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] The present invention, in some embodiments thereof, relates to cell-free protein translation systems, and more particularly, but not exclusively, to methods and uses for synthesizing proteins and their enantiomers without aminoacyl-tRNA synthetases. [Background technology]

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

[0003] SEQUENCE LISTING STATEMENT An ASCII file entitled 90912 Sequence Listing.txt, created on Feb. 16, 2022, containing 36,864 bytes, and submitted contemporaneously with the filing of this application is hereby incorporated by reference into this disclosure.

[0004] Cell-free protein synthesis is an important tool for molecular biologists in basic and applied sciences. It is increasingly used in high-throughput functional genomics and proteomics, and offers great advantages compared to protein expression in living cells. Cell-free protein synthesis is essential for the creation of protein arrays, such as nucleic acid programmable protein arrays (NAPPA), and for enzyme engineering using display technologies. Cell-free approaches provide the fastest way to correlate phenotype (function of expressed protein) with genotype. Protein synthesis can be performed in a few 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 made from cells engaged in protein synthesis at high rates. The most frequently used cell-free expression systems require macromolecular components for translation, such as ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation factors, elongation factors, and termination factors. To ensure efficient translation, commercial 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 serve as the energy regeneration system, while phosphoenolpyruvate and pyruvate kinase are supplemented for prokaryotic systems. For coupled transcription and translation systems, RNA polymerases from phage are supplemented, allowing the expression of genes cloned downstream of the polymerase promoter.

[0006] The emergence of protein enzymes is the key to the transition from RNA-based life to modern biology. The discovery of tRNA-aminoacylation ribozymes suggested the possibility of synthesizing protein enzymes from highly simplified translation systems using tRNAs charged by ribozymes. Other systems using pre-loaded tRNAs prepared by aaRS, urzyme, and chemical acylation have also been reported. Among these, a highly robust and versatile tRNA-aminoacylation ribozyme system, named flexizyme, discovered by in vitro selection, has been shown to be capable of loading a wide variety of amino acids onto tRNAs. Using flexizyme and tRNAs charged by aaRS, the incorporation of multiple unnatural amino acids into translated peptides has been achieved, enabling the selection of peptide drugs in practical settings. However, in part due to low translation yields, only short peptides (less than seven amino acid residues in length) are translated using only flexizyme-loaded tRNA in the absence of an aaRS (hereafter referred to as "aaRS-free"), whereas ribosomal production of a full-length functional protein enzyme with all 20 proteinogenic amino acids under aaRS-free conditions remains to be demonstrated so far.

[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] report an engineered system that uses a pair of rRNA and tRNA with compensatory mutations, which specifically uses a genetic code programmed differently from the natural genetic code and can therefore synthesize peptides orthogonal to their wild-type counterparts. With these translation mechanisms, a single mRNA produces two different peptides according to an artificially programmed genetic code. Summary of the Invention

[0008] Aspects of the present invention are directed to cell-free and aaRS-free protein translation / expression / synthesis systems and methods, and uses thereof. The present disclosure relates to a method for the synthesis of Mg using only tRNAs charged with flexizymes. 2+ Through improved translation yields by lowering the concentration of cations and increasing the concentration of tRNA, successful translation of multiple proteins including active enzymes with different functions is provided. An aaRS-free translation system is demonstrated that produces an active aaRS (TrpRS), which further catalyzed a larger cargo of tRNA. Also demonstrated is a mirror-image tRNA charged with D-amino acids by a synthetic L-flexizyme. 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 mirror-image translation. The cation-depleted flexizyme-loaded tRNA is useful for translation of complete or partial non-natural peptides when used in combination with or without other aaRS proteins.

[0009] Thus, according to an aspect of some embodiments of the present invention, mRNA molecules that code for proteins; A plurality of loaded tRNA molecules; and Cell-free translation mix, Mg in the system +2 is less than 100 mM; A system for producing a protein is provided.

[0010] According to some embodiments, the system is essentially devoid of aminoacyl-tRNA synthetases. 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 molecules is greater than 160 μM and Mg +2 The concentration of is less than 100 mM.

[0011] According to some embodiments, at least one tRNA molecule of the plurality of loaded tRNA molecules is loaded by a flexizyme. In some embodiments, the tRNA molecule is loaded with an unnatural amino acid residue. According to some embodiments, the unnatural amino acid residue is a D-amino acid residue.

[0012] According to some embodiments, the tRNA molecule comprises an L-ribonucleic acid residue (L-tRNA). According to some embodiments, the L-tRNA is prepared using a 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 (SEQ ID NO: 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 an active L protein enzyme and an active D protein enzyme. According to certain other aspects of some embodiments of the present invention, Mg below a certain concentration +2 providing a plurality of loaded tRNA molecules having a concentration; and Contacting the loaded tRNA molecule with an mRNA molecule encoding a protein in a cell-free translation mix, thereby obtaining a protein. A method for producing a protein using the system provided herein is provided, comprising:

[0014] According to some embodiments, the system used in the method is essentially devoid of aminoacyl-tRNA synthetases. According to some embodiments, providing a plurality of loaded tRNA molecules comprises, prior to said contacting step, adding Mg +2 This includes adjusting (reducing or depleting) the concentration of According to some embodiments, Mg +2 Adjusting the concentration of includes, for example, using techniques such as chromatography, alcohol precipitation and pellet washing, ultrafiltration, and dialysis. According to some embodiments, providing a plurality of loaded tRNA molecules further comprises adjusting a concentration of the loaded tRNA molecules to a concentration that is more than twice the loaded tRNA concentration in other protein translation systems comprising an aaRS enzyme. According to some embodiments, the concentration of the loaded tRNA molecules is greater than 160 μM.

[0015] According to certain other aspects of some embodiments of the present invention, there is provided a method for loading an L-tRNA with a D-amino acid, the method comprising: preparing said L-tRNA molecule using a D-polymerase; Preparing activated D-amino acids; Providing an L-aminoacylated ribozyme; and contacting the L-tRNA, the L-aminoacylating ribozyme, and the activated D-amino acid, thereby obtaining a D-amino acid loaded L-tRNA molecule; It is executed by.

[0016] According to some embodiments, the L-aminoacylation ribozyme is L-flexizyme. In some embodiments, the method can be analyzed by PAGE analysis of a reaction mixture of D-amino acid loaded L-tRNA molecules, where the PAGE gel features distinct peaks of loaded tRNA species and distinct peaks of unloaded tRNA species.

[0017] According to another aspect of some embodiments of the present invention there is provided an L-flexizyme comprising an L-ribonucleotide residue. In some embodiments, the L-flexizyme comprises 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more L-ribonucleotide residues. In some embodiments, said L-flexizyme is comprised of L-ribonucleotide residues.

[0018] In some embodiments, L-flexizyme has a sequence that shows 80% or more identity to 5'-ggaucgaaagauuuccgcauccccgaaaggguacauggcguuaggu-3' (SEQ ID NO: 82). According to certain other aspects of some embodiments of the present invention there is provided a protein prepared by the methods provided herein. In some embodiments, the protein is selected from the group consisting of a protein that includes at least one non-canonical amino acid residue, a protein that includes 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 E. coli TrpRS. In some embodiments, the proteins have sequences that can be decoded into textual and / or numerical information and include natural and / or unnatural amino acids. In some embodiments, the protein is encoded by mRNA#6. According to certain other aspects of some embodiments of the invention, there is provided a library of randomized or partially randomized peptides obtained by the methods described, at least one of the peptides comprising at least one unnatural amino acid.

[0020] Unless otherwise specified, all technical and / or scientific terms used in this disclosure have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described in this disclosure can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. [Brief description of the drawings]

[0021] Some embodiments of the present invention are described in this disclosure, by way of example only, with reference to the accompanying drawings. With specific and detailed reference now to the drawings, it should be stressed that the matter shown is by way of example and for the purposes of illustrative illustration of embodiments of the invention. In this regard, the description made with the aid of the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings:

[0022] [Figure 1] FIG. 1 shows a schematic diagram of some embodiments of the invention, in particular the aaRS-free translation of a protein using flexizyme-loaded tRNA (10), in which tRNA 11 is loaded by the flexizyme system 12 to generate a population of loaded tRNA 13 representing proteinogenic amino acids for translation of a protein enzyme, and the aaRS-free translation includes a step 14a in which the loaded tRNA is purified by HPLC to reduce Mg2+ 14b contamination, and a step 15 in which the loaded tRNA 13 is enriched for aaRS-free translation of mRNA 16 in ribosome 17, and the translated polypeptide 18 can fold into an active protein enzyme 19a containing aaRS 19b, which can be used to load tRNA, thereby completing the cycle. [Diagram 2]FIG. 2 presents an acidic PAGE analysis of tRNA loading yield before and after HPLC purification, where "U" represents unloaded tRNA, "C" represents crude loaded tRNA, and "P" represents purified loaded tRNA; tRNA loading yield was determined by the software package IMAGEJ using the integrated peak area of ​​loaded tRNA relative to total tRNA. [Figure 3A] 3A-E present the concept and results of flexizyme-catalyzed tRNA loading en route to aaRS-free loading of the mirror-image tRNA according to some embodiments of the present invention. These figures show D-tRNA cargo catalyzed by D-flexizyme and its mirror-image version, i.e., mirror-image tRNA cargo catalyzed by L-flexizyme (PDB source: 1EHZ (tRNA), 3CUL (flexizyme) (FIG. 3A), loading of D-alanine onto enzymatically transcribed mirror-image tRNAAla by L-flexizyme (with the natural chirality counterpart for comparison) (FIG. 3B), loading of glycine onto enzymatically transcribed mirror-image tRNAGly by L-flexizyme (with the natural chirality counterpart for comparison) (FIG. 3C). The loading of D-lysine onto the enzymatically transcribed mirror-image tRNALys by L-flexizyme (along with its natural chirality counterpart for comparison) (Figure 3C), loading of D-phenylalanine onto the enzymatically transcribed mirror-image tRNAPhe by L-flexizyme (along with its natural chirality counterpart for comparison) (Figure 3D), and loading of D-phenylalanine onto the enzymatically transcribed mirror-image tRNAPhe by L-flexizyme (along with its natural chirality counterpart for comparison) (Figure 3E), where the tRNA loading yield was determined using the integrated peak area of ​​the loaded tRNA relative to the total tRNA using the software package IMAGEJ. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 4A]4A-G show the results of aaRS-free translation of multiple short peptides according to some embodiments of the present invention. Shown are MALDI-TOF-MS analysis of translated short peptides from mRNA#1 (Figure 4A); aaRS-free translation yields of short peptides analyzed by Tricine-SDS-PAGE, where unloaded tRNA concentrations ranged from 160 to 540 µM, but flexizyme-loaded tRNA concentrations remained at 70 µM, resulting in loading yields ranging from 44 to 13% (top of Figure 4B); total tRNA concentrations ranged from 16 to 1003 µM, resulting in loading yields remaining at 56% (bottom of Figure 4B) (error bars represent standard deviations 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). [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. Shown are that the loading yield remained at 44% when the total tRNA concentration ranged from 20 to 644 μM (Figure 5A), that the total flexizyme concentration ranged from 240 to 525 μM and that the total tRNA concentration remained at 160 μM (Figure 5B), and that flexizyme and 0-380 μM unloaded tRNA were mixed in 10 mM MgCl2 (Figure 5C) and in 100 mM MgCl2 (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 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 Tricine-SDS-PAGE gel analysis for calculating aaRS-free translation yield, and show gel images corresponding to Figures 4B, 5A, 5B, 5C, and 5D (Figures 6A-E, respectively), for calculating aaRS-free translation yield, where "M" is a synthetic peptide standard (Fph-KYDKYD (sequence number 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-B show the results of in vitro translation experiments in the presence of LysRS, TyrRS, and AspRS, showing Tricine-SDS-PAGE analysis of translation products and Fph-tRNAfMet preloaded by enhanced flexizyme using unloaded, unmodified total tRNA concentrations ranging from 22 to 680 μM in the presence of LysRS, TyrRS, and AspRS (Figure 7A), as well as the calculated translation yields (Figure 7B) (error bars represent standard deviation from three independent experiments). [Figure 7B] Same as above. [Figure 8A] 8A-B show the flexizyme cargo yields of 21 tRNAs with their cognate proteinogenic amino acids, with cargo yields determined after ethanol precipitation (FIG. 8A) and after HPLC purification of 14 flexizyme-loaded tRNAs, where N / A indicates that no purification of the flexizyme-loaded tRNAs was performed (FIG. 8B). [Figure 8B] Same as above. [Figure 9]Figure 9 shows MALDI-TOF MS analysis of aaRS-free translated mRNA #6, showing that at higher total tRNA concentrations (520 μM) in the aaRS-free translation system, a mistranslated product with a molecular weight of 2,252.7 Da was observed, whereas the correctly translated product had a molecular weight of 2,240.7 Da, where au stands for arbitrary unit, and C and O stand for 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]11A-G show SDS-PAGE analysis of aaRS-free translated protein enzymes, with the entire gel image shown in FIG. 12A (FIG. 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 (FIG. 11B); the entire gel image shown in FIG. 12C (FIG. 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 (FIG. 11D); the entire gel image shown in FIG. 14A (FIG. 11E); A 300 ng sample of recombinant E. coli TrpRS expressed and purified from E. coli BL21 strain analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue (FIG. 11F), 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 min and scanned by a Typhoon FLA 9500 under Cy2 mode (FIG. 11G), where M is a 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]12A-D show the results of an experimental proof of concept of aaRS-free translation of protein enzymes according to some embodiments of the present invention, including aaRS-free translation of N-terminal FAM-labeled chicken lysozyme analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode (M stands for benchmark fluorescent protein standard) (FIG. 12A); enzymatic assay of crude aaRS-free translated chicken lysozyme with fluorescently labeled bacterial (Micrococcus lysodeikticus) cell wall material as substrate (FIG. 12B); and aaRS-free translation of N-terminal FAM-labeled chicken lysozyme analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode (FIG. 12C). aaRS-free translation of N-terminal FAM-labeled Gaussia luciferase scanned by the 9500 (FIG. 12C) and enzymatic assay of crude aaRS-free translated Gaussia luciferase using coelenterazine as substrate (FIG. 12D) (RFU stands for relative fluorescence units, RLU stands for relative luminescence units). [Figure 12B] Same as above [Figure 12C] Same as above [Figure 12D] Same as above [Figure 13] FIG. 13 shows the yield estimate of aaRS-free translated Gaussia luciferase, where a standard curve was plotted using 0, 25 nM, 50 nM, 100 nM, and 250 nM of recombinant Gaussia luciferase (shown as squares) and the yield of translated Gaussia luciferase was estimated to be approximately 25 nM (shown as triangles). [Figure 14A]14A-C show aaRS-free translation of TrpRS, aaRS-free translation of N-terminally FAM-labeled E. coli TrpRS (M represents a benchmark fluorescent protein standard) analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode (FIG. 14A), sequence and secondary structure of internal Cy5-labeled tRNATrp (FIG. 14B), and enzymatic assay of crude aaRS-free translated TrpRS using Cy5-tRNATrp as substrate, analyzed by 8% acidic PAGE and scanned by Typhoon FLA 9500 under Cy5 mode (FIG. 14C). [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 15A] Figures 15A-B show the results of transcription of mirror-image tRNALys by D-Dpo4-5m-Y12S, showing the extension of 5'-FAM-labeled L-universal primer on L-ssDNA template, where polymerization was performed by synthetic D-Dpo4-5m-Y12S polymerase, and reaction aliquots were terminated at different time points and analyzed by 12% denaturing PAGE gel in 7 M urea (Figure 15A), as well as the mirror-image transcription and I2-mediated cleavage of tRNALys transcripts analyzed by 10% denaturing PAGE gel in 7 M urea and stained by SYBR-Green II (Thermo Fisher Scientific, MA, USA) (Figure 15B). [Figure 15B] Same as above. [Figure 16A] Figures 16A-B show the results of biochemical characterization of enzymatically transcribed native tRNA and mirror image tRNA, showing 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, including synthetic DNA-RNA chimeric oligos cleaved at phosphorothioate modification sites by I2 (Figure 17A), MALDI-TOF MS spectrum of uncleaved oligos under negative linear mode (Figure 17B), and MALDI-TOF MS spectrum of I2-cleaved oligos under negative linear mode (m / z>4000) and negative reflectron mode (m / z<4000) (Figure 17C), where capital letters indicate DNA nucleotides, lowercase letters indicate RNA nucleotides, and "*" indicates phosphorothioate modification. au represents arbitrary units, and C, O represent calculated and observed m / z values, respectively. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 18A] 18A-B show the translation of complete or partial non-natural peptides using cation-depleted flexizyme-loaded tRNAs, translation of peptide drugs and non-natural proteins using cation-depleted flexizyme-loaded tRNAs in an in vitro translation system (FIG. 18A), and translation, data storage, and ribosome / mRNA display of complete or partial non-natural proteins using cation-depleted flexizyme-loaded tRNAs in an in vitro translation system (FIG. 18B). [Figure 18B] Same as above. [Figure 19A] Figures 19A-B show 8% acid PAGE photographs and analysis of the experimental proof of concept of loading pre-activated amino acids onto fully functional L-tRNA molecules enzymatically transcribed by the mirror enzyme (D-Dpo4-5m-Y12S), where Figure 19A shows the results of loading onto enzymatically transcribed L-tRNA and Figure 19B shows the results of loading onto synthetically produced L-tRNA. [Figure 19B] Same as above. [Figure 20A]20A-C show the results of in vitro translation of short peptides containing two consecutive D-phenylalanines, where FIG. 20A shows MALDI-TOF-MS analysis of the short peptide translated from mRNA#7, FIG. 20B shows MALDI-TOF-MS analysis of the short peptide translated from mRNA#8, and FIG. 20C shows Tricine-SDS-PAGE analysis of the translation products of mRNA#7 or mRNA#8 with 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 Typhoon FLA 9500 under Cy2 mode. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 21A] 21A-B show the results of in vitro translation of short peptides containing three consecutive D-phenylalanines, where FIG. 21A shows MALDI-TOF-MS analysis of short peptides translated from mRNA#9, and FIG. 21B shows Tricine-SDS-PAGE analysis of the translation products of mRNA#9 using only unloaded tRNAPhe, 30 μM LPhe-tRNAPhe, 30 μM DPhe-tRNAGluE2CUA, or 300 μM DPhe-tRNAGluE2CUA, scanned by Typhoon FLA 9500 under Cy2 mode. [Figure 21B] Same as above. [Figure 22] FIG. 22 shows the results of in vitro translation of a short peptide containing three consecutive β-Glns, showing Tricine-SDS-PAGE analysis of the translation products of mRNA#10 using uncargoed tRNA alone, 30 μM βGln-tRNAGluE2CUA, or 300 μM βGln-tRNAGluE2CUA, scanned by Typhoon FLA 9500 under Cy2 mode. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention, in some embodiments thereof, relates to cell-free protein translation systems, more specifically, but not limited to, methods for synthesizing proteins and their enantiomers that are free of aminoacyl-tRNA synthetases, and uses thereof.

[0024] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0025] As mentioned above, despite the discovery of tRNA-aminoacylation ribozymes such as flexizyme, the synthesis of protein enzymes from highly simplified translation systems in the absence of aaRS remains undemonstrated. One of the main reasons for the low yield of aaRS-free translation is the lack of recycling of cargo to tRNA by flexizyme compared to tRNA aminoacylation by aaRS. In addition, the use of in vitro transcribed unmodified tRNA for aaRS-free cargo may also contribute to the low translation yield.

[0026] While devising the present invention, the inventors set out to test the ability of the aaRS-free system to translate protein enzymes with all 20 proteinogenic amino acids using tRNA loaded only by flexizyme. Preliminary results showed that by increasing the concentration of flexizyme-loaded tRNA through purification and decreasing the concentration of cation Mg2+, multiple protein enzymes with different functions, such as lysozyme, luciferase, and even aaRS itself, could be synthesized. The loading of mirror-L-tRNA with mirror-D-amino acids by synthetic mirror-L-flexizyme has also been demonstrated, which ultimately allows the realization of a mirror-translation apparatus.

[0027] FIG. 1 shows a schematic diagram of some embodiments of the invention, in particular the aaRS-free translation of proteins using loaded tRNAs by flexizyme (10), in which tRNAs 11 are loaded by the flexizyme system 12 to generate a population of loaded tRNAs 13 representing proteinogenic amino acids for the translation of protein enzymes, and the aaRS-free translation is carried out by HPLC purification of the loaded tRNAs using MgCl2. 2+ It includes a step 14 to reduce contamination and a step 15 in which loaded tRNA 13 is enriched for aaRS-free translation of mRNA 16 in ribosomes 17, and the translated polypeptide 18 can fold into an active protein enzyme 19a containing aaRS 19b, which can be used to load tRNA to complete the cycle.

[0028] This disclosure demonstrates aaRS-free translation of protein enzymes using an exclusive set of ribozyme-charged tRNAs. The finding that neither aaRS-catalyzed tRNA charging nor tRNA recycling is required for processive and reliable ribosomal translation reveals that protein enzymes with more structural motifs and therefore more catalytic functions than short peptides can be translated from a highly simplified aaRS-free translation system. Notably, the average size of modern natural proteins is about 270-470 amino acid residues. aaRS-free translation of proteins as large as TrpRS suggests the possibility of producing other important protein enzymes, such as tRNA-modifying enzymes, to further improve translation efficiency and fidelity. Also presented in the present disclosure is the discovery that high concentrations of ribozyme-loaded tRNAs significantly improve the yield of aaRS-free translation, which may shed light on possible conditions for the emergence of protein enzymes on prebiotic Earth, where an abundant feedstock of ribozyme-loaded tRNAs would have been important for primitive translation systems to operate efficiently.

[0029] One of the limitations of current aaRS-free translation systems is that the tRNA cargo must be uncoupled from translation in that the tRNA is preloaded before being added to the translation system. This is because flexizyme is a non-specific catalyst that loads a variety of amino acids onto tRNA. The methodology of 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 preloaded tRNA to produce peptides or proteins from a set of amino acids that are all or partly unnatural, allowing for immediate applications in many areas of synthetic biology and drug discovery.

[0030] The realization of aaRS-free translation of a protein enzyme establishes the way to aaRS-free translation apparatus as a more viable model for realizing mirror-image translation, because all aaRS proteins collectively represent 29% (about 1.4 MDa) of the molecular weight of the E. coli translation apparatus, including ribosomes, translation factors, aaRS, and tRNAs (total molecular weight of about 4.9 MDa). Furthermore, the translation of the small 169 amino acid residue Gaussia luciferase demonstrated in this disclosure provides a sensitive and chiral-specific assay for testing mirror-image translation.

[0031] aaRS-free cell-free translation system: As discussed above in this disclosure, cell-free protein synthesis provides an easy and rapid method for synthesizing, monitoring, analyzing and purifying proteins from DNA templates while paving the way for genetic code expansion, which allows, among other things, site-specific incorporation of unnatural amino acids (UAAs; also known as nonstandard amino acids) into proteins via ribosomal translation. While known systems are based on the exogenous addition of an orthogonal translation system (OTS) comprising an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase (aaRS) to a cell-free reaction mixture, the protein translation system provided in this disclosure further extends this concept by allowing efficient production of proteins without the presence of an aminoacyl-tRNA synthetase (aaRS), and an aaRS-free translation system is provided in this disclosure.

[0032] In the context of this disclosure, the term "aaRS-free" as used herein refers to a ribosomal translation system and / or method and / or platform for preparing a protein from a transcription template (e.g., a ribonucleic acid molecule) that is essentially devoid of an aminoacyl-tRNA synthetase (aaRS). Essentially devoid of an aminoacyl-tRNA synthetase means that no step in protein production involves the use or presence of an aaRS. According to some embodiments of the present invention, the only exception to the definition of an aaRS-free translation system / method / platform is an embodiment in which an aminoacyl-tRNA synthetase is the protein product produced by said system / method / platform. Essentially devoid of a tRNA synthetase enzyme means that the system does not include a 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 pre-loaded tRNA molecules.

[0033] Thus, according to an aspect of some embodiments of the present invention there is provided a system for producing a protein, the system comprising: mRNA molecules that code for proteins; A plurality of loaded tRNA molecules; and Cell-free translation mix, The system essentially lacks aminoacyl-tRNA synthetase, and the system is +2 The concentration of is less than 100 mM.

[0034] The term "system" as used in this disclosure refers to the essential reaction mixture (i.e., solvents, solutes, reactants, and optional detectable markers) and reaction conditions (concentrations, temperature, and mixing) that are conducive to carrying out complex chemical reactions such as protein synthesis.

[0035] In the context of some embodiments of the present invention, the phrase "cell-free translation mix" refers to an in vitro protein translation mixture that does not include 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 phrase "aaRS-free translation mix" refers to a cell-free translation mix as known in the art, except that the cell-free (in vitro) translation mix is ​​essentially devoid of aaRS protein, unless otherwise specified.

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

[0037] The protein translation system comprises a plurality of loaded tRNA molecules, which in the present disclosure are also referred to as pre-loaded tRNA transcripts in the context of some embodiments of the present invention. In some embodiments, the tRNA molecules are synthetically prepared polynucleotides, while in other embodiments, the tRNA molecules are enzymatically prepared transcripts, with important differences between these two categories being described below.

[0038] According to some embodiments of the invention, the plurality of loaded tRNA molecules includes at least tRNA molecules loaded with amino acid residues that are encoded by the mRNA and that will be present in the protein sequence as encoded by the mRNA molecule. The plurality of loaded 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 of the individual loaded tRNA molecules matches 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 loaded tRNA molecules in the system will reflect that frequency, and the tRNA molecules Met Approximately eight times more tRNA than Ser In some embodiments, the tRNA molecule is made from L-nucleotides, resulting in a tRNA molecule mirror image of a natural tRNA molecule. In some embodiments, the tRNA molecule is composed of L-nucleotides and is further loaded with residues of D-amino acids.

[0039] As used in this disclosure, the terms "residue" and / or "moiety" refer to a part of a molecule, typically a major portion thereof, or a group of atoms involved in a particular function. For example, the term "amino acid residue" refers to an amino acid in the context of the compound to which it is bound, a peptide is a chain of amino acid residues linked together, and a tRNA molecule loaded with a ribonucleic acid residue is a ribonucleic acid bound to a tRNA molecule.

[0040] Tables A-B show some optional amino acid residues that are relevant in the context of some embodiments of the present invention, which are examples and should not be viewed as 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 producing proteins according to embodiments of the present invention is effective at low cation concentrations, more specifically low magnesium ion concentrations. Magnesium is present in most cell-free protein translation mixtures, including commercial mixtures, at relatively high concentrations. Magnesium is also present in most loaded tRNA preparations, particularly flexizyme loaded tRNA preparations. As shown in the Examples section below, the inventors have surprisingly found that reducing the magnesium concentration to a practical minimum in the cell-free aaRS-free protein translation system significantly improves the efficiency and fidelity of protein production. Therefore, to reach the improved performance of the system disclosed in this disclosure, the inherent presence of magnesium ions carried over from various components in known protein translation systems had to be intentionally reduced by the inventors.

[0044] Thus, according to some embodiments of the present invention, the system for producing proteins requires lower Mg content compared to any previously known cell-free protein translation system. +2 More specifically, the magnesium concentration in the system according to the present invention is characterized by the Mg concentration in the loaded tRNA preparation. +2 In absolute terms, the Mg concentration in the system is +2 is 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 practically obtainable 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 of the disclosed system may be pre-loaded by any method known in the art, but in some preferred embodiments, the tRNA is loaded by flexizyme. The concentration of loaded tRNA molecules present in the system is also subject to modification compared to their concentration in known cell-free protein translation systems.

[0047] According to some embodiments, the concentration of the loaded tRNA is at least 2-fold higher than the concentration in other known cell-free protein translation systems. According to some embodiments, the concentration of the 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] Reversed Chirality Elements: Because the system provided in this disclosure does not require an aaRS enzyme, the system is particularly suitable for translating proteins with unnatural / 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 translating mRNA into an all-D amino acid chain. As demonstrated below, the system was used to translate a complete mirror image protein.

[0049] As shown later in this disclosure, the system can be successfully used with tRNA molecules (L-tRNA) that contain or consist of L-ribonucleic acid residues. Thus, according to some embodiments of the present invention, the system includes an L-tRNA molecule. The L-tRNA is prepared using a D-polymerase, such as, but not limited to, D-Dpo4-5m-Y12S. However, other methods of producing L-tRNA molecules are also contemplated within the scope of the present invention.

[0050] In some embodiments of the invention, the system comprises L-tRNA molecules preloaded with D-amino acid residues by L-flexizyme for translating D-proteins (mirror image proteins).

[0051] L-aminoacylated ribozymes: According to some embodiments of the present invention, the system comprises an L-tRNA pre-loaded 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, a ribozyme composed entirely or substantially of L-ribonucleotides.

[0052] Thus, according to some aspects of some embodiments of the present invention, there is provided a polyribonucleic acid molecule (RNA) comprising L-ribonucleotides (mirror image to comparable natural RNA molecules), which has catalytic activity (tRNA cargo activity) of aminoacylation of RNA by using activated amino acids (ribozyme). That is, the present disclosure provides an L-flexizyme.

[0053] As demonstrated in the Examples section below, loading of D-amino acid residues into L-tRNA molecules is more efficient and more consistent using L-flexizyme. The L-flexizyme provided in the present disclosure has substantially the same sequence as its mirror image (D-aaRS ribozyme; D-flexizyme) or has 80% or more sequence identity to D-flexizyme known in the art. For example, in some embodiments, the L-flexizyme has a sequence that shows 80% or more identity to 5'-ggaucgaaagauuuccgcauccccgaaaggguacauggcguuaggu-3' (SEQ ID NO: 82).

[0054] An aspect of the L-flexizyme is the use of the L-flexizyme to load pre-activated D-amino acid residues onto an L-tRNA molecule.Accordingly, according to another aspect of some embodiments of the present invention, a method for loading an L-tRNA with a D-amino acid is provided, the method comprising: Preparing activated D-amino acids; Preparing an L-tRNA molecule; Providing L-Flexizyme; and reacting the L-tRNA, the L-flexizyme, and an activated D-amino acid, thereby obtaining an L-tRNA molecule loaded with a D-amino acid; This is done by.

[0055] According to some embodiments of the present invention, the L-tRNA molecules are prepared using D-polymerase, rather than the product of a synthetic machine. 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 source of L-tRNA is enzymatic rather than synthetic (see the explanations for Figures 3A-3E and 19A-19B below). This advantage can be shown and identified using PAGE analysis of the reaction mixture of D-amino acid loaded L-tRNA molecules, which is characterized by clear peaks of loaded tRNA species and clear peaks of unloaded tRNA species. In contrast, in the reaction with machine-synthesized L-tRNA molecules, the reaction mixture shows a continuous large peak, which indicates multiple intermediates, mismatches, and other side reactions due to the use of low-quality L-tRNA as starting material.

[0056] Method of using the system: Use of the system provided in the present disclosure differs from use of other known cell-free protein translation systems, and even from previously known aaRS-free protein translation systems, in that at least the concentration of pre-loaded tRNA molecules is higher than that used in the known systems, and Mg+2 The concentration of Mg used in a system with known +2 This is in the sense that it is lower than the concentration.

[0057] Thus, according to certain other aspects of some embodiments of the present invention, there is provided a method for producing a protein using the cell-free aaRS-free protein translation system provided herein, the method comprising: The Mg +2 Concentration below Mg +2 providing a plurality of pre-loaded tRNA molecules having a concentration (less than half the concentration of other known cell-free protein translation systems, or less than 100 mM); and contacting said plurality of loaded tRNA molecules with an mRNA molecule encoding a desired protein in a cell-free translation mix, thereby obtaining the protein of interest; This is done by.

[0058] In some embodiments, the method further comprises contacting the pre-loaded tRNA preparation with the cell-free translation mix with Mg. +2 The depletion of ions, particularly cations, from a system containing macromolecules, particularly sensitive biopolymers, can be accomplished by any procedure known in the art. For example, Mg +2 The concentration can be reduced by, but is not limited to, chromatography (e.g., HPLC), alcohol precipitation followed by washing of the precipitated pellet, ultrafiltration, and dialysis. Other procedures are also contemplated within the scope of the present invention.

[0059] In some embodiments, the method further comprises adjusting the concentration of the pre-loaded tRNA molecules to a desired high concentration, as this feature is discussed above in this disclosure, before contacting the pre-loaded tRNA preparation with the cell-free translation mix.Thus, in some embodiments, the method further comprises concentrating the loaded tRNA molecules to a concentration at least 2-fold higher than the concentration in the system containing the aaRS.In some embodiments, the concentration of the pre-loaded tRNA molecules is 160 μM or more.

[0060] The Examples section below provides a detailed presentation of several embodiments of the systems disclosed in this disclosure and methods of using the systems to produce proteins in the cell-free and aaRS-free protein translation systems disclosed in this disclosure.

[0061] Products of the disclosed systems and methods: As demonstrated by the experimental proof of concept 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 natural system. Proteins produced by the provisions of the present invention can also be mirror image proteins produced from chirality-inverting elements, such as fully active enzymes that catalyze a reaction from a mirror image starting material to produce a mirror image product.

[0062] Thus, according to an aspect of some embodiments of the present invention there is provided a protein produced by the systems and / or methods provided herein, in some embodiments the protein is a mirror image protein (a D-protein consisting essentially 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 E. coli TrpRS.

[0064] Libraries: According to some embodiments, the systems and methods provided in the present disclosure can be used to generate libraries of randomized or partially randomized peptides, at least one of the peptides comprising at least one unnatural amino acid.

[0065] One advantage of the aaRS-free system provided in the present disclosure is that it requires 21 tRNAs to operate efficiently. There are more than 20 other natural tRNA transcripts available for assigning unnatural amino acids (genetic code reprogramming). In other protein translation systems, these tRNAs are not available because the aaRS loads them with natural amino acids. Thus, the present invention can provide a means to translate randomized peptides containing multiple unnatural amino acids without running into the problem of misloaded tRNA molecules.

[0066] The protein translation system provided in the present 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 flexizyme and cannot be loaded by any aaRS protein, and suffers from the problem of inefficiency, which is solved by the provisions of the present invention. For example, known aaRS-free systems have low yields for translating heptapeptides, such as (Fph)-Lys-Tyr-Asp-Lys-Tyr-Asp (SEQ ID NO: 125), with a yield of about 0.15 μM and low processivity (7-amino acid residues). Under the improved conditions provided by the system according to embodiments of the present invention, the yield is about 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 more suitable for peptide drug discovery due to better yields (higher concentration of peptide pool) and longer products (higher sequence diversity).

[0067] Non-biological use: In the search for ultra-high density, high fidelity information storage facilities, the inventors have contemplated the use of the systems and methods disclosed in this disclosure in the manufacture of proteinaceous macromolecules having sequences that can be coded and decoded using known procedures, but cannot be degraded by natural biochemical elements. The use of unnatural amino acid residues in the proteins provides protection from biodegradation.

[0068] Additionally, the present rules can be used to maximize data density by incorporating unnatural amino acids, which in a textual analogy are essentially letters as character-modifiers. Thus, in some embodiments of the present invention, proteins that are products of the use of the systems provided in this disclosure are characterized as having amino acid sequences that can be decoded into textual and / or numerical information and that include at least some unnatural amino acid residues. Realization of this concept requires translation of peptides of arbitrary sequences. The inventors have demonstrated this using the 20 proteinogenic amino acids in Figures 4C-4G.

[0069] This concept was realized in a demonstrative 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 FIG. 4G) and successfully translated the full-length information-bearing peptide.

[0070] As used in this disclosure, the term "about" refers to ±10%. The terms "comprise," "comprising," "include," "including," "having" and their conjugations mean "including, but not limited to."

[0071] The term "consisting of" means "including and limited to." The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0072] As used in this disclosure, the phrases "substantially devoid of" and / or "essentially devoid of" in the context of a particular substance refer to a composition that is completely devoid of said substance or that contains less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% of said substance based on the total weight or volume of the composition. Alternatively, the phrases "substantially devoid of" and / or "essentially devoid of" in the context of a process, method, property, or characteristic refer to a process, composition, structure, or article that is completely devoid of a particular process / method step or a particular property or characteristic, or a process / method in which a particular process / method step is achieved with less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% of a property or characteristic compared to a given standard.

[0073] As used in this disclosure, the term "substantially maintain" when applied to an original or desired or given property of an object or composition means that the property is not changed by more than 20%, by more than 10%, or by more than 5% in the treated object or composition.

[0074] The word "exemplary" is used in this disclosure to mean "serving as an example, instance, or illustration." Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments, and / or as excluding the incorporation of features from other embodiments.

[0075] The words "optionally" or "alternatively" are used in this disclosure to mean "is provided in some embodiments and is not provided in other embodiments." Any particular embodiment of the invention may include more than one "optional" feature, unless such features are inconsistent.

[0076] As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" encompasses a plurality of compounds, including mixtures thereof.

[0077] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This is true regardless of the breadth of the range.

[0078] Whenever a numerical range is given in this disclosure, it is meant to include any recited numbers (fractional or integer) within the range given. The phrases "ranging / range" a first designated number and a second designated number, and "ranging / range" "from" a first designated number to a second designated number, are used interchangeably in this disclosure and are meant to include the first and second designated numbers and all fractional numerals and integers therebetween.

[0079] As used in this disclosure, the terms "process" and "method" refer to ways, means, techniques, and procedures for accomplishing a given task, including but not limited to ways, means, techniques, and procedures known to practitioners of chemical, materials, mechanical, computational, and digital technologies or readily developed from known methods, means, techniques, and procedures.

[0080] It is expected that many related methods for aaRS-free protein translation systems will be developed during the life of any patent resulting from this application, and the scope of the phrase "aaRS-free protein translation system" is intended to include a priori all such new technologies.

[0081] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or 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 construed as essential features of those embodiments, unless the embodiment is inoperable without those elements.

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

[0083] Reference is now made to the following examples, which together with the above descriptions illustrate, in a non-limiting manner, some embodiments of the present invention.

[0084] Example 1 Experimental procedure material: Amino acid substrates for cargo by flexizyme were prepared as 3,5-dinitrobenzyl esters (DBEs), except for Asn and fluorescein (FAM)-labeled Phe (Fph), which were synthesized as 4-chlorobenzylthioesters (CBT) and cyanomethyl esters (CME), respectively. Amino acid DBEs were either ordered 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 using the following procedure: 1 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) phosphorodiamidyl 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 extracted with anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of 19:1 (v / v) trifluoroacetic acid (TFA) / ddH2O and concentrated by rotary evaporation. 2 2 ml of a solution containing 20O was added and stirred at room temperature for 4 h. The solution was saturated with NaHCO3 The mixture was neutralized with hexanes, extracted with dichloromethane, and concentrated by rotary evaporation. The crude product was dissolved in methanol and purified by C18 HPLC column (Inertsil ODS-3, 5 μm, 10 × 150 mm, GL Sciences, Japan) using a gradient of 30-80% acetonitrile in 0.1% TFA. The fractions containing the product were pooled, lyophilized, and verified: 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.40 (s, 3H), 7.74 (d, J = 9.3 Hz, 1H), 7.44-7.35 (m, 4H), 7.33 (d, J = 9.5 Hz, 1H), 4.54-4.46 (m, 1H), 4.25 (d, J = 9.4 Hz, 2H), 2.78 (dh, J = 14.3, 7.5, 6.4 Hz, 2H). D-DNA oligos were ordered and obtained from Genewiz (Jiangsu, China).

[0086] RNA oligos and DNA-RNA chimera oligos were ordered from Tsingke (Beijing, China). L-DNA oligos and L-Flexizyme were synthesized on an H-8 DNA synthesizer (K&A Laborgeraete, Germany) using L-deoxynucleosides and L-2'-t-butyldimethylsilyl (TBDMS) phosphoramidites (Chemgenes, MA, USA). Phosphorothioate modifications were introduced using Sulfur 42 reagent (Sigma-Aldrich, MO, USA). The synthesized L-oligos were cleaved from CPG by concentrated ammonium hydroxide at 65°C for 2 hours. For the synthesis of L-Flexizyme, 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 oligos and L-NTPs were purified by denaturing PAGE and HPLC, respectively. L-Flexizyme was precipitated by ethanol and purified by HPLC.

[0088] The genes of AlaRS, AspRS, LysRS, TrpRS and TyrRS were amplified and cloned from E. coli K12 MG1655 genomic DNA. The gene of Gaussia luciferase was synthesized by Genewiz (Jiangsu, China). Recombinant aaRS proteins 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 TEV protease. Purified hen 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-extension of two overlapping primers (1F and 2R) (2 μM forward primer 1F, 3 μM reverse primer 2R, 0.2 mM each dNTP, and 5 U EasyTaq (TransGen Biotech, Beijing, China) per 100 μl reaction) in a five-cycle PCR or by 25-cycle assembly PCR with four primers (1F, 2R, 3F, and 4R) (2 μM each primer 1F and 4R, 0.05 μM each primer 2R and 3F, 0.2 mM each dNTP, and 5 U EasyTaq per 100 μl reaction).

[0090] The PCR products were purified by phenol-chloroform followed by ultrafiltration. For tRNA sequences beginning with a 5' nucleotide other than guanosine, a self-cleaving hammerhead motif is placed 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 dsDNA templates 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] The 1 ml transcription reaction system contained 20 μg of purified dsDNA template, 2 mM each of NTPs, 0.1 mg / ml of T7 RNA polymerase, 400 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, MA, USA), and 25 mM MgCl 2 The transcription reaction products were incubated for 2 h at 37°C, treated with 10 μl of DNase I (New England Biolabs, MA, USA), and incubated for an additional 0.5 h before being quenched by the addition of 60 μl of 0.5 M EDTA, followed by ethanol precipitation. The transcribed RNA was purified using the "crush and soak" method. 7 The tRNAs were gel purified using HPLC, desalted, and concentrated by ultrafiltration. Using this method, both 5'-triphosphate (tRNA sequences beginning with G) and 5'-hydroxyl terminal tRNAs (tRNA sequences beginning with A / U / C) were prepared; His With the exception of , it has been shown previously that these tRNAs are functionally equivalent to physiological 5'-monophosphate-terminated tRNAs [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]. Thus, the two versions of tRNA HisThe following was prepared: 5'-triphosphate-terminated tRNA His was used in all flexizyme-based cargo assays; a 5'-monophosphate-terminated tRNA with an additional G at position -1 His was prepared by a previously reported method and used as a control requiring aaRS activity.

[0093] Flexizyme-catalyzed tRNA-aminoacylation: The procedure for flexizyme-catalyzed tRNA cargo 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, pH 7.5, containing 50 mM HEPES-KOH and 100 mM KCl. The mixture was heated to 95°C for 2 min and cooled to 25°C for 10 min, followed by addition of 100 mM MgCl. 2 The mixture was incubated at room temperature for 10 min and on a refrigerated metal block for 3 min. 5 mM DBE substrate was added to the system on the cold metal block to initiate the cargo reaction. The reaction products were incubated at 4°C for 6 h, quenched with 2x 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, the substrate was supplemented with 5 mM DTT; for Pro-DBE, the substrate concentration was increased from 5 mM to 40 mM; for Fph-CME, 30 μM enhanced flexizyme was used, the Fph-CME substrate concentration was reduced from 5 mM to 1 mM, and MgCl 2The concentration was increased from 100 mM to 400 mM; for Asn-CBT, the substrate concentration was increased from 5 mM to 25 mM; for Trp-DBE, 20% DMSO was additionally added. Cargo yields were determined by acidic PAGE analysis: precipitated cargo reaction products were dissolved in loading buffer containing 93% formamide, 100 mM NaOAc at pH 5.2, 10 mM EDTA, and a trace of bromophenol blue. Acidic gels were prepared with 8% acrylamide, 100 mM NaOAc at pH 5.2, and 7 M urea. Gels were run for 16 h in a 4 °C refrigerator with an aluminum cooling plate using 100 mM NaOAc as the running buffer at pH 5.2. Gels were stained with SYBR-Green II (Thermo Fisher Scientific, MA, USA), scanned on a Typhoon FLA 9500 operated under Cy2 mode, and analyzed with the software package ImageJ [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 The peak areas were integrated to calculate the yield of loaded tRNA, excluding Asp-tRNA. Asp The peak height was used to estimate the yield of Asp-tRNA. Asp is an unloaded tRNA Asp (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 the 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 by the software package ImageJ 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, reaction mixtures were adjusted so that each codon was decoded by a cognate flexizyme-loaded tRNA ranging from 1.25 to 80 μM. The tRNA concentrations corresponded to 16 to 1000 μM total tRNA. HPLC purification of flexizyme-loaded tRNA was performed as described in 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 remained stable as a dry pellet at -80°C for up to 3 days as judged by acidic PAGE analysis. The loaded tRNA pellet was dissolved in 0.5x translation volume of 1 mM NaOAc, pH 5.2. Serial dilutions were performed to generate 2x concentrated tRNA with the concentrations shown in Table 3. Table 3 shows the total tRNA concentrations used for aaRS-free translation.

[0098] [Table 3]

[0099] The translation reaction was started by mixing tRNA with an equal volume of 2x aaRS-free translation mix, preincubated at 37 °C for 5 min. All translation reactions were incubated at 37 °C for 2 h. 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 Fph-labeled peptides. Peptide standards (Fph-KYDKYD (SEQ ID NO: 125)) at 0.125–4 μM, custom synthesized by Genscript (Jiangsu, China), were also loaded for calibration. Titration of unloaded tRNA was performed using tRNA fMet :tRNA Lys :tRNA Tyr:tRNA Asp =1:2.5:2.5:2.5 molar ratio, mixed with flexizyme-loaded tRNA, incubated briefly at room temperature and then added to the aaRS-free translation system. The final concentration of unloaded tRNA was 90-470 μM. Flexizyme titrations were performed by mixing dinitro-flexizyme with flexizyme-loaded tRNA in 1 mM NaOAc before mixing with an equal volume of 2x aaRS-free mix to start the translation reaction. The final concentration of flexizyme was 240-520 μM. Titrations of folded flexizyme and tRNA complexes were performed with 50 μM dinitro-flexizyme, 6 μM tRNA fMet , 15 μM tRNA each Lys , tRNA Tyr , and tRNA Asp The mixture was then slowly cooled to 25°C and heated to 95°C for 2 min in the presence of 50 mM HEPES-KOH at pH 7.5 and 100 mM KCl. 2 or 10 mM MgCl 2 Either 100% or 100% ethanol 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 diluted with a small amount of ddH 2 The flexizyme-tRNA complexes were dissolved in ddHO and serially diluted to reach 2x concentrations of 750, 375, and 188 µM, respectively. 2 An equal amount of the aaRS-free translation system was added along with a control containing O only.

[0100] MALDI-TOF MS: MALDI-TOF MS was used to analyze aaRS-free translation of mRNA#2-#6. To reduce peptide shedding, the scale of each loading reaction was adjusted according to codon abundance in each gene, such that each codon was matched with an equal concentration of flexizyme-loaded tRNA (10 μM per codon for mRNA#2-#5 and 5 μM for mRNA#6). Controls containing unloaded tRNA contained 30 μM (respectively) of tRNA. Asn, tRNA Glu , tRNA Lys , tRNA Ile , and 5 μM of other tRNAs (each), and 100 μM of each amino acid. The loading reaction was quenched, precipitated, and washed once with 70% ethanol. The washed pellets of the different 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 tRNA with an equal volume of 2x aaRS-free translation mix. After 2 h translation at 37°C, TFA was added to the translation system to reduce the pH to less than 4, the sample was briefly centrifuged, and the supernatant was desalted with a C18 spin column (Thermo Fisher Scientific, MA, USA). After elution, the sample volume was reduced to approximately 2–3 μl by a centrifugal vacuum concentrator (Eppendorf, Germany), of which 0.5 μl was used for MALDI-TOF analysis (Applied Biosystems 4800 plus MALDI TOF / TOF analyzer, CA, USA) under positive reflectron mode. Control experiments with unloaded tRNA and free amino acids (100 μM for each amino acid type) were performed, desalted, and analyzed by MALDI-TOF MS in parallel. The concentration of unloaded tRNA used in the control experiment was 100 μM for tRNA. Asn , tRNA Glu , tRNA Lys , tRNA Ile for each tRNA, it was 30 μM (each), and for the other tRNAs, it was 5 μM (each).

[0101] Protein Identification by LC-MS / MS: A volume of 20 μl of crude aaRS-free translated N-terminal FAM-labeled E. coli TrpRS was separated by 15% SDS-PAGE and silver stained with ProteoSilver silver staining kit (Sigma-Aldrich, MO, USA). Protein bands between EF-Tu (43 kDa) and MTF (34 kDa) were excised from the gel, reduced with 5 mM dithiothreitol, and alkylated with 11 mM iodoacetamide. In-gel digestion was performed overnight at 37°C with sequencing-grade trypsin in 50 mM ammonium bicarbonate. Peptides were extracted twice with 0.1% TFA in 50% aqueous acetonitrile for 30 min. The extracts were then concentrated by centrifugal vacuum concentrator. Peptides resulting from tryptic digestion were 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 mirror image tRNA: The synthesis and folding of D-polymerase D-Dpo4-5m-Y12S for mirror-image transcription was reported previously [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-nucleic acid oligo sequences are listed in Table 4. In the table, "*" indicates phosphorothioate modification, capital letters indicate L-DNA nucleotides, and lowercase letters indicate L-RNA nucleotides.

[0103] [Table 4]

[0104] Mirror transcription was performed with a 24 nt primer binding site tethered to the 3' end of the mirror single-stranded DNA (L-ssDNA) template to facilitate RNA purification by PAGE due to the different product lengths (so that the L-RNA transcripts were 23 nucleotides shorter than the 99 nucleotide L-ssDNA template, which could be separated by 12% denaturing PAGE in 7M urea). The L-primer was designed to contain a phosphorothioate modified L-RNA nucleotide at the 3' end. Mirror tRNA Ala , tRNA Gly , tRNA Lys and tRNA Phe After mirror image transcription, the L-primer was cleaved at the phosphorothioate site with 100 μM I in ethanol at 70 °C for 10 min. 2 For RNase A digestion, 0.4 μM D- or L-tRNA was used. Ala was mixed with 4 μM RNase A, incubated at 37° C. for 15 min, and analyzed by 10% denaturing PAGE in 7 M urea. Ala 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] Mirror tRNA cargo: Mirror tRNA cargo was prepared using the same aminoacylation method as above, except that the L-tRNA and L-flexizyme concentrations were scaled down to 2 μM and 10 μM, respectively. Mirror tRNA was transcribed by synthetic D-Dpo4-5m-Y12S polymerase, and natural tRNA was transcribed by 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 Phe) The tRNA loading yield was determined by the software package ImageJ 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 their cognate amino acids by the 46-nucleotide dinitro-flexizyme or the 45-nucleotide enhanced flexizyme. The loading reaction was quenched with 0.3 M NaOAc and precipitated. The pellet was purified by either a 70% ethanol wash or a Shimadzu Prominence HPLC system (Japan) as appropriate (see Figures 8A and 8B).

[0107] 3A-3E show the concept and results of flexizyme-mediated loading of tRNA en route to aaRS-free loading of mirror-image tRNA according to some embodiments of the present invention, including D-tRNA cargo catalyzed by D-flexizyme and its mirror-image version, i.e., mirror-image tRNA cargo catalyzed by L-flexizyme (PDB source: 1EHZ (tRNA), 3CUL (flexizyme)) (FIG. 3A); loading of D-alanine by L-flexizyme onto enzymatically transcribed mirror-image tRNAAla, natural chiral tRNA, and the like. The loading of glycine by L-flexizyme onto the enzymatically transcribed mirror-image tRNAGly, the native chirality counterpart is also shown for comparison (Figure 3B), the loading of D-lysine by L-flexizyme onto the enzymatically transcribed mirror-image tRNALys, the native chirality counterpart is also shown for comparison (Figure 3D), and the loading of D-phenylalanine by L-flexizyme onto the enzymatically transcribed mirror-image tRNAPhe, the native chirality counterpart is also shown for comparison (Figure 3E). The tRNA loading yield was determined using the software package ImageJ using the integrated peak area of ​​the loaded tRNA relative to the total tRNA.

[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 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]. Fractions containing flexizyme-loaded tRNA were precipitated and dissolved in 10 mM NaOAc at pH 5.2, and the concentration was measured by Nanodrop spectrophotometer (Thermo Fisher Scientific, MA, USA). The desired amount of tRNA was then mixed and precipitated again by ethanol. The pellet was air-dried and stored at -80 °C until use.

[0109] Example 3 Cell-free in vitro translation The cell-free in vitro translation mix was 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 protein, IF2 protein, IF3 protein, EF-Ts protein, EF-Tu protein, EF-G protein, RF-2 protein, RF-3 protein, RRF protein, and MTF protein were expressed in Escherichia coli BL21 strain with an N-terminal TEV-cleavable His tag, 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 diluted with 50 mM NaCl at pH 7.6. The mixture was exchanged into a buffer containing HEPES, 100 mM potassium glutamate, 10 mM magnesium acetate, 7 mM β-mercaptoethanol, and 30% glycerol. Buffer components 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 aaRS-free translation of protein enzymes The 20-codon DNA templates for chicken lysozyme, Gaussia luciferase, and E. coli TrpRS were synthesized and assembled by Genewiz (Jiangsu, China) and cloned into 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. Upon 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 dissolved tRNA mix was then added to the aaRS-free translation mix pre-incubated at 37°C for 5 min, where the final DNA template concentration was approximately 10 ng / μl. For translation of lysozyme and luciferase, approximately 1 μM of flexizyme-loaded tRNA was used for each codon to be translated. For translation of TrpRS, 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 codons Cys, Pro, and Cys, and approximately 0.2 μM of flexizyme-loaded tRNA for each of the remaining codons (total tRNA concentrations are shown in Table 3 above). Control experiments without DNA template were performed using the same flexizyme-loaded tRNA concentrations, while control experiments with unloaded tRNA were performed using tRNA concentrations of 0.1 μM. Asn , tRNA Glu , tRNA Lys , tRNA IletRNAs were used at 30 μM (each), other tRNAs at 5 μM (each), and free amino acids at 100 μM (each). Translation reactions were incubated at 37°C for 2 h for lysozyme and luciferase, and 4 h 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 min for loading. Alexa Fluor 488-labeled Benchmark fluorescent protein standards were purchased from Thermo Fisher Scientific (MA, USA). Gels were scanned by a Typhoon FLA 9500 (GE Healthcare, UK) operated in Cy2 mode.

[0113] Example 5 Biochemical characterization of aaRS-free translated protein enzymes aaRS-free translation of chicken lysozyme, Gaussia luciferase, and Escherichia coli TrpRS was performed using Met-tRNA fMetThe chicken lysozyme translation mix was diluted with an equal volume of 2× folding buffer (pH 7.5) containing 0.1 M sodium phosphate and 0.1 M NaCl, incubated at room temperature for 24 h, and assayed by the EnzChek Lysozyme Assay Kit (Thermo Fisher Scientific, MA, USA). The translation mix for Gaussia luciferase, previously shown to promote disulfide bond formation in recombinant Gaussia luciferase [Yu, T., Laird, JR, Prescher, JA, and Thorpe, C. (2018). Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding. Protein Science 27, 1509-1517], was diluted with an equal volume of 2× folding buffer (pH 7.3) containing 6 mM reduced glutathione and 4 mM oxidized glutathione, incubated at room temperature for 16 h, and assayed by Pierce Gaussia Luciferase Glow Assay Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. For translation of E. coli TrpRS, Cy5-tRNA was used. Trp was prepared by enzymatic ligation of two synthetic oligos and purified by 10% denaturing 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 internal Cy5-labeled tRNAs. Trp 1 shows the RNA oligo sequence for enzymatic ligation of

[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 was completed, incubated at 37° C. for 1 h, quenched with 0.3 M NaOAc and extracted with phenol-chloroform. The loaded samples were analyzed by 8% acid PAGE (Supplementary Information) and scanned by a Typhoon FLA 9500 operated in Cy5 mode. 2 μM unloaded Cy5-tRNA Trp , and 2 μM Cy5-tRNA loaded with 100 nM recombinant E. coli TrpRS. Trp Samples were used as standards. All control experiments lacking DNA template or using uncargoed tRNA and free amino acids were assayed under identical 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 investigated an aaRS-free translation system. The rationale was that increasing the concentration of flexizyme-loaded tRNA to compensate for the lack of tRNA recycling might improve the yield of aaRS-free translation. Previous studies have shown that in an aaRS-based E. coli translation system, the addition of excess tRNA inhibits translation [Rojiani, MV, Jakubowski, H., and Goldman, E. (1990). Relationship between protein synthesis and concentrations of charged and uncharged tRNA Trpin 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 tRNAs outcompeting loaded tRNAs by occupying the ribosomal A site, or to a cation imbalance resulting from the addition of large amounts of tRNA. However, since all these experiments were performed in the presence of aaRS, the exact loading yield was not determined, and inefficient loading and altered cations (e.g., Mg 2+ ) concentration effects were difficult to discern.

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

[0118] 4A-4G show the results of aaRS-free translation of multiple short peptides according to some embodiments of the present invention. Shown are MALDI-TOF-MS analysis of short peptides translated from mRNA#1 (FIG. 4A); aaRS-free translation yields of short peptides analyzed by Tricine-SDS-PAGE, where unloaded tRNA concentrations ranged from 160 to 540 μM and flexizyme-loaded tRNA concentrations were 70 μM, resulting in loading yields ranging from 44 to 13% (top of FIG. 4B); and total tRNA concentrations ranged from 16 to 1003 μM, with loading yields remaining at 56% (FIG. 4C). Bottom of B) (error bars represent 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 control of translation was performed with 55-135 μM total amount of unloaded tRNA and 100 μM of each amino acid (for mRNA#1, FAM-labeled Fph-tRNA loaded by 5 μM flexizyme). fMet added to both control and aaRS-free translation experiments), and also perform aaRS-free translation of aaRS-free translation with 170-414 µM total flexizyme-loaded tRNA (see Table 3). au represents arbitrary units, and C, O represent calculated and observed m / z values ​​(Figure 4C-G, respectively).

[0119] The mRNA template is converted to tRNA fMet , tRNA Lys , tRNA Tyr and tRNA Asp Decoded by tRNA fMet The 45-nucleotide enhancer flexizyme is used to synthesize Fph-tRNA. fMetwere loaded, and others were loaded with their cognate amino acids by the 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 work in ribosomal peptide synthesis assays. Individual loading yields for each tRNA were determined by polyacrylamide gel electrophoresis under acidic conditions (acid-PAGE) and used to estimate the weighted average (overall) loading yield of the translation system (see Table 3). Titration of tRNAs was performed to obtain a total loading yield of approximately 44% (Fph-tRNA mixed at a molar ratio of 1:2:2:2). fMet :Lys-tRNA Lys :Tyr-tRNA Tyr :Asp-tRNA Asp ), first by adding loaded total tRNA such that the total tRNA concentration in the final translation system ranged from 20 to 644 μM, and found that the translation yield reached its highest level when the total tRNA concentration was approximately 160 μM, without plateauing, and that further increases in tRNA concentration resulted in a decrease in translation yield (see Figure 5A ).

[0120] Figures 5A-5D show the results of aaRS-free translation of mRNA#1 under various conditions. Shown are that the loading yield remained at 44% when the total tRNA concentration ranged from 20 to 644 μM (Figure 5A), the total flexizyme concentration ranged from 240 to 525 μM, the total tRNA concentration remained at 160 μM (Figure 5B), and the loading yield remained at 44% when the total flexizyme concentration ranged from 240 to 525 μM (Figure 5C). Flexizyme and 0 to 380 μM unloaded tRNA were used in the presence of 10 mM MgCl. 2 In Fig. 5D, 100 mM MgCl 2The flexizyme-loaded tRNA was mixed in PBS, desalted by ethanol precipitation, and added to the aaRS-free translation mix, leaving the concentration of flexizyme-loaded tRNA at 70 μM (error bars represent standard deviation from three independent experiments).

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

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

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

[0124] Another factor that may be responsible for the observed translation inhibition is the release of Mg from flexizyme-loaded tRNA. 2+ It was speculated that this was due to carryover of cations. To test this theory, we added exogenous MgCl 2 When MgCl was added to the flexizyme-loaded tRNA, translation was indeed accelerated. 2 It was found that the inhibition was accompanied by increased 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 depleted with Mg2+ The concentration was reduced (Fph-tRNA was instead treated by ultrafiltration to minimize fluorescence quenching). fMet This process also removed most of the flexizyme and modestly improved the overall cargo yield from 44 to 56% (see Figure 2).

[0126] The purified flexizyme-loaded tRNA was then added to the aaRS-free translation system, and it was observed that the translation yield was significantly improved by 5-fold when only the flexizyme-loaded tRNA was concentrated. 2+ A further 2-fold improvement was observed upon reducing contamination, resulting in an overall approximately 10-fold improvement in translation yield (see Figures 4B and 5A) and shifting the optimal total tRNA concentration from 160 μM to 500 μM.

[0127] However, when the tRNA concentration was further increased from 500 to 1000 μM, the overall translation yield decreased by about 50%, which was due to the Fph-tRNA fMet Mg 2+ Moreover, a similar titration assay using high concentrations of unloaded tRNA in the presence of aaRS did not result in improved translation yields (see Figures 7A-B), suggesting that the improvement in aaRS-free translation yields is likely due to the increased concentration of flexizyme-loaded tRNA per se.

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

[0129] Example 7

[0130] aaRS-free translation of multiple short peptides Having found that increasing the tRNA concentration improves the yield of aaRS-free translation, the inventors sought to test aaRS-free translation of several short peptides and determine the translation fidelity at high flexizyme-loaded tRNA concentrations. fMet A minimal set of 21 E. coli tRNAs, including tRNA 1, tRNA 2, tRNA 3, tRNA 4, tRNA 5, tRNA 6, tRNA 7, tRNA 8, tRNA 9, tRNA 10, tRNA 11, tRNA 12, tRNA 13, tRNA 14, tRNA 15, tRNA 16, tRNA 17, tRNA 18, tRNA 19, tRNA 20, tRNA 21, tRNA 22, tRNA 23, tRNA 24, tRNA 25, tRNA 26, tRNA 27, tRNA 28, tRNA 29, tRNA 30, tRNA 31, tRNA 32, tRNA 33, tRNA 34, tRNA 35, tRNA

[0131] [Table 7-1] [Table 7-2]

[0132] Each tRNA was loaded separately by flexizyme, with loading yields ranging from 20 to 60% (see Figure 8A). Figures 8A-8B show the loading yields of 21 tRNAs with their cognate proteinogenic amino acids by flexizyme, determined after ethanol precipitation (Figure 8A) and after HPLC purification of 14 flexizyme-loaded tRNAs. N / A indicates that no purification of flexizyme-loaded tRNAs was performed (Figure 8B), and gly-tRNA was used to facilitate purification as previously reported. Gly Reversible N-pentenoylation was carried out on

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

[0134] MALDI-TOF MS results showed that all 21 Flexizyme-loaded tRNAs accurately decoded mRNAs at up to about 200-fold molar excess over ribosomes (e.g., for mRNA#5, 414 μM tRNA vs. 2 μM ribosomes). In control experiments with unloaded tRNAs and free amino acids (see Figures 4C-G), no peptide products were detected, thus minimizing concerns of contamination with aaRS and loaded tRNAs from ribosome preparations.

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

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

[0137] Figure 9 shows MALDI-TOF MS analysis of aaRS-free translated mRNA #6, showing that at higher total tRNA concentrations (520 μM) in the aaRS-free translation system, a mistranslated product with a molecular weight of 2,252.7 Da was observed, whereas the correctly translated product had a molecular weight of 2,240.7 Da. au represents arbitrary units, and C and O represent calculated and observed m / z values, respectively.

[0138] Example 8 aaRS-free translation of protein enzymes The successful translation of short peptides led to aaRS-free translation of protein enzymes using only in vitro transcribed unmodified tRNAs loaded by the Flexizyme system. Two small enzymes, chicken lysozyme with 130 amino acid residues and Gaussia luciferase with 169 amino acid residues, were chosen as models. Neither enzyme is native to E. coli, which minimizes the concern of 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 positions translated by flexizyme-loaded tRNA were purified by either ethanol precipitation or HPLC (underlined).

[0140] A subset of 21 flexizyme-loaded tRNAs (amino acids underlined in Figures 10A-B) was purified by HPLC and incubated with Mg 2+Carryover was reduced and individual cargo yields after HPLC purification were determined by acidic PAGE (see FIG. 8B), resulting in an overall cargo yield of approximately 40%. The total tRNA concentrations of approximately 330 μM for chicken lysozyme and approximately 430 μM for Gaussia luciferase (Table 3) were approximately 10-20 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] aaRS-free translation of full-length protein was confirmed using FAM-labeled Fph-tRNA fMet When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the fluorescently labeled product bands matched the molecular weights of chicken lysozyme and Gaussia luciferase (14.8 kDa and 18.7 kDa, respectively), and the mobilities of the product bands were similar to those of commercial chicken lysozyme and recombinant Gaussia luciferase, respectively (see Figures 11A-11D).

[0142] 11A-11G show SDS-PAGE analysis of aaRS-free translated protein enzymes, including the full gel image shown in FIG. 12A (FIG. 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 (FIG. 11B); a full gel image shown in FIG. 12C (FIG. 11C); a 400 ng sample of recombinant Gaussia luciferase expressed and purified from E. coli strain BL21 analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue (FIG. 11D); and a full gel image shown in FIG. 14A (FIG. 11E); a 400 ng 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 (FIG. 14A). 300 ng sample (FIG. 11F), heated to 98° C. for 3 min or not, analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode, 5 μM Fph-CME, 1 μM Fph-tRNA fMet , and 5 μM Fph-tRNA fMet A sample of (Figure 11G) is shown, where M is a benchmark fluorescent protein standard.

[0143] In contrast, no product bands were present in control experiments lacking a DNA template or using uncargoed tRNA and free amino acids (see Figures 12A and 12C).

[0144] 12A-12D show the results of experimental proof of concept of aaRS-free translation of protein enzymes according to some embodiments of the present invention, including aaRS-free translation of N-terminal FAM-labeled chicken lysozyme analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode (M stands for benchmark fluorescent protein standard) (FIG. 12A); enzymatic assay of crude aaRS-free translated chicken lysozyme using fluorescently labeled bacterial (Micrococcus rhizodeikticus) cell wall material as substrate (FIG. 12B); and enzyme assay of N-terminal FAM-labeled chicken lysozyme analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode (FIG. 12C). aaRS-free translation of N-terminal FAM-labeled Gaussia luciferase scanned by 9500 (FIG. 12C) and enzyme assay of crude aaRS-free translated Gaussia luciferase using coelenterazine as substrate (FIG. 12D) are shown (RFU stands for relative fluorescence units, RLU stands for relative luminescence units).

[0145] These results suggested that aaRS-free translation was sufficiently processive to achieve synthesis of a full-length protein before the flexizyme-loaded tRNA was hydrolyzed. Attempts to characterize the aaRS-free translated protein from the excised product band using liquid chromatography-tandem mass spectrometry (LC-MS / MS) were unsuccessful due to contamination with ribosomal proteins. However, this was addressed by translating a larger protein whose molecular weight was further different from that of the ribosomal proteins, as described in this disclosure. Next, FAM-labeled Fph-tRNA was used to initiate translation. fMet Unlabeled Met-tRNA fMetand enzyme assays were performed to test whether the translated proteins could fold correctly in vitro and have their corresponding catalytic activity. The results showed that after incubation in folding buffer for up to 24 hours, 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 lacking a DNA template or using uncargoed tRNA and free amino acids did not produce detectable signals, thus minimizing concerns of autofluorescence or contaminating luminescence from the aaRS-free translation system.

[0146] Comparison of the emitted bioluminescence of aaRS-free translated Gaussia luciferase with known standards of recombinant luciferase suggested a translation yield of approximately 25 nM (see FIG. 13), which was approximately 80-fold lower than the maximum yield of aaRS-free translation of a seven amino acid residue peptide (see FIG. 4B), likely a result of the low availability of flexizyme-loaded tRNAs for each translation codon and the limited folding efficiency of Gaussia luciferase with multiple disulfide bonds.

[0147] FIG. 13 shows the yield estimate of aaRS-free translated Gaussia luciferase, where a standard curve was plotted using 0, 25 nM, 50 nM, 100 nM and 250 nM recombinant Gaussia luciferase (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 inventors sought to explore the potential of the aaRS-free translation system to produce functional aaRS itself, a key step in establishing a self-replicating translation apparatus. To this end, the 334 amino acid residue E. coli TrpRS was used as a model. The majority of the in vitro transcribed flexizyme-loaded tRNAs (14 out of a total of 21) were purified by HPLC and then incubated with Mg. 2+ (underlined amino acids in FIG. 10A ), resulting in an overall cargo yield of about 42% and a total tRNA concentration of about 170 μM (see Table 3).

[0149] The inventors of the present application have fMet Translation of the full-length protein was tested using the reporter, and a product band representing the 334 amino acid residue E. coli TrpRS (37.8 kDa) was observed by SDS-PAGE (the mobility of the fluorescently labeled protein band was similar to that of recombinant TrpRS) (see Figures 6E and 6F), but this band was absent in control experiments lacking a DNA template or using uncargoed tRNA and free amino acids (see Figure 14A).

[0150] Figures 14A-C show aaRS-free translation of TrpRS, an N-terminal FAM-labeled E. coli TrpRS (M stands for benchmark fluorescent protein standard) analyzed by 15% SDS-PAGE and scanned by Typhoon FLA 9500 under Cy2 mode (Figure 14A), an internal Cy5-labeled tRNA (M stands for benchmark fluorescent protein standard) (Figure 14B). Trp (Figure 14B), and Cy5-tRNA Trp 14C shows an enzymatic assay of crude aaRS-free translated TrpRS using as a substrate and analyzed by 8% acid PAGE and scanned by Typhoon FLA 9500 under Cy5 mode.

[0151] Several faster migrating bands were also observed, which may represent the truncated TrpRS translation product and unused Fph-tRNA. fMet (see FIG. 11G).

[0152] To further confirm the aaRS-free translation of TrpRS, protein content from the excised product bands was analyzed using LC-MS / MS and four non-overlapping peptide segments were identified from E. coli TrpRS, resulting in approximately 16% sequence coverage. On the other hand, no peptides corresponding to E. coli TrpRS were detected in control experiments using uncargoed tRNA and free amino acids, suggesting that the detected TrpRS was not derived from endogenous aaRS contamination (Table 8).

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

[0154] [Table 8]

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

[0156] The placement of the Cy5 label allows for the transport of loaded Cy5-tRNA without interference from other loaded tRNA species. Trp Allows in situ detection of Met-tRNA for translation initiation fMet After translation of TrpRS, Cy5-tRNA was synthesized. Trp was added to the aaRS-free translation system together with tryptophan and adenosine triphosphate (ATP). Trp While tryptophan was successfully loaded onto Cy5-tRNA, in control experiments lacking a DNA template or using unloaded tRNA and free amino acids, Cy5-tRNA was successfully loaded onto Cy5-tRNA. TrpNo cargo was observed (see FIG. 14C). Trp This suggests that the cargo activity is unlikely to be due to endogenous aaRS contamination from the ribosome preparation or residual flexizyme activity.

[0157] Example 10 aaRS-free cargo onto mirror tRNA As a proof-of-concept experiment to test the loading of mirror D-amino acids onto mirror L-tRNA by the synthetic mirror L-flexizyme (see FIG. 3A), we applied a previously established mirror gene transcription system based on an engineered mutant mirror version of Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) to transcribe the mirror tRNA (see FIG. 15A).

[0158] FIG. 15A-FIG. 15B show the mirror image tRNA produced by D-Dpo4-5m-Y12S. Lys Figure 15A shows the results of transcription of tRNA, extension of 5'-FAM-labeled L-universal primer on L-ssDNA template polymerized by synthetic Dpo4-5m-Y12S polymerase, and reaction aliquots terminated at different time points and analyzed by 12% denaturing PAGE gel in 7 M urea (Figure 15B), and tRNA, analyzed by 10% denaturing PAGE gel in 7 M urea and stained with SYBR-Green II (Thermo Fisher Scientific, MA, US). Lys Mirror image transfer of the transcript and I 2 mediated cleavage (Figure 15B).

[0159] To avoid the high cost of synthesizing 21 different L-RNA primers, we applied a universal primer for transcription of the mirror image tRNA (see FIG. 15B).

[0160] The fully extended primer is then ligated via a previously reported mechanism. 2The universal primer was modified with a phosphorothioate near the 3' end so that it could be efficiently cleaved by RNase A to produce a full-length mirror-image tRNA (see Figure 15B), which, as expected, was resistant to native RNase A digestion and could not be loaded by native aaRS (see Figures 16A-B).

[0161] 16A-16B show the results of biochemical characterization of enzymatically transcribed native tRNAs and mirror image tRNAs, including enzymatically transcribed D- and L-tRNAs. Ala RNase A digestion of (Figure 16A) and enzymatically transcribed D- and L-tRNA Ala 16B shows the AaRS-catalyzed aminoacylation of

[0162] I 2 The mediated cleavage results in RNA with a hydroxyl-terminated 5'-end, as verified by MALDI-TOF MS (see Figures 17A-C). 17A to 17C are diagrams showing the I 2 1 shows MALDI-TOF MS analysis of I-mediated cleavage. 2 Synthetic DNA-RNA chimeric oligos cleaved at the phosphorothioate modification sites by I (Figure 17A), MALDI-TOF MS spectra of the uncleaved oligos in negative linear mode (Figure 17B), and I in negative linear mode (m / z>4000) and negative reflectron mode (m / z<4000). 2 The MALDI-TOF MS spectrum of the oligo cleaved with , is shown (Figure 17C), where uppercase letters indicate DNA nucleotides, lowercase letters indicate RNA nucleotides, and "*" indicates phosphorothioate modification. au represents arbitrary units, and C, O represent calculated and observed m / z values, respectively.

[0163] Next, a chemically synthesized 46-nucleotide L-flexizyme (dinitro-flexizyme) was successfully used to load four representative D-amino acids (lysine, alanine, glycine, and phenylalanine) belonging to different amino acid categories (polar (Lys), non-polar (Ala), achiral (Gly), and aromatic (Phe) respectively) onto their cognate mirror image tRNAs with similar efficiencies comparable to those of the natural system (Figures 3B-E).

[0164] Example 11 Translation of complete or partial unnatural peptides using cation-depleted flexizyme-loaded tRNAs It is inferred that the flexizyme system can be used to incorporate multiple unnatural amino acids for peptide translation, either in combination with or without other aaRS proteins. The present invention provides the ability to test whether unnatural peptides can be translated using cation-depleted flexizyme-loaded tRNAs, or at least using preparations of flexizyme-loaded tRNAs in which the concentration of Mg+2 has essentially been reduced to the lowest possible level, and whether purification and concentration of cation-depleted flexizyme-loaded tRNAs by means such as HPLC and ultrafiltration can increase the translation yield, especially for difficult-to-translate peptides, e.g., completely or partially unnatural peptides. In the translation system, aaRS proteins may be added to enhance the loading of certain tRNAs that are not loaded by flexizymes (see Figures 18A-B).

[0165] 18A-18B show flow chart translation of complete or partial unnatural peptides using cation-depleted flexizyme-loaded tRNAs, translation of peptide drugs and unnatural proteins using cation-depleted flexizyme-loaded tRNAs in an in vitro translation system (see FIG. 18A), and translation, data storage, and ribosome / mRNA display of complete or partial unnatural proteins using cation-depleted flexizyme-loaded tRNAs in an in vitro translation system (see FIG. 18B).

[0166] In this experiment, unnatural amino acids are first loaded onto unmodified tRNA. Unnatural 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). 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. The flexizyme-loaded tRNA is then concentrated to 100-500 μM and used as a substrate for in vitro translation. The translation products are analyzed by MALDI-TOF MS and Tricine-SDS-PAGE.

[0167] As a proof of concept, a peptide drug is translated using an aaRS-free translation system (see Figure 18A). The amino acid sequence of a peptide drug is Ac yFAYDRR(2-ACHC)LSNN(2-ACHC)RNYcG-NH 2(SEQ ID NO: 124), in which the first amino acid is Acetyl-D-Tyr and the penultimate amino acid is D-Cys, which spontaneously forms a cyclic bond with the Acetyl-D-Tyr residue. This peptide has previously been 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 the 169 amino acid Gaussia luciferase can be synthesized by binding to tRNA Asn , tRNA Ile and tRNA Lys The tRNAs are translated using cation-depleted flexizyme-loaded tRNAs, including but not limited to, tRNAs 10 and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 fMet The translation products are analyzed by measuring bioluminescence and SDS-PAGE. Since the Fph residue makes Gaussia luciferase fluorescent on SDS-PAGE gels, the purity of the Gaussia luciferase translated thereby can be easily determined based on its fluorescence. This application does not require radioisotopes and laborious protein purification procedures and is useful for high-throughput analysis of translation purity.

[0169] Translation of fully or partially unnatural peptides using cation-depleted flexizyme-loaded tRNAs, used 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 data storage with fully or partially unnatural peptides using amino acid letters (see FIG. 18B).

[0170] Example 12 Enzymatically transcribed L-tRNA cargo using L-flexizyme Figures 19A-19B show 8% acid PAGE photographs and analysis of the experimental proof of concept of loading fully functional enzymatically transcribed L-tRNA molecules with preactivated amino acids, where Figure 19A shows the results of loading enzymatically transcribed L-tRNA and Figure 19B shows the results of loading synthetically produced L-tRNA.

[0171] As seen in Figures 19A-B, a band shift is evident when enzymatically transcribed L-tRNA is loaded (Figure 19A), whereas when pre-activated amino acids are loaded onto L-tRNA prepared by a commercial synthesizer with L-flexizyme, no band shift is observed and the loaded L-tRNA molecules cannot be distinguished from unloaded tRNA molecules. This is probably due to the low quality of synthetically prepared tRNA.

[0172] This experiment clearly demonstrates the advantage of obtaining enzymatically transcribed L-tRNA molecules and also clearly shows the advantage of using L-flexizyme and obtaining a D-enzyme capable of enzymatically transcribing L-RNA molecules.

[0173] Example 13 Translation of peptides containing two or three consecutive D-phenylalanines To test the effect of increasing the concentration of cation-depleted tRNA and whether it can improve the translation yield of difficult unnatural amino acids such as D-amino acids, the inventors synthesized a short peptide (mRNA#7): Fph-KKK D F D We attempted to translate FDYKDDDDK (SEQ ID NO: 127) and loaded its fluorescein-labeled L-phenylalanine (Fph) and L-lysine (K) onto their cognate tRNAs by flexizyme, while L-aspartate (D) and L-tyrosine (Y) onto their cognate tRNAs by 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] According to the teachings 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) a modified tRNA having a sequence optimized for D-amino acid incorporation; GluE2 CUA (see Table 9) was loaded with Flexizyme ( D Phe-tRNA GluE2 CUA ). This peptide contains two consecutive D-phenylalanines, which has previously been shown to be difficult to translate, with a yield of less than 15%, compared to a peptide of the same sequence but containing two consecutive L-phenylalanines [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 the present application designed and in vitro transcribed mRNA#7 capable of Watson-Crick base pairing with the anticodon of flexizyme-loaded tRNA (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] For in vitro translation, the inventors used cation depletion at 20 μM or 200 μM. D Phe-tRNA GluE2 CUA In both translation reactions, the overall Mg 2+ The carryover of Mg in the in vitro translation system proposed in this disclosure 2+ Within acceptable limits (<100mM Mg 2+ As a control, the inventors of the present invention also used flexizyme-loaded tRNA Phe ( L Phe-tRNA Phe ) was used to generate 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 (SEQ ID NO: 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 correct incorporation of two consecutive D-phenylalanines in mRNA#7 (FIG. 20A). However, the mass peak was not detected at 200 μM. D Phe-tRNA GluE2 CUA It can only be detected in samples containing 20 μM D Phe-tRNA GluE2 CUA In contrast, in the control experiment, the exact mass peak was not detected at 20 μM. L Phe-tRNA Phe However, it was possible to detect uncargoed tRNA in samples containing PheFurthermore, the results of Tricine-SDS-PAGE showed that the 200 μM D Phe-tRNA GluE2 CUA The translation yield of mRNA#7 when using 20 μM D Phe-tRNA GluE2 CUA The translation yield was approximately two times higher than that obtained with 20 μM L Phe-tRNA Phe The results show that the results were similar to those of the control using

[0181] Figures 20A-C show the results of in vitro translation of short peptides containing two consecutive D-phenylalanines. Figure 20A shows MALDI-TOF-MS analysis of short peptides translated from mRNA#7, Figure 20B shows MALDI-TOF-MS analysis of short peptides translated from mRNA#8, and Figure 20C shows Tricine-SDS-PAGE analysis of the translation products of mRNA#7 or mRNA#8 with 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 Typhoon FLA 9500 under Cy2 mode.

[0182] Encouraged by the successful translation of mRNA#7 with two consecutive D-phenylalanines, the inventors translated mRNA#9 into a short peptide Fph-KKK with three consecutive D-phenylalanines. D F D F D FDYKDDDDK (SEQ ID NO: 127) (see Table 10). Previous attempts to translate short peptides with three consecutive D-phenylalanines showed yields of less than 5% compared to peptides of the same sequence but containing three consecutive L-phenylalanines [Achenbach, J. et al., 2015]. The inventors used cation starvation at 30 μM or 300 μM for in vitro translation.D Phe-tRNAGluE2 CUA The translation reactions were analyzed by MALDI-TOF MS and 20% Tricine-SDS-PAGE. 2+ The carryover of Mg in the in vitro translation system proposed in this disclosure 2+ Within acceptable limits (<100mM Mg 2+ The MALDI-TOF MS results show the correct incorporation of three consecutive D-phenylalanines in mRNA#9 (Figure 21A). Furthermore, the Tricine-SDS-PAGE results show that the 300 μM D Phe-tRNAGluE2 CUA The translation yield of mRNA#9 when using 30 μM D Phe-tRNAGluE2 CUA The translation yield was approximately two times higher than that obtained with 30 μM L Phe-tRNA Phe The results show that the results were similar to those of the control using 100% ethanol (Figure 21B).

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

[0184] Taken together, these results suggest that the efficiency of incorporation of D-amino acids (up to three consecutive D-phenylalanines) was significantly improved by increasing the concentration of cation-depleted flexizyme-loaded tRNA from ∼20–30 μM to ∼200–300 μM.

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

[0186] In both translation reactions, the overall Mg 2+ The carryover of Mg in the in vitro translation system proposed in this disclosure 2+ Within acceptable limits (less than 100 mM Mg 2+ ) The translation products were purified by ANTI-FLAG M2 magnetic beads (Sigma) so that full-length translation products were separated from their truncated translation products.

[0187] Tricine-SDS-PAGE results show that 300 μM βGln-tRNAGluE2 CUA The translation yield of mRNA#10 when using β Gln-tRNAGluE2 CUA The results show that the translation yield was slightly higher than that obtained using the .

[0188] FIG. 22 shows the results of in vitro translation of a short peptide containing three consecutive β-Glns, showing Tricine-SDS-PAGE analysis of the translation products of mRNA#10 using uncargoed tRNA alone, 30 μM βGln-tRNAGluE2CUA, or 300 μM βGln-tRNAGluE2CUA, scanned by Typhoon FLA 9500 under Cy2 mode.

[0189] Taken together, these results suggest that the efficiency of incorporation of β-amino acids (up to three consecutive β-Gln) was improved by increasing the concentration of cation-depleted flexizyme-loaded tRNA from ∼30 μM to ∼300 μM.

[0190] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0191] All publications, patents, and patent applications mentioned in this disclosure are incorporated by reference in their entirety into this disclosure to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference into this disclosure. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

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

Claims

1. 1. A system for producing a protein, comprising: an mRNA molecule encoding said protein; A plurality of loaded tRNA molecules; and Cell-free translation mix Including, The concentration of Mg2+ in the system is less than 100 mM; The system.

2. The system of claim 1 , wherein at least one tRNA molecule of the plurality of loaded tRNA molecules is loaded with a ribozyme.

3. The system of claim 2, wherein the tRNA molecule is loaded with an unnatural amino acid residue, and the unnatural amino acid residue is a D-amino acid residue.

4. The system of claim 3, wherein the tRNA molecule comprises an L-ribonucleic acid residue (L-tRNA).

5. The system according to claim 3 or claim 4, wherein the ribozyme comprises an L-ribonucleic acid residue (L-flexizyme).

6. Providing the plurality of loaded tRNA molecules having Mg2+ at or below the concentration; and contacting the plurality of loaded tRNA molecules 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 according to any one of claims 1 to 5, comprising:

7. 7. The method of claim 6, wherein said preparing comprises adjusting the concentration of said Mg2+ prior to said contacting.

8. The method of claim 6 or claim 7, wherein the preparing further comprises adjusting the loaded tRNA molecule to a concentration greater than twice the loaded tRNA concentration in a protein translation system comprising an aaRS.

9. A method for loading an L-tRNA with a D-amino acid, comprising: preparing said L-tRNA molecule using a D-polymerase; Providing an activated D-amino acid; Providing an L-aminoacylated ribozyme; and contacting the L-tRNA, the L-aminoacylating ribozyme, and the activated D-amino acid, thereby obtaining a D-amino acid loaded L-tRNA molecule; The method comprising:

10. The method of claim 9, wherein the L-aminoacylated ribozyme is L-flexizyme.

11. An L-flexizyme comprising an L-ribonucleotide residue.

12. A protein prepared by the method according to any one of claims 6 to 8.

13. 13. The protein of claim 12, which is selected from the group consisting of a protein comprising at least one non-standard amino acid residue, a protein comprising at least one D-amino acid residue, an L-protein, and a D-protein.

14. The protein of claim 13 , selected from the group consisting of chicken lysozyme, Gaussia luciferase, and E. coli TrpRS.

15. The protein described in claim 12, encoded by mRNA #6.

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