach, J. et al., "Outwitting EF-Tu and the ribosome: translation with D-amino acids", Nucleic Acids Research, 2015, 43, pp. 5687-5698]. The inventors of this application designed mRNA #7 capable of forming a Watson-Crick base pair with the anticodon of flexizyme-loaded tRNA and transcribed it in vitro (see Table 10).

[0177] Table 10 below shows the DNA template sequences for in vitro translation of mRNA #7 to mRNA #10.

[0178] [Table 10]

[0179] The inventors of this application have found that for in vitro translation, 20 μM or 200 μM of cation depletion D Phe-tRNA GluE2 CUA In both translation reactions, the overall Mg was added by the loaded tRNA. 2+ The carryover is Mg of the in vitro translation system proposed in this disclosure. 2+ Within the limits of permissible limits (<100mM Mg) 2+ ) was controlled to be the case. The inventors also used flexizyme-loaded tRNA as a control. Phe ( L Phe-tRNA Phe ) using mRNA#8(Fph- L K L K L K- L F L F L F- L D L Y L K L D L D L D L D L K (Sequence ID 129) (see Table 10) was translated in parallel.

[0180] The translation reaction was incubated at 37°C for 2 hours and analyzed by MALDI-TOF MS and 20% tricine-SDS-PAGE. The MALDI-TOF MS results show the precise incorporation of two consecutive D-phenylalanine molecules in mRNA #7 (Figure 20A). However, the mass peak was 200 μM. D Phe-tRNA GluE2 CUA It can only be detected in samples containing 20 μM D Phe-tRNA GluE2 CUA This could not be done in samples containing (Figure 20A). On the other hand, in the control experiment, the accurate mass peak was obtained at 20 μM. L Phe-tRNA Phe Unloaded tRNA could be detected in samples containing it, but not in samples containing it. PheThis could not be done with samples containing only [the substance]. Furthermore, the results of tricine-SDS-PAGE were [resulting in a 200 μM [component]] D Phe-tRNA GluE2 CUA The translation yield of mRNA #7 when using 20 μM is D Phe-tRNA GluE2 CUA The translation yield was approximately twice as high as when using 20 μM. L Phe-tRNA Phe This indicates similarity to the control using [the specified method].

[0181] Figures 20A–20C show the results of in vitro translation of short peptides containing two consecutive D-phenylalanine molecules. Figure 20A shows the MALDI-TOF-MS analysis of the short peptide translated from mRNA#7, Figure 20B shows the MALDI-TOF-MS analysis of the short peptide translated from mRNA#8, and Figure 20C shows the tricine-SDS-PAGE analysis of the translation products of mRNA#7 or mRNA#8 using unloaded tRNAPhe only (mRNA#7), 20 μM LPhe-tRNAPhe (mRNA#7), 20 μM Dphe-tRNAGluE2CUA (mRNA#8), or 200 μM Dphe-tRNAGluE2CUA (mRNA#8), scanned by a Typhoon FLA 9500 under Cy2 mode.

[0182] Encouraged by the successful translation of mRNA #7, which has two consecutive D-phenylalanine molecules, the inventors of the present invention translated mRNA #9 into a short peptide Fph-KKK, which has three consecutive D-phenylalanine molecules. D F D F D It was translated to FDYKDDDDK (SEQ ID NO: 127) (see Table 10). Previous attempts to translate short peptides containing three consecutive D-phenylalanine molecules showed yields of less than 5% compared to peptides with the same sequence but containing three consecutive L-phenylalanine molecules [Achenbach, J. et al., 2015]. The inventors of this application used 30 μM or 300 μM cation depletion for in vitro translation.D Phe-tRNAGluE2 CUA The substance was added, and the translation reaction was analyzed by MALDI-TOF MS and 20% tricine-SDS-PAGE. In both translation reactions, the overall Mg by loaded tRNA was observed. 2+ The carryover is Mg of the in vitro translation system proposed in this disclosure. 2+ Within the limits of permissible limits (<100mM Mg) 2+ ) was controlled to be the case. MALDI-TOF MS results show the precise incorporation of three consecutive D-phenylalanine molecules in mRNA #9 (Figure 21A). Furthermore, tricine-SDS-PAGE results showed that 300 μM D Phe-tRNAGluE2 CUA The translation yield of mRNA #9 when using this method is 30 μM D Phe-tRNAGluE2 CUA The translation yield was approximately twice as high as when using 30 μM. L Phe-tRNA Phe This shows similarity to the control using (Figure 21B).

[0183] Figures 21A and 21B show the results of in vitro translation of short peptides containing three consecutive D-phenylalanine molecules. Figure 21A shows the MALDI-TOF-MS analysis of the translated short peptide from mRNA#9, and Figure 21B shows the tricine-SDS-PAGE analysis of the translation product of mRNA#9 containing unloaded tRNAPhe only, 30 μM LPhe-tRNAPhe, 30 μM DPhe-tRNAGluE2CUA, or 300 μM DPhe-tRNAGluE2CUA, scanned by a Typhoon FLA 9500 under Cy2 mode.

[0184] In summary, these results suggest that increasing the concentration of cation-depleted flexizyme-loaded tRNA from approximately 20-30 μM to approximately 200-300 μM significantly improved the uptake efficiency of D-amino acids (up to three consecutive D-phenylalanine molecules).

[0185] Example 14 Translation of a peptide containing three consecutive β-amino acids To test whether increasing the concentration of cation-depleted tRNA can improve the translation yield of β-amino acids, the inventors of this invention used the short peptide (mRNA#10):Fph-KKK β Q β Q β We attempted to translate QDYKDDDDK (SEQ ID NO: 129) (see Table 10). The inventors of this application used 30 μM or 300 μM cation depletion for in vitro translation. β Q-tRNAGluE2 CUA The substance was added, and the translation reaction was analyzed by 20% trichine-SDS-PAGE.

[0186] In both translation reactions, the overall Mg by loaded tRNA 2+ The carryover is Mg of the in vitro translation system proposed in this disclosure. 2+ Within the limits of permissible intake (Mg less than 100 mM) 2+ The process was controlled to ensure that the full-length translation products were separated from their cleavage-type translation products. The translation products were purified using ANTI-FLAG M2 magnetic beads (Sigma) to ensure that the full-length translation products were separated from their cleavage-type translation products.

[0187] The results of trichine-SDS-PAGE showed 300 μM βGln-tRNAGluE2 CUA The translation yield of mRNA #10 when using this method is 30 μM. β Gln-tRNAGluE2 CUA This indicates that the translation yield was slightly higher than that obtained when using [the other method] (Figure 22).

[0188] Figure 22 shows the in vitro translation results of a short peptide containing three consecutive β-Gln molecules, and shows tricine-SDS-PAGE analysis of the translation product of mRNA #10 using only unloaded tRNA, 30 μM βGln-tRNAGluE2CUA, or 300 μM βGln-tRNAGluE2CUA, scanned under Cy2 mode by Typhoon FLA 9500.

[0189] In summary, these results suggest that increasing the concentration of cation-depleted flexizyme-loaded tRNA from approximately 30 μM to approximately 300 μM improved the uptake efficiency of β-amino acids (up to three consecutive β-Gln).

[0190] While the present invention has been described in relation to its specific embodiments, it is evident that many alternative, modified, and variant forms are apparent to those skilled in the art. Therefore, it is intended to encompass all such alternative, modified, and variant forms that fall within the spirit and broad scope of the appended claims.

[0191] All publications, patents, and patent applications referenced in this disclosure are incorporated by reference in whole to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference in this disclosure. Furthermore, no citation or specification of any reference in this application should be construed as an acknowledgment that such reference is available as prior art to the invention. Section headings should not necessarily be construed as restrictive insofar as they are used.

[0192] Furthermore, any priority documents of this application are incorporated in their entirety into this disclosure by reference.

Claims

1. A system for producing proteins, mRNA molecule encoding the aforementioned protein; Multiple loaded tRNA molecules, wherein each tRNA molecule comprises an L-ribonucleic acid residue (L-tRNA), and at least one of the multiple loaded tRNA molecules is loaded by an aminoacylated ribozyme; and Cell-free translation mix containing ribosomes and ribosomal translation factors essential for cell-free in vitro protein translation, but free from intact / viable cells. Includes, In the aforementioned system, Mg ２＋ It is present at a concentration of less than 100 mM. The aforementioned system.

2. The system according to claim 1, wherein at least one tRNA molecule among the plurality of loaded tRNA molecules is loaded by flexizyme.

3. The system according to claim 2, wherein a non-natural amino acid residue is loaded onto the tRNA molecule, and the non-natural amino acid residue is a D-amino acid residue.

4. The system according to claim 3, wherein the ribozyme comprises an L-ribonucleic acid residue.

5. Mg at or below the aforementioned concentration ２＋ To prepare the plurality of loaded tRNA molecules having; and The plurality of loaded tRNA molecules are brought into contact with the mRNA molecule encoding the protein in the cell-free translation mix, thereby obtaining the protein. A method for producing a protein using the system described in any one of claims 1 to 4, including the system described in any one of claims 1 to 4.

6. The above preparations are made before the contact, the Mg ２＋ The method according to claim 5, comprising adjusting the concentration of the following.

7. The method according to claim 5 or 6, wherein the preparation further comprises adjusting the loaded tRNA molecules to a concentration greater than 160 μM.

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