Acyl-tRNA synthetase

Abstract

The present invention relates to acyl-tRNA synthetases with altered activity. For example, beta-amino acid-tRNA synthetase, nucleic acids encoding the synthetase, cells expressing the synthetase, and methods for using the same. The present invention also relates to beta-hydroxy-tRNA synthetase, nucleic acids encoding the synthetase, cells expressing the synthetase, and methods for using the same. Furthermore, the present invention relates to α,α-disubstituted amino acid-acyl-tRNA synthetase, nucleic acids encoding the synthetase, cells expressing the synthetase, and methods for using the same. The present invention also relates to methods for producing polymers containing the genetic incorporation of α,α-disubstituted amino acids.

Description

[Technical Field] 【0001】 The present invention relates to acyl-tRNA synthetases with altered activity. For example, beta-amino acid-tRNA synthetases, nucleic acids encoding the synthetases, cells expressing the synthetases, and methods for using the same. The present invention also relates to beta-hydroxy-tRNA synthetases, nucleic acids encoding the synthetases, cells expressing the synthetases, and methods for using the same. Furthermore, the present invention relates to α,α-disubstituted amino acid-acyl-tRNA synthetases, nucleic acids encoding the synthetases, cells expressing the synthetases, and methods for using the same. The present invention also relates to methods for producing polymers containing genetic incorporation of α,α-disubstituted amino acids. [Background technology] 【0002】 The genetic code of living cells is synthesized through site-specific incorporation of hundreds of non-canonical amino acids (ncAAs) into proteins (Dumas, A., Lercher, L., Spicer, CD & Davis, BG Designing logical codon reassignment - Expanding the chemistry in biology. Chem Sci 6, 50-69, doi:10.1039 / c4sc01534g (2015), Young, DD & Schultz, PG Playing with the molecules of life. ACS chemical biology 13, 854-870 (2018)), as well as the encoded synthesis of non-canonical polymers, macrocyclic peptides, and depsipeptides (Chin, JW Expanding and reprogramming the genetic code. Nature 550, 53-60, doi:10.1038 / nature24031 (2017), De La Torre, D. & Chin, JW Reprogramming the genetic code. Nature Reviews Genetics 22, 169-184, doi:10.1038 / s41576-020-00307-7 (2021), Robertson, WE et al. Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021), Spinck, M. et al. Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles. Nature Chemistry 15, 61-69 (2023)) are being reprogrammed to enable this.Despite significant advances, monomers that can be site-specifically incorporated into proteins within cells are essentially limited to α-L amino acids with variant side chains, and closely related hydroxy acids. A wider range of monomers are being incorporated into in vitro translation reactions (Ellman, JA, Mendel, D. & Schultz, PG Site-specific incorporation of novel backbone structures into proteins. Science 255, 197-200 (1992), Mendel, D., Cornish, VW & Schultz, PG Site-directed mutagenesis with an expanded genetic code. Annual review of biophysics and biomolecular structure 24, 435-462 (1995), Hecht, SM Expansion of the genetic code through the use of modified bacterial ribosomes. Journal of molecular biology 434, 167211 (2022), Katoh, T. & Suga, H. In vitro genetic code reprogramming for the expansion of usable noncanonical amino acids. Annual Review of Biochemistry 91, 221-243). (2022)) (mainly for short peptides), these in vitro techniques cannot be extended to living cells. (S)β. 3 -parabromo-homophenylalanine((S)β 3-pBrhF) has been incorporated at very low levels in competition with Phe at Phe codons in Escherichia coli (E. coli) using forced phenylalanine (Phe) starvation conditions (Melo Czekster, C., Robertson, WE, Walker, AS, Soll, D. & Schepartz, A. In Vivo Biosynthesis of a beta-Amino Acid-Containing Protein. J Am Chem Soc 138, 5194-5197, doi:10.1021 / jacs.6b01023 (2016)); this method yields a mixture of amino acids at all Phe codons and (S)β at a single position corresponding to a single codon, which is necessary to reprogram the gene code. 3 - Does not enable site-specific integration of pBrhF. 【0003】 The encoded, site-specific incorporation of non-canonical monomers (ncMs) by cellular translation requires both acylation of orthogonal tRNAs using ncMs by orthogonal synthases and ribosomal polymerization of ncMs into polymer chains (Figure 1). Current methods for modifying aminoacyl-tRNA synthetases to acylate novel monomers rely on translational readouts (Santoro, SW, Wang, L., Herberich, B., King, DS & Schultz, PG An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nature biotechnology 20, 1044-1048 (2002), Chin, JW, Martin, AB, King, DS, Wang, L. & Schultz, PG Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proceedings of the National Academy of Sciences 99, 11020-11024 (2002)), and therefore, for frequent incorporation at specific sites in proteins, the monomer must be a ribosomal substrate. Many of the target noncanonical amino acids (ncMs) are weak ribosomal substrates (Katoh, T. & Suga, H. In vitro genetic code reprogramming for the expansion of usable noncanonical amino acids. Annual Review of Biochemistry 91, 221-243 (2022), Melo Czekster, C., Robertson, WE, Walker, AS, Soll, D. & Schepartz, A. In Vivo Biosynthesis of a beta-Amino Acid-Containing Protein.).J Am Chem Soc 138, 5194-5197, doi:10.1021 / jacs.6b01023 (2016)、Tan, Z., Forster, A. C., Blacklow, S. C. & Cornish, V. W. Amino Acid Backbone Specificity of the Escherichia coli Translation Machinery. Journal of the American Chemical Society 126, 12752-12753 (2004)、Pavlov, M. Y. et al. Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proceedings of the National Academy of Sciences 106, 50-54 (2009)、Katoh, T., Tajima, K. & Suga, H. Consecutive Elongation of D-Amino Acids in Translation. Cell Chem Biol 24, 46-54, doi:10.1016 / j.chembiol.2016.11.012 (2017)、Katoh, T. & Suga, H. Ribosomal Incorporation of Consecutive beta-Amino Acids. J Am Chem Soc 140, 12159-12167, doi:10.1021 / jacs.8b07247 (2018)、Dedkova, L. M. et al. beta-Puromycin selection of modified ribosomes for in vitro incorporation of beta-amino acids. Biochemistry 51, 401-415, doi:10.1021 / bi2016124 (2012)), this leads to an evolutionary deadlock in cells; orthogonal synthases cannot evolve to acylate orthogonal tRNAs using ncM, a weak ribosomal substrate, and ribosomes cannot evolve to polymerize ncM, which cannot acylate on orthogonal tRNAs. 【0004】 The inventors previously described tRNA elongation (tREX), a rapid and scalable method for determining the user-defined aminoacylation state of tRNA from cells (Cervettini, D. et al. Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase-tRNA pairs. Nat Biotechnol, doi:10.1038 / s41587-020-0479-2 (2020)). In this method, total tRNA is isolated from cells, and the 2',3' diols on the 3' end ribose of non-acylated tRNA are selectively oxidized to the dialdehyde, while acylated tRNA is protected from diol oxidation. A fluorophore-supported DNA probe is then annealed to the 3' end of the target tRNA under conditions that facilitate deacylation of acylated tRNA, exposing the free diol at its 3' end. This allows for polymerase-mediated elongation of unoxidized (acylated) tRNA. The resulting mass difference between oxidized, unelongated tRNA and unoxidized, elongated tRNA is broken down by gel electrophoresis, enabling the differentiation of acylated tRNA from free tRNA. 【0005】 Another previous method is disclosed in Saito et al. (the EMBO Journal, Vol. 20, No. 7, pp 1797-1806, 2001). This method relies on a biotinylated substrate and is performed in vitro. [Prior art documents] [Non-patent literature] 【0006】 [Non-licensed document 1] Dumas, A., Lercher, L., Spicer, CD & Davis, BG Designing logical codon reassignment - Expanding the chemistry in biology. Chem Sci 6, 50-69, doi:10.1039 / c4sc01534g (2015) 【Non-licensed Document 2】 Young, DD & Schultz, PG Playing with the molecules of life. ACS chemical biology 13, 854-870 (2018) [Non-licensed document 3] Chin, JW Expanding and reprogramming the genetic code. Nature 550, 53-60, doi:10.1038 / nature24031 (2017) 【Non-licensed Document 4】 De La Torre, D. & Chin, JW Reprogramming the genetic code. Nature Reviews Genetics 22, 169-184, doi:10.1038 / s41576-020-00307-7 (2021) 【Non-licensed Document 5】 Robertson, WE et al. Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021) 【Non-licensed Document 6】 Spinck, M. et al. Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles. Nature Chemistry 15, 61-69 (2023) [Non-Patent Document 7] Ellman, JA, Mendel, D. & Schultz, PG Site-specific incorporation of novel backbone structures into proteins. Science 255, 197-200 (1992) [Non-Patent Document 8] Mendel, D., Cornish, VW & Schultz, PG Site-directed mutagenesis with an expanded genetic code. Annual review of biophysics and biomolecular structure 24, 435-462 (1995) [Non-Patent Document 9] Hecht, SM Expansion of the genetic code through the use of modified bacterial ribosomes. Journal of molecular biology 434, 167211 (2022) [Non-Patent Document 10] Katoh, T. & Suga, H. In vitro genetic code reprogramming for the expansion of usable noncanonical amino acids. Annual Review of Biochemistry 91, 221-243 (2022) [Non-Patent Document 11] Melo Czekster , C. , Robertson , WE , Walker , AS , Soll , D. & Schepartz , A. In Vivo Biosynthesis of a Beta-Amino Acid-Containing Protein . J Am Chem Soc 138, 5194–5197. 【Outdoor Tools 12】 Santoro , SW , Wang , L. , Herberich , B. , King , DS & Schultz , PG An efficient system for the evolution of aminoacyl-tRNA synthetase specificity . Nature biotechnology 20, 1044–1048 (2002). 【Outdoor Tools13】 Chin , JW , Martin , AB , King , DS , Wang , L. & Schultz , PG Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli . Proceedings of the National Academy of Sciences 99, 11020–11024 (2002) 【Outdoor Tools 14】 Tan , Z. , Forster , AC , Blacklow , SC & Cornish , VW Amino Acid Backbone Specificity of the Escherichia coli Translation Machinery . Journal of the American Chemical Society 126, 12752–12753 (2004). 【Outdoor Tools 15】 Pavlov, MY et al. Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proceedings of the National Academy of Sciences 106, 50-54 (2009) [Non-Patent Document 16] Katoh, T., Tajima, K. & Suga, H. Consecutive Elongation of D-Amino Acids in Translation. Cell Chem Biol 24, 46-54, doi:10.1016 / j.chembiol.2016.11.012 (2017) [Non-Patent Document 17] Katoh, T. & Suga, H. Ribosomal Incorporation of Consecutive beta-Amino Acids. J Am Chem Soc 140, 12159-12167, doi:10.1021 / jacs.8b07247 (2018) [Non-Patent Document 18] Dedkova, LM et al. beta-Puromycin selection of modified ribosomes for in vitro incorporation of beta-amino acids. Biochemistry 51, 401-415, doi:10.1021 / bi2016124 (2012) [Non-Patent Document 19] Cervettini, D. et al. Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase-tRNA pairs. Nat Biotechnol, doi:10.1038 / s41587-020-0479-2 (2020) [Non-Patent Document 20] Saito et al. (the EMBO Journal, Vol. 20, No. 7, pp 1797-1806, 2001) [Overview of the Initiative] [Means for solving the problem] 【0007】 In the first embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using a beta amino acid, M300A, M300C, M300D, M300M, or M300S for sequence number 1; A302A, A302C, A302D, A302G, A302H, A302L, A302N, or A302Y for sequence number 1; and N346A, N346C, N346G, N346S, N346T, or N346V for Sequence ID No. 1 An acyl-tRNA synthetase is provided that contains an amino acid sequence including the corresponding mutation. 【0008】 In one embodiment, the acyl-tRNA synthetase includes the M300D mutation for SEQ ID NO: 1, the A302H, A302Y, or A302C mutation for SEQ ID NO: 1, and the N346G, N346A, or N346S mutation for SEQ ID NO: 1. 【0009】 In a second embodiment, the use of acyl-tRNA synthetase in a method for producing a polymer containing beta-amino acids is provided. In one embodiment, the use of acyl-tRNA synthetase disclosed herein in a method for producing a polymer containing beta-amino acids is provided. 【0010】 Also provided is a method for producing a polymer containing a beta-amino acid, comprising i) the use of an acyl-tRNA synthetase disclosed herein for acylation of tRNA using a beta-amino acid, and ii) the incorporation of the beta-amino acid into a polymer chain. 【0011】 In a third embodiment, a nucleic acid encoding the acyl-tRNA synthetase of the first embodiment is provided. 【0012】 In a fourth embodiment, cells are provided that include an acyl-tRNA synthetase according to the first embodiment, a nucleic acid according to the third embodiment, or a vector comprising the nucleic acid according to the third embodiment. 【0013】 In the fifth aspect, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, A302C, A302G, A302H, or A302S for Sequence ID No. 1; and N346A, N346C, N346E, N346G, N346T, or N346V for Sequence ID No. 1 An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0014】 A sixth aspect provides the use of acyl-tRNA synthetase in a method for producing polymers containing α,α-disubstituted amino acids. 【0015】 Also provided is a method for producing a polymer containing an α,α-disubstituted amino acid, comprising i) the use of an acyl-tRNA synthetase disclosed herein for acylation of tRNA using an α,α-disubstituted amino acid, and ii) the incorporation of the α,α-disubstituted amino acid into a polymer chain. 【0016】 In the seventh aspect, a nucleic acid encoding the acyl-tRNA synthetase of the fifth aspect is provided. 【0017】 In the eighth aspect, a cell is provided that includes an acyl-tRNA synthetase according to the fifth aspect, a nucleic acid according to the seventh aspect, or a vector comprising the nucleic acid according to the seventh aspect. 【0018】 In the ninth aspect, an acyl-tRNA synthetase capable of specifically acylating tRNA using a beta-hydroxy acid, M300A, M300D, M300M, M300N, or M300S for Sequence ID No. 1; and A302D, A302G, A302H, or A302N for Sequence ID No. 1 An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0019】 In a tenth aspect, the use of acyl-tRNA synthetase in a method for producing a polymer containing a beta-hydroxy acid is provided. 【0020】 Also provided is a method for producing a polymer containing a beta-hydroxy acid, comprising i) the use of an acyl-tRNA synthetase disclosed herein for acylation of tRNA using a beta-hydroxy acid, and ii) the incorporation of the beta-hydroxy acid into a polymer chain. 【0021】 In the eleventh aspect, a nucleic acid encoding the acyl-tRNA synthetase of the ninth aspect is provided. 【0022】 In the twelfth aspect, a cell is provided that includes an acyl-tRNA synthetase according to the ninth aspect, a nucleic acid according to the eleventh aspect, or a vector comprising the nucleic acid according to the eleventh aspect. 【0023】 Furthermore, the use of acyl-tRNA synthetases disclosed herein in a method for genetically incorporating monomers into polymers is also provided. 【0024】 Also provided are methods for producing polymers, comprising i) the use of an acyl-tRNA synthetase disclosed herein for acylation of tRNA with a monomer, and ii) the incorporation of the monomer into a polymer chain. [Brief explanation of the drawing] 【0025】 [Figure 1]The cellular uptake of encoded non-standard monomers into proteins and non-standard polymers requires both tRNA acylation and ribosomal polymerization. Site-specific incorporation of encoded non-standard monomers (ncM, yellow asterisk) by cellular translation requires both acylation of orthogonal tRNA using ncM by orthogonal synthase and ribosomal polymerization of ncM onto polymer chains. Current methods for modifying aminoacyl-tRNA synthases to acylate novel monomers rely on translational readouts, and therefore require the monomer to be a ribosomal substrate. For ncM, which is a weak ribosomal substrate, this codependency leads to an evolutionary deadlock in the cell; orthogonal synthases cannot evolve to acylate orthogonal tRNA using ncM, which is a weak ribosomal substrate, and ribosomes cannot evolve to polymerize ncM, which cannot be acylated on orthogonal tRNA. To overcome this impasse, the inventors developed a direct selection of orthogonal synthases for aminoacylation of their congeneral orthogonal tRNAs using ncM, regardless of whether ncM is a ribosomal substrate or not. [Figure 2-1] ~ [Figure 2-2]tRNA display enables the direct selection of orthogonal aminoacyl-tRNA synthetases that aminoacylate their congeneral orthogonal tRNAs using ncAAs. a. Schematic diagram of tRNA display, a translation-independent strategy for selecting aaRS enzymes that aminoacylate specific tRNAs using desired monomers. In tRNA display, a library of stmRNAs is transformed into cells and grown in multiple repeats in the presence and absence of the non-standard monomer of interest (indicated by yellow asterisks). The library contains PylRS variants that are active and selective for ncM (yellow), PylRS variants that are inactive in cells for both ncM and standard amino acids (light blue), and PylRS variants that are active in cells for one or more standard amino acids but not selective for ncM (dark blue). Bio-mREX is performed on each repeat, and the cDNA is subjected to next-generation sequencing (NGS). The results were analyzed by plotting selectivity (the ratio of the relative abundance of a particular sequence in the positive sample (+ncM) divided by the relative abundance in the negative sample (-ncM)) against enrichment (the ratio of the same sequence in the positive sample divided by the relative abundance in the input library) for all observed sequences; this yields a fusiform plot (hereafter referred to as the fusiform plot). Active and selective PylRS variants are expected to be highly selective and enriched (yellow dots - upper right quadrant), active but non-selective variants are expected to be non-selective but highly enriched (dark blue dots), and inactive PylRS variants are expected to be non-selective and unenriched (light blue dots). b. Structure of non-standard α-alpha-amino acids used in this study.N6-((benzyloxy)carbonyl)-L-lysine (CbzK)(2), N6-((propa-2-in-1-yloxy)carbonyl)-L-lysine (AlkyneK)(3), N6-benzoyl-L-lysine (BenzK)(4), 3-([2,2'-bipyridine]-5-yl)-2-aminopropanoic acid (BiPyA)(5), Nτ-methyl-L-histidine (NτmH)(6), (S)-2-amino-3-(thiophen-3-yl)propanoic acid (3-ThiA)(7) ), (S)-2-amino-3-(pyridine-3-yl)propanoic acid (PyA)(8), (S)-2-amino-3-(4-iodophenyl)propanoic acid (pIF)(9), (S)-2-amino-3-(4-bromothiophen-2-yl)propanoic acid (BrThiA)(10), (2S)-2-amino-3-(((2-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)thio)ethoxy)carbonyl)amino)propanoic acid (pcDAP)(11). c, tRNA display containing the PylRS library (stmRNAvol2_lib1). This library has three positions mutated to all other standard amino acids (Y306X, L309X, and N346X). The spindle plot shows the results of tRNA display selection for CbzK(2) using a one-step parallel selection outlined in (a). Samples were run in three replicates and data were processed as described in the methods. Red dots indicate 65 clones that were further characterized. d, GFP fluorescence measured in cells containing the corresponding PylRS variant / tRNAPyl CUA pair, GFP(150TAG)His6 and CbzK(2) against ln(enrichment, +2) (red dot in (c)) of the PylRS variant derived from tRNA display. The dotted line represents a linear regression on the displayed data points; R2=0.6611, p<0.0001. e~j, (left) white bars: GFP fluorescence derived from cells containing GFP(150TAG)His6, the displayed PylRS variant / tRNAPyl CUA pair, and the displayed ncAA. Gray bars: wtPylRS / tRNAPyl CUA pairs having the same ncAA.Fluorescence is shown as a fraction of fluorescence produced by wt PylRS / tRNAPyl CUA pairs using 2 mM BocK(1) and GFP(150TAG)His6. (Right) ESI-MS of GFP(150X)His6 (X is the shown ncAA). f, measured mass: 27922.0 Da, expected mass: 27923.3 Da; g, measured mass: 27944.8 Da, expected mass: 27945.5 Da; h, measured mass: 27867.6 Da, expected mass: 27866.4 Da; i, measured mass: 27862.0 Da, expected mass: 27861.4 Da; j, measured mass: 27986.4 Da, expected mass: 27986.2 Da; k, measured mass: 27945.6 Da, expected mass: 27944.3 Da. [Figure 3-1] ~ [Figure 3-3]tRNA display allows for the selection of orthogonal aminoacyl-tRNA synthetases to charge non-standard monomers. a. Structures of the non-standard monomers used in this study: (S)-3-amino-3-(3-bromophenyl)propanoic acid ((S)β3mBrF)(12), (S)-3-amino-6-(((benzyloxy)carbonyl)amino)hexanoic acid ((S)-β3CbzK)(13), (S)-6-acetamido-3-aminohexanoic acid ((S)-β3AcK)(14), BocAhx(15), 6-(((benzyloxy)carbonyl) mino)hexanoic acid (CbzAhx)(16), 3-amino-2-((1-ethyl-1H-imidazole-5-yl)methyl)propanoic acid (β2NeH(17)), 3-amino-4-(4-bromophenyl)butanoic acid (β3pBrhF)(18), 2-benzyl-3-hydroxypropanoic acid (β2OH-F)(19), 3-amino-3-phenylpropanoic acid (β3F)(20). b. Fluoro-tREX for the indicated PylRS variants. The experiment was repeated three times using tRNA extracted from cells carrying the pMB1 plasmid encoding each PylRS variant and TRNAPyl CUA in the presence and absence of 4 mM 15. c-d. Fluoro-tREX for the indicated PylRS variants derived from primary selection (panel c) and after further evolution (panel d). The experiment was repeated three times using tRNA extracted from cells carrying the respective PylRS variants and the pMB1 plasmid encoding tRNAPyl CUA, in the presence and absence of 4 mM 12. The selected PylRS variant acylates tRNAPyl CUA using 12. LC-MS traces on AQC-induced eluate from tRNA pulldowns derived from cells expressing the indicated PylRS variants (scanning ion mode for the AQC adduct of substrate 12). Cells carrying the corresponding PylRS variants and the pMB1 plasmid encoding tRNAPyl CUA (or tRNA(-) only as a control) were grown in the presence of substrate 12, and tRNA pulldowns were performed using a biotinylated probe for tRNAPyl CUA.f. Quantification of relative acylation of tRNAPyl CUA by selected PylRS variants. e. Integrated sub-peak area of ​​LC-MS traces shown. g. GFP fluorescence from cells containing GFP(150TAG)His6, PylRS(12_1) or PylRS(12_1evol1), and tRNAPyl CUA, grown in or without substrate 12. Fluorescence is shown as a fraction of fluorescence produced by wt PylRS / tRNAPyl CUA pairs using 2 mM BocK(1) and GFP(150TAG)His6. h. Intact ESI-MS of GFP(150(S)β3mBrF)His6 purified from cells carrying PylRS(12_1evol1), tRNAPyl CUA, and GFP(150TAG)His6, grown in the presence of 4 mM 12. Measured mass: 27939.0 Da, predicted mass: 27,938.2 Da. i. Close-up of residue 150 of GFP(150(S)β3mBrF)His6, derived from the crystal structure determined at 1.5 Å. The 2Fo-Fc map is shown at the contour level of sigma=2 (PDB code 8OVY). The electron density (blue) confirms the incorporation of 12 at position 150, clearly demonstrating the extension of the peptide backbone by a single methylene group and the stereochemistry of the β-amino acids in the protein. [Figure 4]Deacylation of tRNA after oxidation under alkaline conditions increases the acylation signal in fluoro-tREX, thereby enabling robust detection of acylation by hydroxy acids and carboxylic acids. a. Chemical structures of N6-(tert-butoxycarbonyl)-L-lysine (BocK)1, (S)-6-((tert-butoxycarbonyl)amino)-2-hydroxyhexanoic acid (OH-BocK)21, and 6-((tert-butoxycarbonyl)amino)hexanoic acid (BocAhx)15. MmPylRS is highly active with respect to BocK1 and OH-BocK(Dumas, A., Lercher, L., Spicer, CD & Davis, BG Designing logical codon reassignment - Expanding the chemistry in biology. Chem Sci 6, 50-69, doi:10.1039 / c4sc01534g (2015))21, and exhibits acylation activity with respect to BocAhx15 (Young, DD & Schultz, PG Playing with the molecules of life. ACS chemical biology 13, 854-870 (2018)). The free acids b, 1, 21, and 15 range in terms of estimated pKa. The rate constant for alkaline hydrolysis of esters to obtain fixed alcohols and variable carboxylic acids increases as the pKa of the obtained carboxylic acid decreases. Therefore, the inventors predict that when the acylation monomer is a simple carboxylic acid, such as an α-hydroxy acid (and β-amino acid), the hydrolysis rate of the acylated tRNA will be slower than when the acylation monomer is an α-amino acid. c, MmPylRS acylates Mm tRNAPyl in cells using BocK1, OH-BocK21, and BocAhx15, respectively. Northern blots of Mm tRNAPyl derived from tRNA isolated from cells carrying the pMB1 plasmid encoding the MmPylRS / Mm tRNAPyl pair in the presence and absence of BocK, OH-BocK, or BocAhx. Experiments were performed in three biological replicates and similar results were obtained.d. To robustly detect the acylation activity of MmPylRS by fluoro-tREX using a non-alpha amino acid substrate after oxidation, a deacylation step under alkaline conditions is necessary. Fluoro-tREX was performed using the tRNA samples described in panel c, with and without 45 minutes of tRNA incubation with 50 mM bicine at pH 9.6 after oxidation. The acylation signals from OH-BocK and BocAhx were dependent on tRNA deacylation prior to the creno-(exo-) extension step of the protocol. The experiment was performed in three biological replicates, and similar results were obtained. [Figure 5] Relationship between acylation signals measured by bio-mREX for stmRNA and GFP fluorescence signals measured for intact, translatable tRNA. For stmRNA, active aminoacyl-tRNA synthetase (aaRS) results in acylation of the stmRNA they encode, which is then extended, separated, and ultimately reverse transcribed by bio-mREX. This yields cDNA of the active synthetase, which can be quantified by qPCR. In the case of inactive aaRS, stmRNA is not acylated, and no cDNA is produced in the bio-mREX experiment. Therefore, the activity of the synthetase in bio-mREX correlates with the number of cDNA molecules measured by qPCR. In standard translation, the active aaRS enzyme yields acylated, intact, congeneral tRNACUA, which is used in protein translation. The inactive aaRS enzyme yields unacylated tRNA, which is not used in protein translation. The production of GFP protein from GFP150TAGHi6, as measured by GFP fluorescence, reports tRNACUA acylation and other steps in protein production. [Figure 6]The PylRS libraries used in this study. a. Overview of the seven libraries designed and created. These libraries target a total of 11 amino acid residues and 7 types of degenerate codons used in the PylRS active site. NNK codons are depicted in dark red, DBK codons (+lysine codons) in blue, NDT codons in dark green, and NRT codons in yellow. For certain sites, custom residue mixes containing the most commonly observed mutations were used (1-7 mixes, depicted as gray spheres). All libraries were created using at least 10⁹ independent transformants. N=A,T,G,C;K=G,T;D=G,A,T;B=G,T,C;R=G,A. Custom mixes are described in the methods. b. The 11 amino acid residues targeted for mutagenesis in the PylRS active site are shown in red. Images were rendered using Pymol based on the PDB structure 2ZIN. [Figure 7]tRNA display identifies active and selective orthogonal aaRS variants from stmRNA libraries. a. Spindle plot derived from tRNA display selection using stmRNA libraries 1 and ncAA1. b. Identification of regions in the spindle plot enriched with active and selective clones. The inventors anticipate that the upper right quadrant of the spindle plot region is enriched with active and selective clones. Since selectivity is derived from the ratio of sequence counts +ncAA to -ncAA, enriched clones exhibiting negative selectivity values ​​correspond to specific enrichment of the clone under the -ncAA condition with respect to the +ncAA condition. The inventors hypothesized that many apparent enrichments of this type are spurious, and therefore regions of the spindle plot where positive selectivity values ​​are reflected by negative selectivity values ​​of the same magnitude may contain substantial noise. Based on this assumption, the inventors anticipated that active and ncAA-selective clones are most enriched in regions of the plot where selectivity is asymmetrical for a given positive enrichment value. To enrich this asymmetric population, the inventors binned the average selectivity values: the 5350 points in the spindle plot (Figure 2c) were divided into 500 equal bins along the enrichment + 1 dimension (163 bins contained data). The average selectivity of each bin was plotted against the natural logarithm of enrichment + 1. From this, an enrichment value was defined where the spindle plot is asymmetric along the selectivity axis, using a threshold of approximately 7.4 (corresponding to the logarithmic score value of 2). c, Experimental GFP fluorescence values ​​of 100 clones plotted against the natural logarithm of the enrichment score in the presence of 1. GFP was expressed from GFP150TAGHis6 in the presence of the MmPylRS variant clone, congener Mm tRNAPyl CUA, and ncAA(1). Points above the symmetry threshold are colored red (65 points), and points below it are colored blue (21 points).A strong positive correlation was observed between the tRNA display sequence data and the experimental expression data for the red dots (R² = 0.6611, p < 0.0001), but no significant correlation was found for the blue dots (R² = 0.0392, p = 0.397), which is consistent with our assumptions. The red dots are also shown in Figure 2c. In subsequent selection, based on this analysis, we focused primarily on identifying clones on the right-hand side of the spindle plot where, for a given enrichment value, the magnitude of the positive selectivity for a clone was greater than the magnitude of the negative selectivity for clones with the same enrichment value. d. The tRNA display identifies ncAA-specific PylRS variants. The plot, color-coded as shown in panel b, shows the selectivity derived from the spindle plot relative to the experimental selectivity for the clones shown in panel b. The experimental selectivity, as shown in panel b, is derived from GFP expression experiments but is + / -ncAA. [Figure 8]Schematic diagram of a tRNA display-based strategy for selecting PylRS variants that instruct ncAA incorporation into proteins. In the first round, naive stmRNA vol2 libraries 2, 13, 14, 3D, 4D, and 5D were transformed into BL21 cells and grown overnight. Libraries 3D and 4D were combined to produce library 3D4D. The five libraries were grown to an OD600 of 0.3–0.4, and 2.6 mL of the cell culture from each library was added to the stock solution of each ncAA; this yielded 50 samples (5 libraries × 10 ncAAs). Cells were grown for 40 minutes to induce stmRNA, and cells were grown for a further 20 minutes. Bio-mREX was performed on the isolated RNA for each of the 50 samples. For each reaction, cDNA was amplified using primers appropriate for Golden Gate assembly. Next, all amplicons from the selected libraries for the same ncAA were combined in equimolar ratios (resulting in a total of 10 combined libraries) and cloned into a fresh ColE1 vector backbone. This produced 10 pre-selected libraries. The 10 pre-selected libraries were transformed into BL21 cells and grown overnight. The pre-selected libraries of ncAA2-6 were combined to create a single cluster library. Similarly, the pre-selected libraries of ncAA7-11 were combined to create a second cluster library. 2.6 mL of the first cluster library was added to a solution of ncAA2-6. Also, 2.6 mL of the first cluster library was added to a sample without ncAA as a control. Cells were grown for 40 minutes to induce stmRNA, and the cells were grown for a further 20 minutes to isolate the RNA. Three RNA samples were converted to cDNA as bio-mREX input controls. Bio-mREX was performed on the isolated RNA. This generated seven samples (bio-mREX input, -ncAA control for bio-mREX, and five bio-mREX samples for ncAA2-6). The experiment was repeated three times, generating 21 samples.The cDNA from each sample was sequenced and analyzed by NGS to generate spindle plots and sequence listings. The second cluster was processed in the same way as the first cluster, using ncAA7-11 instead of 2-6. [Figure 9] Selection of PylRS variants of CbzK2 by tRNA display. a. Chemical structure of CbzK2. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+2) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Bordered regions indicate selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity obtained as a result of selection with respect to substrate 2, showing selectivity of 5 or more and enrichment of 5 or more. [Figure 10-1] ~ [Figure 10-2]Selection of PylRS variants of N6-((propa-2-in-1-yloxy)carbonyl)-L-lysine (AlkyneK)3 by tRNA display. a. Chemical structure of AlkyneK3. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+3) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity resulting from selection for substrate 3, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150AlkyneKHis6 from cells carrying wild-type (wt) Mm PylRS or the pMB1 plasmid encoding the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM of substrate 3. Fluorescence is shown compared to cells carrying wt Mm PylRS and Mm tRNAPyl CUA expressing GFP derived from GFP150TAGHis6 in the presence of 2 mM of substrate 1. e. Raw mass spectrum of purified GFP150AlkyneKHis6 produced using PylRS 3_1. f. Analyzed mass spectrum obtained from the major spectrum shown in panel e. Predicted mass 27923.3 Da, measured mass 27922.0 Da. The small peak labeled -met corresponds to the cleavage of the N-terminal methionine residue. This is the same as g~h, e and f, but relates to GFP150AlkyneKHis6 produced using PylRS 3_2. Predicted mass 27923.3 Da, measured mass 27923.6 Da.i-j, e, and f are the same, but this concerns GFP150AlkyneKHis6 produced using PylRS 3_3. Predicted mass: 27923.3Da, measured mass: 27923.2Da. [Figure 11-1] ~ [Figure 11-2]Selection of PylRS variants of N6-benzoyl-L-lysine (BenzK)4 by tRNA display. a. Chemical structure of BenzK4. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+4) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Bordered regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity obtained as a result of selection for substrate 4, showing selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150BenzKHis6 derived from GFP150TAGHis6 from cells carrying wild-type (wt) Mm PylRS or the pMB1 plasmid encoding the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM of substrate 4. Fluorescence is shown compared to cells carrying wt Mm PylRS and Mm tRNAPyl CUA expressing GFP derived from GFP150TAGHis6 in the presence of 2 mM of substrate 1. e. Raw mass spectrum for purified GFP150BenzKHis6 produced using PylRS 4_1. f. Analyzed mass spectrum obtained from the major spectrum shown in panel e. Predicted mass 27945.5 Da, measured mass 27944.8 Da. Small peaks labeled -met correspond to cleavage of the N-terminal methionine residue. g~h, e, and f are the same, but this concerns GFP150BenzKHis6 produced using PylRS 4_2. Predicted mass 27945.5Da, measured mass 27944.8Da. i~j, e, and f are the same, but this concerns GFP150BenzKHis6 produced using PylRS 4_3.Predicted mass 27945.5 Da, measured mass 27944.4 Da. Same as k~l, e, and f, but this concerns GFP150BenzKHis6 produced using PylRS 4_4. Predicted mass 27945.5 Da, measured mass 27944.4 Da. [Figure 12] Selection of PylRS variants of 3-([2,2'-bipyridine]-5-yl)-2-aminopropanoic acid (BiPyA) 5 by tRNA display. a. Chemical structure of BiPyA 5. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+5) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity obtained as a result of selection for substrate 5, showing selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150BiPyAHis6 derived from GFP150TAGHis6 from cells carrying the pMB1 plasmid encoding wild-type (wt) Mm PylRS or the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM 5. Fluorescence is shown compared to cells carrying Mm tRNAPyl CUA expressing wt Mm PylRS and GFP150TAGHis6 in the presence of 2 mM substrate 1. [Figure 13]Selection of PylRS variants of Nτ-methyl-L-histidine (NτmH)6 by tRNA display. a. Chemical structure of NτmH6. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+6) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. 16 sequences ordered by selectivity obtained as a result of selection for substrate 6, showing selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150NτmHHis6 derived from GFP150TAGHis6 from cells carrying wild-type (wt) Mm PylRS or the pMB1 plasmid encoding the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM 6. Fluorescence is shown compared to cells carrying wt Mm PylRS and Mm tRNAPyl CUA expressing GFP derived from GFP150TAGHis6 in the presence of 2 mM substrate 1. [Figure 14-1] ~ [Figure 14-2]Selection of PylRS variants of (S)-2-amino-3-(thiophen-3-yl)propanoic acid (3-ThiA)7 by tRNA display. a. Chemical structure of 3-ThiA7. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+7) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by the selectivity obtained as a result of selection for substrate 7, showing selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150-3-ThiAHis6 derived from GFP150TAGHis6 from cells carrying wild-type (wt) Mm PylRS or the pMB1 plasmid encoding the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM 7. Fluorescence is shown compared to cells carrying wt Mm PylRS and Mm tRNAPyl CUA expressing GFP derived from GFP150TAGHis6 in the presence of 2 mM substrate 1. e. Raw mass spectrum for purified GFP150-3-ThiA KHis6 produced using PylRS 7_1. f. Analyzed mass spectrum obtained from the major spectrum shown in panel e. Predicted mass 27866.4 Da, measured mass 27867.2 Da. The small peak labeled -met corresponds to the cleavage of the N-terminal methionine residue. This is the same as g~h, e, and f, but relates to GFP150-3-ThiAHis6 produced using PylRS 7_2. Predicted mass 27866.4Da, measured mass 27866.8Da.i~j, e, and f are the same, but this concerns GFP150-3-ThiAHis6 produced using PylRS 7_3. Predicted mass 27866.4Da, measured mass 27866.8Da. k~l, e, and f are the same, but this concerns GFP150-3-ThiAHis6 produced using PylRS 7_4. Predicted mass 27866.4Da, measured mass 27866.4Da. m~n, e, and f are the same, but this concerns GFP150-3-ThiAHis6 produced using PylRS 7_5. Predicted mass 27866.4Da, measured mass 27867.6Da. o~p, e, and f are the same, but this concerns GFP150-3-ThiAHis6 produced using PylRS 7_6. Predicted mass 27866.4Da, measured mass 27867.6Da. Same as q~r, e and f, but concerns GFP150-3-ThiAHis6 produced using PylRS 7_7. Predicted mass 27866.4Da, measured mass 27867.6Da. Same as s~t, e and f, but concerns GFP150-3-ThiAHis6 produced using PylRS 7_8. Predicted mass 27866.4Da, measured mass 27868.0Da. [Figure 15]Selection of PylRS variants of (S)-2-amino-3-(pyridine-3-yl)propanoic acid (PyA)8 by tRNA display. a. Chemical structure of PyA8. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+8) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. Five sequences ordered by selectivity obtained as a result of selection for substrate 8, showing selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150PyAHis6 derived from GFP150TAGHis6 from cells carrying wild-type (wt) Mm PylRS or the pMB1 plasmid encoding the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM 8. Fluorescence is shown compared to cells carrying wt Mm PylRS and Mm tRNAPyl CUA expressing GFP derived from GFP150TAGHis in the presence of 2 mM substrate 1. e. Raw mass spectrum for purified GFP150PyAHis6 produced using PylRS 8_1. f. Analyzed mass spectrum obtained from the major spectrum shown in panel e. Predicted mass 27861.4 Da, measured mass 27860.4 Da. Small peaks labeled -met correspond to cleavage of the N-terminal methionine residue. g~h, e, and f are the same, but this concerns GFP150PyAHis6 produced using PylRS 8_2. Predicted mass 27861.4Da, measured mass 27862.0Da. i~j, e, and f are the same, but this concerns GFP150PyAHis6 produced using PylRS 8_3.Predicted mass 27861.4Da, measured mass 27859.6Da. Same as k~l, e and f, but this concerns GFP150PyAHis6 produced using PylRS 8_4. Predicted mass 27861.4Da, measured mass 27860.8Da. [Figure 16-1] ~ [Figure 16-2]Selection of PylRS variants of (S)-2-amino-3-(4-iodophenyl)propanoic acid (pIF)9 by tRNA display. a. Chemical structure of pIF9. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+9) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity obtained as a result of selection for substrate 9, showing selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150pIFHis6 derived from GFP150TAGHis6 from cells carrying wild-type (wt) Mm PylRS or the pMB1 plasmid encoding the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM 9. Fluorescence is shown compared to cells carrying wt Mm PylRS and Mm tRNAPyl CUA expressing GFP derived from GFP150TAGHis6 in the presence of 2 mM substrate 1. e. Raw mass spectrum for purified GFP150pIFHis6 produced using PylRS 9_1. f. Analyzed mass spectrum obtained from the major spectrum shown in panel e. Predicted mass 27986.2 Da, measured mass 27987.6 Da. Small peaks labeled -met correspond to cleavage of the N-terminal methionine residue. g~h, e, and f are the same, but this concerns GFP150pIFKHis6 produced using PylRS 9_2. Predicted mass: 27986.2Da, measured mass: 27986.0Da.i~j, e, and f are the same, but this concerns GFP150pIFKHis6 produced using PylRS 9_3. Predicted mass 27986.2Da, measured mass 27986.4Da. k~l, e, and f are the same, but this concerns GFP150pIFKHis6 produced using PylRS 9_4. Predicted mass 27986.2Da, measured mass 27986.4Da. m~n, e, and f are the same, but this concerns GFP150pIFKHis6 produced using PylRS 9_5. Predicted mass 27986.2Da, measured mass 27986.4Da. o~p, e, and f are the same, but this concerns GFP150pIFKHis6 produced using PylRS 9_6. Predicted mass 27986.2Da, measured mass 27987.2Da. [Figure 17-1] ~ [Figure 17-2]Selection of PylRS variants of (S)-2-amino-3-(4-bromothiophen-2-yl)propanoic acid (BrThiA)10 by tRNA display. a. Chemical structure of BrThiA10. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+10) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity resulting from selection for substrate 10, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150BrThiAHis6 from cells carrying wild-type (wt) Mm PylRS or pMB1 plasmid encoding the indicated Mm PylRS variant, and p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM 10. Fluorescence is shown compared to cells carrying wt Mm PylRS and Mm tRNAPyl CUA expressing GFP derived from GFP150TAGHis6 in the presence of 2 mM substrate 1. e. Raw mass spectrum of purified GFP150BrThiAHis6 produced using PylRS 10_1. f. Analyzed mass spectrum obtained from the major spectrum shown in panel e. Predicted mass 27944.3 Da, measured mass 27945.6 Da. The small peak labeled -met corresponds to the cleavage of the N-terminal methionine residue. This is the same as g~h, e and f, but relates to GFP150BrThiAHis6 produced using PylRS 10_2. Predicted mass 27944.3 Da, measured mass 27945.2 Da.i~j, e, and f are the same, but this concerns GFP150BrThiAHis6 produced using PylRS 10_3. Predicted mass 27944.3Da, measured mass 27945.6Da. k~l, e, and f are the same, but this concerns GFP150BrThiAHis6 produced using PylRS 10_4. Predicted mass 27944.3Da, measured mass 27945.2Da. m~n, e, and f are the same, but this concerns GFP150BrThiAHis6 produced using PylRS 10_5. Predicted mass 27944.3Da, measured mass 27944.4Da. o~p, e, and f are the same, but this concerns GFP150BrThiAHis6 produced using PylRS 10_6. Predicted mass: 27944.3 Da, measured mass: 27944.8 Da. [Figure 18-1] ~ [Figure 18-2]Selection of PylRS variants of (2S)-2-amino-3-(((2-((1-(6-nitrobenzo[d][1,3]dioxol-5yl)ethyl)thio)ethoxy)carbonyl)amino)propanoic acid (pcDAP) 11 by tRNA display. a. Chemical structure of pcDAP 11. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 8. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+11) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity resulting from selection for substrate 11, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Amber repression activity data for selected PylRS variants, measured by the production of GFP150pcDAPHis6 derived from GFP150TAGHis6 from cells carrying the pMB1 plasmid encoding wild-type (wt) Mm PylRS or the indicated Mm PylRS variant, and the p15A plasmid encoding GFP150TAGHis6, in the presence and absence of 4 mM 11. Fluorescence is shown compared to cells carrying Mm tRNAPyl CUA expressing wt Mm PylRS and GFP150TAGHis6 in the presence of 2 mM substrate 1. e. Raw mass spectrum of purified GFP150pcDAPHis6 produced using PylRS 11_1. f. Analyzed mass spectrum obtained from the major spectrum shown in panel e. Predicted mass 28096.4 Da, measured mass 28093.2 Da. The small peak labeled -met corresponds to the cleavage of the N-terminal methionine residue. This is the same as g~h, e and f, but relates to GFP150pcDAPHis6 produced using PylRS 11_2.Predicted mass 28096.4Da, measured mass 28096.4Da. Same as i~j, e and f, but concerns GFP150pcDAPHis6 produced using PylRS 11_3. Predicted mass 28096.4Da, measured mass 28097.2Da. Same as k~l, e and f, but concerns GFP150pcDAPHis6 produced using PylRS 11_4. Predicted mass 28096.4Da, measured mass 28093.6Da. [Figure 19] Schematic diagram of the selection strategy for non-standard monomers. Library 14 was transformed into BL21 cells and grown overnight. Cells were grown to an OD600 of 0.3–0.4. 4 mL of library culture was added to the stock solution of each ncM. 4 mL of library culture was also added to wells without ncM. Cells were grown for 40 minutes to induce stmRNA, and then grown for a further 20 minutes to isolate the RNA. Bio-mREX was performed on the isolated RNA for each sample. The experiment was repeated four times to obtain 40 cDNA samples. Six more cDNA samples were generated for six of the RNA inputs to bio-mREX. The 46 obtained cDNA samples were sequenced by NGS and analyzed to generate spindle plots and sequence listings. [Figure 20]Selection of PylRS variants of (S)-3-amino-3-(3-bromophenyl)propanoic acid ((S)β3mBrF)12 by tRNA display. a. Chemical structure of (S)β3mBrF12. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+12) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c) The 14 sequences were ordered by the selectivity obtained as a result of selection for substrate 12, exhibiting selectivity of 5 or more and enrichment of 5 or more. d) Fluoro-tREX for PylRS variants 12_1 to 12_6. Experiments were performed using tRNAs extracted from cells carrying pMB1 plasmids encoding Mm PylRS and Mm tRNAPyl, in the presence and absence of 4 mM 12. The experiment was repeated three times. [Figure 21]Selection of PylRS variants of (S)-3-amino-6-(((benzyloxy)carbonyl)amino)hexanoic acid ((S)-β3CbzK)13 by tRNA display. a. Chemical structure of (S)-β3CbzK 13. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+13) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c) The top 25 sequences were ordered by the selectivity obtained as a result of selection for substrate 13, exhibiting selectivity of 5 or more and enrichment of 5 or more. d) Fluoro-tREX for PylRS variants 13_1 to 13_6. Experiments were performed using tRNAs extracted from cells carrying pMB1 plasmids encoding Mm PylRS and Mm tRNAPyl, in the presence and absence of 4 mM 13. The experiment was repeated three times. [Figure 22]Selection of PylRS variants of (S)-6-acetamido-3-aminohexanoic acid ((S)-β3AcK)14 by tRNA display. a. Chemical structure of (S)-β3AcK14. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+14) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c) Eight sequences are ordered according to the selectivity obtained as a result of selection with respect to substrate 14, exhibiting selectivity of 5 or more and enrichment of 5 or more. [Figure 23]Selection of PylRS variants of BocAhx 15 by tRNA display. a. Chemical structure of BocAhx 15. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+15) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Bordered regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The top 25 sequences are ordered by selectivity obtained as a result of selection for substrate 15, showing selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants 15_1~15_4 and wt PylRS. Experiments were performed using tRNAs extracted from cells carrying pMB1 plasmids encoding Mm PylRS and Mm tRNAPyl, in the presence and absence of 4 mM 15. A control sample for wt PylRS was performed in the presence and absence of 4 mM 15 and 2 mM 1. The experiment was repeated three times. [Figure 24]Selection of PylRS variants of 6-(((benzyloxy)carbonyl)amino)hexanoic acid (CbzAhx)16 by tRNA display. a. Chemical structure of CbzAhx 16. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+16) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. Five sequences are ordered by the selectivity obtained as a result of selection for substrate 16, showing selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variant 16_1. Experiments were performed using tRNAs extracted from cells carrying pMB1 plasmids encoding Mm PylRS and Mm tRNAPyl, respectively, in the presence and absence of 4 mM 16. The experiment was repeated three times. [Figure 25]Selection of PylRS variants of 3-amino-2-((1-ethyl-1H-imidazole-5-yl)methyl)propanoic acid (β2NeH)17 by tRNA display. a. Chemical structure of β2NeH17. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+17) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c) Four sequences are ordered by the selectivity obtained as a result of selection with respect to substrate 17, exhibiting a selectivity of 5 or more and an enrichment of 5 or more. [Figure 26]Selection of PylRS variants of 3-amino-4-(4-bromophenyl)butanoic acid (β3pBrhF)18 by tRNA display. a. Chemical structure of β3pBrhF18. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+18) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. Five sequences are ordered by the selectivity obtained as a result of selection for substrate 18, showing selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants 18_1 to 18_2. Experiments were performed using tRNAs extracted from cells carrying pMB1 plasmids encoding Mm PylRS and Mm tRNAPyl, respectively, in the presence and absence of 2 mM of 18 S or R enantiomers. The experiment was repeated twice. [Figure 27]Selection of PylRS variants of 2-benzyl-3-hydroxypropanoic acid (β2OH-F)19 by tRNA display. a. Chemical structure of β2OH-F19. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+19) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. Three sequences ordered by selectivity obtained as a result of selection for substrate 19, showing selectivity of 5 or more and enrichment of 5 or more. [Figure 28] Selection of PylRS variants of 3-amino-3-phenylpropanoic acid (β3F) 20 by tRNA display. a. Chemical structure of β3F 20. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 19. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+20) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. Five sequences ordered by selectivity obtained as a result of selection for substrate 20, showing selectivity of 5 or more and enrichment of 5 or more. [Figure 29]A schematic diagram of tRNA pulldown followed by LC-MS analysis to determine monomer identity on a target tRNA. tRNA is extracted from cells expressing the target tRNA and its congenerally orthogonal aaRS, and grown in the presence of ncM. Biotinylated probes are annealed to pull down the target tRNA. After washing, ncM is eluted by alkaline deacylation, derivatized by AQC, and detected using LC-MS. [Figure 30] Schematic diagram of a selection strategy for random mutagenesis selection using tRNA display. The inventors performed mutagenesis PCR reactions across the active site sequences of PylRS variants 12_1 and 12_2 using the GeneMorph II (Agilent) kit. Diversified PCR amplicons were cloned into fresh ColE1 plasmid backbone by Golden Gate assembly, and the mutagenesis stmRNA library was transformed into BL21 cells and grown overnight. Cells were grown to an OD600 of 0.3–0.4. 2.6 mL of library culture was added to 12 stock solutions to a concentration of 4 mM. 2.6 mL of library culture was also added to wells without ncM. Cells were grown for 40 minutes to induce stmRNA, and cells were grown for a further 20 minutes to isolate the RNA. Bio-mREX was performed on the isolated RNA for each sample. The experiment was repeated four times to obtain eight cDNA samples. Furthermore, four cDNA samples were generated for four of the RNA inputs to bio-mREX. The resulting 12 cDNA samples were sequenced and analyzed by NGS, and spindle plots and sequence listings were generated. [Figure 31]Selection of improved PylRS variants of ((S)β3mBrF)12 using a random mutagenesis library with tRNA display. a. Parental active site sequences of (S)β3mBrF PylRS 12_1 and 12_2 used as templates for random mutagenesis. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 30. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+12) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 3 or more and enrichment of 3 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. Spindle plot obtained as a result of tRNA display selection as shown in Figure 30. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+6) divided by its relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by its relative abundance in the input library. Enclosed regions indicate sequences exhibiting selectivity of 3 or more and enrichment of 3 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. Table of unprogrammed mutations of evolved PylRS variants, ordered by selectivity calculated from NGS analysis. The inventors characterized the most selective, most enriched, and most abundant variants from sequences exhibiting selectivity of 3 or more and enrichment of 3 or more. All sequences were based on the parent PylRS variant 12_1. d. Fluoro-tREX data for PylRS variants 12_1evol~12_1evol8.tRNAs were isolated from DH10β cells carrying pMB1 plasmids encoding Mm PylRS and Mm tRNAPyl, respectively, in the presence and absence of 4 mM 12. The inventors performed fluoro-tREX on the isolated tRNAs. All experiments were repeated three times, and similar results were obtained. [Figure 32] LC-MS assay for detecting (S)β3mBrF 12 derivatized on Mm tRNAPyl in a PylRS variant-dependent manner. a. Full LC-MS spectra of data presented in Figures 3e and 3f. Additional controls characterizing only the derivatizing agent 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) are shown in black. b. Overlay of LC-MS traces of the true derivatized standard corresponding to 24 nM of 12, along with 12 isolated from the most active PylRS 12_1evol1 acylated tRNA. [Figure 33-1] ~ [Figure 33-3]Characterization of protein production by amber read-through using PylRS(12_1evol1) / tRNAPyl CUA pair. a. Attempts to produce GFP3(S)β3mBrFHis6 protein from GFP3TAGHis6 from cells carrying the pMB1 plasmid encoding the PylRS(12_1evol1) / tRNAPyl CUA pair and the p15A plasmid encoding GFP3TAGHis6, in the presence and absence of 4 mM 12. The modified protein could not be produced by amber read-through at position 3 in GFP. The dots represent the mean of the two biological replicates, and the error bars indicate ±sd. b. Raw mass spectrum of purified GFP150(S)β3mBrFHis6 produced using PylRS 12_1. The analyzed mass spectrum is shown in Figure 3h. c. MS / MS spectrum of ncAA-containing peptide obtained after trypsin digestion of GFP150(S)β3mBrFHis6 produced using PylRS 12_1evol1. The precursor ion confirms the incorporation of 12 at position 150 of GFP. Fragmentation of each peptide is expected to result in a series of β ions (red) and a series of γ ions (blue), as well as the ion corresponding to the full-length peptide (green). d. Raw mass spectrum of purified GFP150(S)β3mBrFHis6 produced using PylRS 12_1evol1. e. Intact ESI-MS of purified GFP(150(S)β3mBrF)His6 from cells carrying PylRS(12_1evol1), Mm tRNAPyl CUA, and GFP(150TAG)His6, grown in the presence of 4 mM 12. Measured mass: 27940.0 Da, predicted mass: 27,938.2 Da. e. MS / MS spectrum of ncAA-containing peptide obtained after trypsin digestion of GFP150(S)β3mBrFHis6 produced using PylRS 12_1. The precursor ion confirms the incorporation of 12 at position 150 of GFP. Fragmentation of each peptide is predicted to result in a series of β ions (red) and a series of γ ions (blue), as well as the ion corresponding to the full-length peptide (green). [Figure 34]Detailed structure of GFP150(S)β3mBrFHis6 (PDB code 8OVY). a. Detailed protein chain at position 150. The obtained structure of GFP150(S)β3mBrF-His6 (yellow) is superimposed on the structure of wt GFP used for refinement (PDB:2B3P), showing the twist in the main chain induced by the β3-amino acid. b. Detailed hydrogen bond network in the beta barrel at position 150. The GFP150(S)β3mBrF-His6 structure (yellow) is superimposed on the structure of wt GFP (PDB:2B3P). The twist induced by the incorporation of (S)β3mBrF(12) affects the partial hydrogen bonding at that position, but the remaining contacts of the corresponding beta chain remain intact. [Figure 35-1] ~ [Figure 35-2]This provides sequence alignments for various aminoacyl-tRNA synthetases (aaRS). "*" indicates a residue found in all aligned aaRS sequences, ":" indicates a residue with strong similarity between aligned sequences, and "." indicates a residue with some similarity between aligned sequences. Mm is Methanosarcina mazei; Mb is Methanosarcina barkeri; 1R26 is Methanomethylophilus sp. 1R26; Lum 1 is Methanomassiliicoccus luminyensis 1; Nitro is Nitrososphaeria archaeon; Tron is Methanonatronarchaeum thermophilum; Gemm is a bacterium of the phylum Gemmatimonadetes; PGA8 is a bacterium of the family Peptostreptococcaceae pGA-8; I2 is Desulfosporosinus sp.)I2; Clos is a bacterium of the order Clostridiales; D121 is a bacterium of the class Deltaproteobacteria; and D146 is another Deltaproteobacteria. Non-conserved residues may be regions that allow for variability. In addition, in embodiments of acyl-tRNA synthetases disclosed herein, along with the percentage of identity with respect to the main chain, these regions have higher tolerance to variation from the main chain. [Figure 36-1] ~ [Figure 36-2]tRNA display selection of O-synthase charging ncM. a. ncMs searched for selective PylRS variants. b. Representative gels of fluoro-tREX for each PylRS variant. Experiments were performed in three independent replicates and similar results were obtained. c. Selected PylRS variants acylate tRNAPylCUA using 12. LC-MS traces of AQC derivatives eluted from tRNA pulldown (scanning ion mode on the 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) adduct of 12). Cells carrying the corresponding PylRS variants and tRNAPyl CUA (or tRNAPyl CUA(-) only) were grown with 12; pulldowns used biotinylated probes for tRNAPyl CUA. Representative traces are shown. d, f, h, j, l, n, p are similar to b but relate to the PylRS variants and ncMs shown. e, g, i, k, m, o, q are similar to c, but relate to the indicated PylRS variant and ncM; e, g, i were performed in two replicates, and c, k, m, o, q were performed in three replicates, with similar results obtained for all replicates. Fluorescence from cells grown in the presence or absence of ncM(12, A1, A2, A3, A4, A5, A6, A7, 4 mM) containing r, GFP(150TAG)His6, the indicated PylRS variant and tRNAPyl CUA. Fluorescence is shown as a fraction of what is produced by wt PylRS / tRNAPyl CUA pairs using 4 mM BocK(1) and GFP(150TAG)His6. The bar graph represents the mean of three independent measurements, with individual data points shown as dots and error bars of + / - sd. ESI-MS of GFP(150(S)α-Me-pIF)His6 purified from cells carrying PylRS(A6_1), tRNAPyl CUA, and GFP(150TAG)His6, grown together with s and A6. Measured mass: 28000.5 Da, predicted mass: 28000.2 Da. A spectrum was obtained once. [Figure 37-1] ~ [Figure 37-2]LC-MS assay for detecting ncM 12 and A1-A7 derivatized with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) eluted from Mm tRNAPyl acylated in a PylRS variant-dependent manner (see Figure 29 for a schematic diagram of the assay). a. Full LC-MS spectrum of the data presented in Figure 36c. The true derivatized standard of ncM 12 is shown in gray. The experiment was repeated three times with similar results. b. Zoomed-in LC-MS spectrum of a. c. Full LC-MS spectrum of the data presented in Figure 36e. The true derivatized standard of ncM A1 is shown in gray. The experiment was repeated twice with similar results. d. Zoomed-in LC-MS spectrum of c. d. Full LC-MS spectrum of the data presented in Figure 36g. The true derivatized standard of ncM A2 is shown in gray. The experiment was repeated twice, and similar results were obtained. Zoomed-in LC-MS spectra shown in e and d. f, full LC-MS spectra of the data presented in Figure 36i. The true derivatized standard of ncM A3 is shown in gray. The experiment was repeated twice, and similar results were obtained. Zoomed-in LC-MS spectra shown in g and f. h, full LC-MS spectra of the data presented in Figure 36k. The true derivatized standard of ncM A4 is shown in gray. The experiment was repeated three times, and similar results were obtained. Zoomed-in LC-MS spectra shown in i and h. j, full LC-MS spectra of the data presented in Figure 36m. The true derivatized standard of ncM A5 is shown in gray. The experiment was repeated three times, and similar results were obtained. Zoomed-in LC-MS spectra shown in k and j. l, full LC-MS spectra of the data presented in Figure 36o. The true derivatized standard of ncM A6 is shown in gray. The experiment was repeated three times, and similar results were obtained. Zoomed-in LC-MS spectra are shown in m and l. Full LC-MS spectra of the data presented in n and Figure 36q. The true derivatized standard of ncM A7 is shown in gray. The experiment was repeated three times, and similar results were obtained. [Figure 38]Schematic diagram of a two-step selection strategy for non-standard monomers. Library 14 was transformed into BL21 cells and grown overnight. Cells were grown to an OD600 of 0.3–0.4. 4 mL of library culture was added to the stock solution of each ncM. Cells were grown for 40 minutes to induce stmRNA, and then grown for a further 20 minutes to isolate the RNA. Bio-mREX was performed on the isolated RNA for each sample. The experiment was repeated four times. For each repeat, cDNA was amplified using primers appropriate for Golden Gate assembly. Then, all amplicons from the selected libraries for the same ncAA were combined in equimolar ratios and cloned into a fresh ColE1 vector backbone. This created one pre-selected library for each ncM. The pre-selected libraries were transformed into BL21 cells and grown overnight. Cells were grown to an OD600 of 0.3–0.4. For each ncM, 4 mL of the respective pre-selected library culture was added to the stock solution of each ncM. For each pre-selected library, 4 mL of the library culture was also added to the wells that did not contain an ncM. Cells were grown for 40 minutes to induce stmRNA, and then grown for a further 20 minutes to isolate the RNA. Bio-mREX was performed on the isolated RNA for each sample. The experiment was repeated three times to obtain six cDNA samples per ncM. Three more cDNA samples were generated for each ncM using three RNA inputs for bio-mREX of each pre-selected library. For each ncM, the nine obtained cDNA samples were sequenced and analyzed by NGS to generate spindle plots and sequence listings. [Figure 39-1] ~ [Figure 39-2]Selection of PylRS variants of (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β3MDF)(A1) by tRNA display. a. Chemical structure of (S)β3MDF A1. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 38. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+A1) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The 25 sequences were ordered by the selectivity obtained as a result of selection for substrate A1, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants A1_1 to A1_12. Experiments were performed using tRNA extracted from cells carrying pMB1 plasmids encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A1. The experiment for A1_1 was repeated three times. [Figure 40-1] ~ [Figure 40-2]Selection of PylRS variants of (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β3pBrF)(A2) by tRNA display. a. Chemical structure of (S)β3pBrF A2. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 38. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+A2) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The 25 sequences were ordered by the selectivity obtained as a result of selection for substrate A2, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants A2_1 to A2_12. Experiments were performed using tRNA extracted from cells carrying pMB1 plasmids encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A2. The experiment for A2_1 was repeated three times. [Figure 41-1] ~ [Figure 41-2]Selection of PylRS variants of (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β3pmFF)(A3) by tRNA display. a. Chemical structure of (S)β3pmFF A3. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 38. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+A3) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The 25 sequences were ordered by the selectivity obtained as a result of selection for substrate A3, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants A3_1 to A3_12. Experiments were performed using tRNA extracted from cells carrying pMB1 plasmids encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A3. The experiment for A3_1 was repeated three times. [Figure 42-1] ~ [Figure 42-2]Selection of PylRS variants of (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β3oBrF)(A4) by tRNA display. a. Chemical structure of (S)β3oBrF A4. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 38. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+A4) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The 12 sequences were ordered by the selectivity obtained as a result of selection for substrate A4, exhibiting a selectivity of 5 or more and an enrichment of 5 or more. d. Fluoro-tREX for PylRS variants A4_1 to A4_7. Experiments were performed using tRNAs extracted from cells carrying pMB1 plasmids encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A4. The experiment for A4_1 was repeated three times. [Figure 43-1] ~ [Figure 43-2]Selection of PylRS variants of (S)-3-amino-3-(trifluoromethyl)phenyl)propanoic acid (A5) ((S)β3mCF3F) by tRNA display. a. Chemical structure of (S)β3mCF3F A5. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 38. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+A5) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The 25 sequences were ordered by the selectivity obtained as a result of selection for substrate A5, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants A5_1 to A5_8. Experiments were performed using tRNA extracted from cells carrying pMB1 plasmids encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A5. The experiment for A5_1 was repeated three times. [Figure 44-1] ~ [Figure 44-3]Selection of PylRS variants of (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid (A6) ((S)α-Me-pIF) by tRNA display. a. Chemical structure of (S)α-Me-pIF A6. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 38. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+A6) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. The 25 sequences were ordered by the selectivity obtained as a result of selection for substrate A6, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants A6_1 to A6_9. Experiments were performed using tRNA extracted from cells carrying pMB1 plasmids encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A6. The experiment for A6_1 was repeated three times. [Figure 45-1] ~ [Figure 45-2]Selection of PylRS variants of (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (A7) (OH-(S)β3mClF) by tRNA display. a. Chemical structure of OH-(S)β3mClF A7. b. Spindle plot obtained as a result of tRNA display selection as shown in Figure 38. Selectivity is defined as the ratio of the relative abundance of a particular sequence in the positive sample (+A7) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive sample divided by the relative abundance in the input library. Enclosed regions indicate sequences showing selectivity of 5 or more and enrichment of 5 or more. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and at least one control sample. Red dots are sequences observed only in positive samples. c. Fourteen sequences were ordered by the selectivity obtained as a result of selection for substrate A7, exhibiting selectivity of 5 or more and enrichment of 5 or more. d. Fluoro-tREX for PylRS variants A7_1 to A7_7. Experiments were performed using tRNA extracted from cells carrying pMB1 plasmids encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A7. The experiment for A7_1 was repeated three times. [Figure 46-1] ~ [Figure 46-2]Characterization of the in vivo acylation activity of evolved ncM-specific PylRS variants by tREX. We have previously shown that this assay quantitatively reports on in vivo acylation. a. tREX for evolved PylRS variants. tREX was performed four times on Mm tRNAPyl extracted from cells carrying the pMB1 plasmid encoding each Mm PylRS variant and Mm tRNAPyl, grown in the presence of the indicated ncMs of 0, 1, 2, or 4 mM. b. Quantification of the in vivo acylation activity of evolved PylRS variants. The acylation level was quantified by taking the ratio of the upper band (acylated tRNA) divided by the sum of the signal intensities of the upper and lower bands (acylated and non-acylated tRNA) in the tREX gel shown in a. The data are shown as a fraction of the acylation activity of wt PylRS using 4 mM BocK (55±1%), set to 1. Points represent individual data points, bars represent the mean, and error bars represent standard deviation (sd). The inventors note that this assay is performed under conditions where acyl-tRNA complexes are not actively consumed by translation in cells and can accumulate over time. Under these conditions, enzymes with low activity in cells may yield acylation levels comparable to those of enzymes with high activity in cells. The inventors note that 12, A2, A5, and A6, thus incorporated into ribosomes, must be acylated to a level that supports ribosomal translation; this suggests that if other monomers are efficient substrates for other parts of the translation mechanism, they can be well acylated to a translation-supporting level. In future research, measuring time-dependent acylation may be useful; however, this is difficult to evaluate in vivo because tRNA acylation in vivo is likely to be governed more by the rate of ncM incorporation than by the rate of acylation.Currently, published in vitro acylation assays for rate measurement require access to radioactive monomers or radioactive tRNAs; reagents for generating these tRNAs are currently unavailable, purification of active soluble synthases for in vitro measurements is difficult, and tRNAs for in vitro measurements typically lack post-transcriptional modifications. Consequently, in vitro measurements of acylation using this system cannot provide insights into in vivo behavior. [Figure 47-1] ~ [Figure 47-2]Mass spectrometry of ncM containing GFP150XHis6. a. Raw mass spectrum of purified GFP150(S)α-Me-pIFHis6 produced from cells containing PylRS A6_1, Mm tRNAPyl CUA, GFP(150TAG)His6, and 4 mM A6. The analyzed mass spectrum is shown in Figure 36s. b. Raw mass spectrum of purified GFP150(S)β3pBrFHis6 produced from cells containing PylRS A2_1, Mm tRNAPyl CUA, GFP(150TAG)His6, and 4 mM A2. c. Intact ESI-MS of GFP(150(S)β3pBrF)His6 purified from cells carrying PylRS(A2_1), Mm tRNAPyl CUA, and GFP(150TAG)His6, grown in the presence of 4 mM A5. Measured mass: 27,939.0 Da, predicted mass: 27,938.2 Da. d. Raw mass spectrum of purified GFP150((S)β3mCF3F)His6 produced using PylRS A5_1. e. Intact ESI-MS of GFP150((S)β3mCF3F)His6 purified from cells carrying PylRS(A5_1), Mm tRNAPyl CUA, and GFP(150TAG)His6, grown in the presence of 4 mM A5. Measured mass: 27,927.0 Da, predicted mass: 27,928.3 Da. f are MS / MS spectra of ncAA-containing peptides obtained after trypsin digestion of GFP150(S)β3mBrFHis6 produced using PylRS A5_1, or GFP150(S)α-Me-pIFHis6 produced using PylRS A6_1, respectively. The precursor ions confirm the incorporation of A5 or A6 at position 150 of GFP. Fragmentation of each peptide is predicted to result in a series of β ions (red) and a series of γ ions (blue), as well as ions corresponding to the full-length peptide. [Figure 48]GFP Production. a. Production of GFPHis6 (or GFPHis6 incorporating 1 at position 150) was measured by GFP fluorescence. Cells contained the pMB1 plasmid encoding wt PylRS / tRNAPyl CUA pair and the p15A plasmid encoding GFP3TAGHis6 or GFPHis6 in the presence and absence of 4 mM BocK(1). The yield of GFPHis6 incorporating 1 produced from GFP3TAGHis6, wt PylRS / tRNAPyl CUA pair, and 1 was 80% of the wtGFPHis6 protein produced from wtGFPHis6. The fluorescence values ​​shown are normalized by OD. Bars represent the mean of three biological replicates, individual replicates are shown as dots, and error bars indicate ±sd. The yields based on the fluorescence of GFPHis6 produced by each ncM incorporation are reported for the GFPHis6 fluorescence produced by incorporation 1 in Figure 36r; the yield percentages are 6.6% (for PylRS variant 12_1 and ncM 12), 14.4% (for PylRS variant 12_1evol1 and ncM 12), 3.7% (for PylRS variant A2_1 and ncM A2), 4.1% (for PylRS variant A5_1 and ncM A5), and 37.0% (for PylRS variant A6_1 and ncM A6). For reference, the Ni-NTA purified yield of GFPHis6 expressed from cells containing the pMB1 plasmid encoding a wt PylRS / tRNAPyl CUA pair and the p15A plasmid encoding GFP3TAGHis6 in the presence of 4 mM BocK(1) was 105 ± 8 mg per liter of culture. The purified yields of GFP150XHis6 using the relevant ncM were 8.4±0.2 mg per liter of culture for PylRS12_1; 16.2±1.2 mg per liter of culture for PylRS12_1evol1; 3.6±0.4 mg per liter of culture for PylRSA2_1; 3.1±0.7 mg per liter of culture for PylRSA5_1; and 35.5±0.9 mg per liter of culture for PylRSA6_1.b. Attempts to produce GFP3(12, A1, A2, A3, A4, A5, A6, A7)His6 protein from cells carrying the pMB1 plasmid encoding the shown PylRS variant / tRNAPyl CUA pair and the p15A plasmid encoding GFP3TAGHis6, in the presence and absence of 4 mM of each ncM. Fluorescence is shown as fractions of fluorescence produced by wt PylRS / tRNAPyl CUA pair using 4 mM BocK(1) and GFP(3TAG)His6. Bars represent the average of three biological replicates, individual replicates are shown as dots, and error bars indicate ±sd. [Modes for carrying out the invention] 【0026】 The present inventors provide herein methods for measuring the degree of tRNA acylation. These methods enable the measurement of the effect of a drug on the tRNA acylation state. For example, the methods can be used to determine: i) whether a drug can charge tRNA; ii) to what extent or how efficiently a drug can charge tRNA; iii) whether a drug has an indirect effect on tRNA charging; or iv) whether a drug can chemically alter a substrate in a manner that affects charging or deacylation. The drug may be any polypeptide, nucleic acid, or condition that has, or is suspected to have, an effect on tRNA acylation. 【0027】 The method disclosed herein does not require the charged tRNA to be active in the ribosome. Therefore, the method enables the measurement of tRNA acylation using substrates that are incompatible with ribosome translation, substrates for which compatible ribosomes have not yet been identified, or substrates for which compatible ribosomes have not yet been developed. 【0028】 The inventors also provide molecules, tRNA, nucleic acid constructs, and labeled tRNA for use in conjunction with such methods. 【0029】 Methods involving split tRNA One aspect of this disclosure relates to the inventors' demonstration that tRNA can be split into two or more parts, functionally expressed in this form, and acylated in this split form. One advantage of splitting tRNA in this manner is that the inventors demonstrate that additional sequences can be covalently ligated to at least one part of tRNA. This then enables a method for measuring the identity of individual drugs capable of tRNA charging, even in large parallel libraries. 【0030】 As an example, the present inventors fuse an acyl-tRNA synthetase gene to a portion of tRNA. Under conditions in which tRNA is expressed and acyl-tRNA synthetase is expressed as a polypeptide, the acylation of tRNA can then be measured, and a direct link between the acylation state in question and acyl-tRNA synthetase can be established. 【0031】 These methods are illustrated in the Examples section of this specification. Further details are provided in the priority documents (European Patent Application No. 2306393.6 filed on 28 April 2023 and European Patent Application No. 2400299.0 filed on 9 January 2024, respectively, which are incorporated herein by reference). These methods have enabled the development of acyl-tRNA synthetases that could not be developed previously. 【0032】 Split tRNA The inventors have developed split tRNAs useful in methods for determining the acylation state of tRNA or the efficiency of tRNA acylation. These products are illustrated in the Examples section of this specification. Further details are provided in the priority documents (European Patent Application No. 2306393.6 filed on 28 April 2023 and European Patent Application No. 2400299.0 filed on 9 January 2024, respectively, which are incorporated herein by reference). These products have contributed to the development of acyl-tRNA synthetases that could not be developed previously. 【0033】 RNA fusion molecules The inventors have developed split tRNAs that can accommodate additional fused sequences and can be further charged. For example, the split tRNAs may contain additional nucleic acid sequences encoding a protein or nucleic acid. These split RNAs may include an RNA molecule containing a portion of the tRNA and a sequence encoding the polypeptide of interest / nucleic acid of interest. These products are illustrated in the Examples section of this specification. Further details are provided in the priority documents (European Patent Application No. 2306393.6 filed on 28 April 2023 and European Patent Application No. 2400299.0 filed on 9 January 2024, respectively, which are incorporated herein by reference). These products have contributed to the development of acyl-tRNA synthetases that could not be developed previously. 【0034】 Method for determining the acylation state or efficiency of tRNA acylation The inventors have further developed methods for labeling acylated tRNAs. These methods enable the identification of charged tRNAs and are particularly sensitive. Embodiments of these methods are referred herein to as fluoro-tREX. This method allows specific acylated tRNAs from a pool of tRNAs isolated from cells to be labeled by primer extension using fluorescent dNTPs. Another embodiment of these methods is referred herein to as bio-tREX. Bio-tREX enables the selective isolation of specific acylated tRNAs by using primer extension with biotinylated dNTPs and streptavidin pulldown. These methods are illustrated in the Examples section herein. Further details are provided in the priority documents (European Patent Application No. 2306393.6 filed on 28 April 2023 and European Patent Application No. 2400299.0 filed on 9 January 2024, respectively, which are incorporated herein by reference). 【0035】 Nucleic acid construct The inventors hereby demonstrate that split-tRNA can be produced from a single gene in which two halves of tRNA are circularly permuted and linked by an intervening sequence. The primary transcript of this gene is processed in cells to obtain a functional split-tRNA that is acylated by acyl-tRNA synthetase. These constructs are illustrated in the Examples section of this specification. Further details are provided in priority documents (European Patent Application No. 2306393.6 filed on 28 April 2023 and European Patent Application No. 2400299.0 filed on 9 January 2024, respectively, which are incorporated herein by reference). These constructs have contributed to the development of acyl-tRNA synthetases that could not be developed previously. 【0036】 Beta-amino acid-acyl-tRNA synthetase The present inventors have identified an acyl-tRNA synthetase capable of acylating tRNA using beta-amino acids, utilizing the methods and means disclosed herein. 【0037】 Therefore, in the first embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using beta amino acids, M300A, M300C, M300D, M300M, or M300S for sequence number 1; A302A, A302C, A302D, A302G, A302H, A302L, A302N, or A302Y for sequence number 1; and N346A, N346C, N346G, N346S, N346T, or N346V for Sequence ID No. 1 An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0038】 In one embodiment, the acyl-tRNA synthetase includes mutations corresponding to M300A, M300C, M300D, or M300S mutations for SEQ ID NO: 1; A302C, A302D, A302G, A302H, A302L, A302N, or A302Y mutations for SEQ ID NO: 1; and mutations corresponding to N346A, N346C, N346G, N346S, N346T, or N346V mutations for SEQ ID NO: 1. 【0039】 When an acyl-tRNA synthetase preferentially acylates tRNA using a specific substrate in the presence of standard amino acids, the synthetase can "specifically" acylate tRNA using the substrate. In certain embodiments, the acyl-tRNA synthetase can be expressed in cells in the presence of a substrate and target tRNA, and at least 60%, 70%, 80%, 90%, 95%, or 99% of the acylated target tRNA in the cell is acylated using the specific substrate. For example, the cells may be bacterial cells such as E. coli cells. 【0040】 The beta-amino acid may be an analog of S-beta-3-phenylalanine. In particular, the beta-amino acid may be (S)-3-amino-3-(3-bromophenyl)propanoic acid. The beta-amino acid may be (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3 It may also be MDF. The beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 The beta amino acid may be (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3 It may also be pmFF. The beta amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 It may also be (oBrF). The beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 (mCF3F) is also acceptable. 【0041】 In certain embodiments, the acyl-tRNA synthetase includes mutations corresponding to M300D and A302H. M300D and A302H were the most common mutations found for screening any of the beta amino acids tested. In certain embodiments, the acyl-tRNA synthetase includes mutations corresponding to M300D;A302H; and N346G, N346A, or N346S. In certain embodiments, the acyl-tRNA synthetase includes mutations corresponding to M300D;A302H; and N346G / N346A. In certain embodiments, the M300 mutation is M300D, the A302 mutation is A302H, and the N346 mutation is N346G. 【0042】 In the embodiment, in particular, when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the acyl-tRNA synthetase includes an amino acid sequence containing mutations corresponding to the M300D mutation for SEQ ID NO: 1; the A302H, A302Y, or A302C mutation for SEQ ID NO: 1; and the N346G, N346A, or N346S mutation for SEQ ID NO: 1. 【0043】 In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(3 - bromophenyl)propanoic acid, the M300 mutation is M330D. In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(benzo[d][1,3]dioxol - 5 - yl)propanoic acid ((S)β 3 MDF), the M300 mutations are M300A, M300C, M300D, or the wild - type residue (M300M). M300D was the most common in the screening. In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(4 - bromophenyl)propanoic acid ((S)β 3 pBrF), the M300 mutations are M300C, M300D, M300S, or the wild - type residue (M300M). M300D was the most common in the screening. In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(3,4 - difluorophenyl)propanoic acid ((S)β 3 pmFF), the M300 residues are M300A, M300C, M300D, or the wild - type M300M. M300D was the most common in the screening. In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(2 - bromophenyl)propanoic acid ((S)β 3 oBrF), the M300 residues are M300D, M300S, or the wild - type M300M. M300D was the most common in the screening. In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 mCF3F), the mutation is M300D. 【0044】 In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(3 - bromophenyl)propanoic acid, the A302 mutations are A302C, A302H, or A302Y. In the example, particularly when the beta - amino acid is (S)-3 - amino - 3-(benzo[d][1,3]dioxol - 5 - yl)propanoic acid ((S)β 3If the MDF is present, the A302 mutation is wild-type (A302A), A302C, or A302H. A302H was the most common in screening. For example, the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 If pBrF is present, the A302 mutation is either A302G or A302H. A302H was the most common in the screening. For example, the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3 If pmFF, the A302 mutation is A302D, A302H, A302L, or A302N. A302H was the most common in screening. For example, the beta amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 If oBrF, the A302 mutation is either A302D or A302H. A302H was the most common in the screening. For example, the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 If the case is mCF3F, the A302 mutation is A302H, A302N, or A302Y; or in particular, A302H or A302Y. 【0045】 The amino acid sequence may include a mutation at the position corresponding to L305 in SEQ ID NO: 1. In the example, the mutation is L305C, L305F, L305G, L305H, L305I, L305N, or L305V, or a conserved substitution or wild-type residue of the said residue. In particular, in the example, when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is L305H or L305C, or a conserved substitution or wild-type residue of the said residue. In particular, in the example, when the beta-amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3If the residue is MDF, the mutation is L305C, L305G, L305H, L305I, or L305N, or a wild-type residue; in particular, the mutation is L305C, L305I, or L305L. For example, in particular, the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 If pBrF is present, the mutations are L305C, L305F, L305I, L305N, L305V, or wild-type (L305L); or in particular, L305F, L305I, L305L, or L305N. For example, in particular, the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3 If pmFF, the mutations are L305C, L305F, L305H, L305I, L305V, or wild type (L305L); or in particular, L305C, L305I, L305L, or L305V. For example, the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 If mCF3F, the mutation is L305F, L305I, or L305V, or wild-type (L305L). The inventors have identified a functional beta-amino acid-tRNA synthetase that does not contain the L305 mutation, which may be referred to as containing L305L. Therefore, this mutation is not present in all embodiments. In L305L, the beta-amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 This is particularly relevant to the example of oBrF. 【0046】 The amino acid sequence may include a mutation at the position corresponding to Y306 in SEQ ID NO: 1. In the example, the mutation is Y306C, Y306F, Y306H, Y306I, Y306L, Y306N, Y306R, or Y306V, or a conserved substitution or wild-type residue of the said residue. In particular, in the example, when the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is Y306L, Y306F, Y306R, or Y306C, or a conserved substitution or wild-type residue of the said residue. In particular, in the example, when the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3 If the result is MDF, the mutation is Y306H, Y306I, or Y306L, or a wild-type residue. For example, the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 If pBrF is present, the mutation is either Y306C or Y306I, or wild-type (Y306Y); or in particular, Y306I or Y306Y. For example, in particular, the beta-amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3 If pmFF, then Y306F, Y306I, Y306L, Y306R, or wild-type (Y306Y); or in particular Y306I, Y306L, or Y306Y. For example, in particular, the beta-amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 If oBrF), the mutations are Y306H, Y306I, Y306L, Y306R, or wild-type (Y306Y); or in particular Y306H, Y306L, Y306R, or Y306Y. For example, in particular the beta-amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3If the gene is mCF3F, the mutations are Y306F, Y306I, Y306L, Y306N, Y306R, or Y306V, or the wild type (Y306Y); or in particular, Y306F, Y306L, or Y306Y. The inventors have identified a functional beta-amino acid-tRNA synthetase that does not contain the Y306 mutation, which may be described as containing Y306Y. Therefore, this mutation is not present in all embodiments. 【0047】 The amino acid sequence may include a mutation at the position corresponding to L309 in SEQ ID NO: 1. In the example, the mutation is L309C, L309F, L309G, L309H, L309I, L309N, L309S, L309V, or L309Y, or a conserved substitution or wild-type residue of the said residue. In particular, in the example, when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is L309V or L309C, or a conserved substitution or wild-type residue of the said residue. In particular, in the example, when the beta-amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3 If the result is MDF, the mutation is L309C, L309F, L309H, L309N, or L309S, or a wild-type residue; or, in particular, L309C, L309F, L309H, L309N, or a wild-type residue. For example, in particular, the beta amino acid is (S)-3-amino-3-(4-(bromophenyl)propanoic acid ((S)β 3 If pBrF is present, the mutation is L309C, L309F, L309G, L309I, L309N, L309S, L309V, or L309Y, or wild-type (L309L); or in particular, L309C, L309F, L309L, or L309N. For example, in particular, the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3If pmFF, the mutations are L309C, L309G, L309H, L309N, L309S, L309V, or wild type (L309L); or in particular L309G, L309H, L309L, L309N, L309S, or L309V. For example, in particular the beta amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 If oBrF, the mutation is L309C, L309G, L309H, or wild-type (L309L); or in particular L309G, L309H, or L309L. For example, in particular the beta amino acid is (S)-3-amino-3-(3-trifluoromethyl)phenyl)propanoic acid ((S)β 3 In the case of mCF3F), the mutations are L309C, L309G, L309H, L309N, L309V, or wild-type (L309L); or more specifically, L309C, L309L, L309N, or L309V. The inventors have identified a functional beta-amino acid-tRNA synthetase that does not contain the L309 mutation, which may be described as containing L309L. Therefore, this mutation is not present in all embodiments. 【0048】 The amino acid sequence may include a mutation at the position corresponding to M344 in SEQ ID NO: 1. In the example, the mutation is M344E, M344G, or M344Q, or a conserved substitution or wild-type residue of the aforementioned residue. In particular, in the example, the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3 If the result is MDF, the mutation is M344E or M344Q, or a wild-type residue. For example, the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 If pBrF is present, the mutation is M344Q or wild-type. For example, the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3If the result is pmFF, the mutation is either M344Q or wild-type (M344M). M344Q was the most common in the screening. For example, the beta amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 If oBrF, the mutation is M344G, M344Q, or wild-type (M344M). M344Q was the most common in screening. For example, the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 If the sequence is mCF3F, it is either M344Q or wild-type (M344M). M344M was the most common in the screening. The inventors identified a functional beta-amino acid-tRNA synthetase containing a wild-type residue at the position corresponding to M344 in SEQ ID NO: 1. This can be described as containing M344M. For example, the sequence contains M344M, particularly when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid. 【0049】 In particular, the N346 mutation is N346G when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid. 3 If the MDF is present, then the N346 mutation is N346A, N346C, N346G, N346S, N346T, or N346V; or in particular, N346G or N346A. For example, in particular, the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 If pBrF is present, then the N346 mutations are N346A, N346C, N346G, N346S, N346T, N346V; or in particular, N346A or N346G. For example, in particular, the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3If the result is pmFF, the N346 mutation is N346A, N346C, N346G, N346S, or N346T. N346G was the most common in the screening. For example, the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 If oBrF)), then the N346 mutation is N346A, N346G, N346S, or N346T; or more specifically, N346A, N346G, or N346S; or more specifically, N346G or N346S. For example, in particular, the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 If the case is mCF3F, the N346 mutation is N346G. 【0050】 The amino acid sequence may contain a mutation at the position corresponding to C348 in SEQ ID NO: 1. In the example, the mutation is C348A, C348F, C348G, C348H, C348I, C348L, C348M, C348N, C348R, C348S, C348T, C348V, or C348Y, or a conserved substitution or wild-type residue of the said residue. In the example, in particular when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is C348I, C348L, C348V, or C348F, or a conserved substitution or wild-type residue of the said residue. In a specific example, in particular when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is C348F, C348I, or C348L. In particular, the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3 If the MDF is present, the mutations are C348G, C348I, C348L, C348M, C348N, C348S, C348V, or C348Y; or in particular C348G, C348N, C348S, or C348V. For example, in particular the beta amino acid is (S)-3-amino-3-(4-(bromophenyl)propanoic acid ((S)β 3If pBrF, the mutation is C348F, C348G, C348H, C348I, C348N, or C348S, or wild-type (C348C); or in particular, C348C, C348G, C348I, or C348S. For example, in particular, the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3 If pmFF, the mutations are C348A, C348F, C348H, C348I, C348L, C348R, C348S, C348T, C348V, or wild type (C348C); or in particular C348F, C348L, C348S, or C348V. For example, in particular the case where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 If oBrF)), the mutation is C348G, C348I, C348S, C348V, C348Y, or wild type (C348C); or in particular, the mutation is C348C, C348I, C348S, or C348Y. For example, in particular, the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 In the case of mCF3F), the mutations are C348F, C348H, C348I, C348L, C348S, C348V, or C348Y, or wild-type (C348C); or in particular, C348F, C348H, or C348V. The inventors have identified a functional beta-amino acid-tRNA synthetase that does not contain the C348 mutation, which may be said to contain C348C. Therefore, this mutation is not present in all embodiments. 【0051】 The amino acid sequence may include a wild-type residue at the position corresponding to Y384 in SEQ ID NO: 1. This can be referred to as containing Y384Y. 【0052】 The amino acid sequence may contain a mutation at the position corresponding to S399 in SEQ ID NO: 1. This can be referred to as containing S399S. 【0053】 The amino acid sequence may contain a mutation at the position corresponding to V401 in SEQ ID NO: 1. In the example, this residue is one of 20 standard amino acids (i.e., V401X). In the example, the mutation is V401A, V401C, V401F, V401K, V401L, V401S, or V401T, or a conserved substitution or wild-type residue of the said residue. In the example, the mutation is V401A, V401C, V401K, V401L, V401S, or V401T, or a conserved substitution or wild-type residue of the said residue. In certain examples, in particular, when the beta-amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is V401C, V401S, V401L, V401K, or V401A, or a conserved substitution or wild-type residue of the said residue. In particular, the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3 If the MDF is present, the mutation is V401A, V401C, V401K, or V401L, or the wild-type residue (V401V); in particular, V401C, V401L, or V401V. For example, in particular, the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 If pBrF is present, the mutation is V401A, V401C, V401K, or V401L, or wild-type (V401V); or in particular V401C, V401L, or V401V. For example, in particular the beta-amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3 If pmFF, the mutations are V401C, V401F, V401K, V401L, V401T, or wild-type (V401V); or in particular V401C, V401L, or V401V. For example, in particular the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3If oBrF)), the mutation is V401A, V401C, V401K, V401L, or wild-type (V401V); or in particular V401C or V401V. For example, in particular the beta-amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 In the case of mCF3F), the mutation is V401C, V401K, V401L, V401S, or V401T, or a wild-type residue; or more specifically, V401S, V401T, or V401V. The inventors have identified a functional beta-amino acid-tRNA synthetase that does not contain the V401 mutation, which may be said to contain V401V. Therefore, this mutation is not present in all embodiments. 【0054】 In certain embodiments, the acyl-tRNA synthetase includes residues corresponding to M300D, A302H, M344M / M344Q, and N346G / N346A, and optionally the C348 mutation. In some embodiments, C348 may be any of C348A, C348F, C348G, C348H, C348I, C348L, C348M, C348N, C348R, C348S, C348T, C348V, or C348Y, or more specifically, any of C348F, C348G, C348H, C348I, C348L, C348N, C348S, C348V, or C348Y. 【0055】 The inventors provide specific embodiments in Table 1. These embodiments are defined for SEQ ID NO: 1, for example, 12_1 includes M300D, A302H, L305H, etc. The inventors tested each of the following sets of mutations when applied to the Mm PylRS backbone (SEQ ID NO: 1) and found that each is functional with respect to charging tRNA using beta amino acids. In particular, these embodiments can charge tRNA using (S)-3-amino-3-(3-bromophenyl)propanoic acid. [Table 1] 【0056】 Acyl-tRNA synthetases may contain amino acid sequences that include any of the sets of mutations in Table 1. Acyl-tRNA synthetases may contain amino acid sequences that include either the sets of mutations and wild-type residues in Table 1. These mutations are particularly suitable for beta-amino acid-tRNA synthetases that can incorporate the beta-amino acids listed in the table headings. 【0057】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing mutations corresponding to M300D, A302H, L305H, N346G, C348F, and V401C. In another embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing residues corresponding to M300D, A302H, L305H, Y306Y, L309L, M344M, N346G, C348F, Y384Y, S399S, and V401C. 【0058】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing mutations corresponding to M300D, A302H, Y306L, N346G, C348L, and V401S. In another embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing residues corresponding to M300D, A302H, L305L, Y306L, L309L, M344M, N346G, C348L, Y384Y, S399S, and V401S. 【0059】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing mutations corresponding to M300D, A302H, Y306L, N346G, C348L, and V401L. In another embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing residues corresponding to M300D, A302H, L305L, Y306L, L309L, M344M, N346G, C348L, Y384Y, S399S, and V401L. 【0060】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing mutations corresponding to M300D, A302C, L305C, Y306F, L309V, N346G, C348L, and V401C. In another embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing residues corresponding to M300D, A302C, L305C, Y306F, L309V, M344M, N346G, C348L, Y384Y, S399S, and V401C. 【0061】 In addition to the mutations discussed above and optionally the set of wild-type residues, the acyl-tRNA synthetase may further include mutations or combinations of mutations at the positions corresponding to the following positions in SEQ ID NO: F295, N307, T364, F366, K375, T387, D414, and G421. In certain embodiments, the mutations may be F295L / F295I, N307K, T364A, F366L, K375R, T387I / T387S, D414V, and G421A, or any one or any combination of conservative substitutions or wild-type residues of the said residues. 【0062】 [Table 2] 【0063】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations in Table 2. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutations and / or wild-type residues in Table 2. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with an identifier in Table 2 (e.g., A1_2) and / or optionally a set of wild-type residues. These mutations are particularly suitable for beta-amino acid-tRNA synthetases that can incorporate the beta-amino acids listed in the table headings. 【0064】 [Table 3] 【0065】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations in Table 3. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutations and / or wild-type residues in Table 3. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with an identifier in Table 3 (e.g., A2_2) and optionally a set of wild-type residues. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with A2_1, A2_2, A2_3, A2_5, A2_6, A2_8, A2_9, A2_10, A2_11, or A2_12 in Table 3 and optionally a set of wild-type residues. These mutations are particularly suitable for beta-amino acid-tRNA synthetases that can incorporate the beta-amino acids listed in the table headings. 【0066】 [Table 4] 【0067】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations in Table 4. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutations and wild-type residues in Table 4. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with an identifier in Table 4 (e.g., A3_2) and optionally the set of wild-type residues. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with any one of A3_1, A3_2, A3_3, A3_4, A3_5, A3_7, A3_8, A3_9, A3_10, or A3_11 in Table 4 and optionally the set of wild-type residues. These mutations are particularly suitable for beta-amino acid-tRNA synthetases that can incorporate the beta-amino acids listed in the table headings. 【0068】 [Table 5] 【0069】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations in Table 5. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutations and / or wild-type residues in Table 5. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with an identifier in Table 5 (e.g., A4_3) and optionally a set of wild-type residues. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with any one of A4_1, A4_3, A4_6, or A4_7 in Table 5 and optionally a set of wild-type residues. These mutations are particularly suitable for beta-amino acid-tRNA synthetases that can incorporate the beta-amino acids listed in the table headings. 【0070】 [Table 6] 【0071】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations in Table 6. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutations and / or sets of wild-type residues in Table 6. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with an identifier in Table 6 (e.g., A5_2) and optionally a set of wild-type residues. Acyl-tRNA synthetase may contain an amino acid sequence that includes either the mutation associated with any one of A5_1, A5_2, A5_3, A5_4, A5_5, A5_6, or A5_8 in Table 6 and optionally a set of wild-type residues. These mutations are particularly suitable for beta-amino acid-tRNA synthetases that can incorporate the beta-amino acids listed in the table headings. 【0072】 In some embodiments, the acyl-tRNA synthetase may further include one of the following sets of mutations: a) N307K and F366L b) F295I and T387I c) G421A d) T364A and T387I e) T387S and D414V f) F295L and K375R. 【0073】 In the experimental data, the mutation referred to as "evol1" is set "a)", the mutation referred to as "evol2" is set "b)", the mutation referred to as "evol3" is set "c)", the mutation referred to as "evol5" is set "d)", the mutation referred to as "evol7" is set "e)", and the mutation referred to as "evol8" is set "f)". For example, "12_1 evol1 This has the mutation "12_1" in Table 1 and the mutation in the above set "a)". 【0074】 In certain embodiments, sets a), b), c), d), e), or f) may each exist in combination with any set of mutations in any of Tables 1, 2, 3, 4, 5, or 6 and optionally with any set of wild-type residues. In preferred embodiments, sets a), b), c), d), e), or f) may each exist in combination with any set of mutations associated with any set of identifiers (e.g., 12_1) in any of Tables 1, 2, 3, 4, 5, or 6 and optionally with any set of wild-type residues. 【0075】 The mutation in the first embodiment is the pyrrolidyl-tRNA synthetase (Mm PylRS) sequence of wild-type metanosarsinina mazei, provided below with reference to Sequence ID No. 1. MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARA PKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKD LQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL(Sequence No. 1) 【0076】 To assist with alignment, a version of the Mm PylRS sequence is provided below, with "X" marked at positions 300, 302, 305, 306, 309, 346, 348, 384, and 401. MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARA PKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKD LQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPXLXPNXXNYXRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLXFXQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVXGDTLDVMHGDLELSSAXVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL(Sequence ID 2) 【0077】 Accordingly, in one embodiment, there is an acyl-tRNA synthetase capable of specifically acyling tRNA using beta amino acids, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid sequence comprises any of the mutations or sets of mutations disclosed in connection with the first embodiment. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the amino acid sequence may be identical to SEQ ID NO: 1 or SEQ ID NO: 2, apart from the described mutations. 【0078】 Acyl-tRNA synthetase may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may include amino acid sequences containing the M300D mutation, A302H, A302Y, or A302C mutation, and the N346G, N346A, or N346S mutation. The amino acid sequence may include mutations corresponding to any one of the mutations 12_1, 12_2, 12_3, 12_4, 12_A, 12_B, 12_C, or 12_D in Table 1. The amino acid sequence may also include residues corresponding to any one of the mutations 12_1, 12_2, 12_3, 12_4, 12_A, 12_B, 12_C, or 12_D in Table 1 and wild-type residues. The amino acid sequence may further include mutations corresponding to the mutations listed herein as set a), b), c), d), e), or f). 【0079】 Acyl-tRNA synthetase may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may include an amino acid sequence containing any of the sets of mutations in Tables 1 to 6. Acyl-tRNA synthetase may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may include an amino acid sequence containing any of the sets of mutations and wild-type residues in Tables 1 to 6. Acyl-tRNA synthetases have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and are A1_1, A1_2, A1_3, A1_4, A1_5, A1_6, A1_7, A1_8, A1_9, A1_10, A1_11, A1_12, A2_1, A2_2, A2_3, A2_5, A2_6, A2_7 in Tables 2-6. The amino acid sequence may include either a set of mutations labeled as A2_8, A2_9, A2_10, A2_11, A2_12, A3_1, A3_2, A3_3, A3_4, A3_5, A3_7, A3_8, A3_9, A3_10, A3_11, A4_1, A4_3, A4_6, A4_7, A5_1, A5_2, A5_3, A5_4, A5_5, A5_6, or A5_8, or an amino acid sequence containing either a set of mutations and a set of wild-type residues. The amino acid sequence may further include mutations corresponding to the mutations listed as sets a), b), c), d), e), or f) herein. 【0080】 In a preferred embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing mutations corresponding to M300D, A302H, L305H, N307K, N346G, C348F, F366L, and V401C relative to SEQ ID NO: 1. The acyl-tRNA synthetase may also comprise an amino acid sequence containing residues corresponding to M300D, A302H, L305H, Y306Y, N307K, L309L, M344M, N346G, C348F, F366L, Y384Y, S399S, and V401C relative to SEQ ID NO: 1. The amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the amino acid sequence may be identical to that of SEQ ID NO: 1 or SEQ ID NO: 2, apart from the described mutations. 【0081】 Those skilled in the art can also apply the identified mutations to other PylRS backchains, including those not disclosed herein. This is not limited to the fact that other PylRS may have low sequence identity with respect to Mm PylRS. To apply the mutations described with reference to Mm PylRS to other synthases, the sequences should be aligned to identify the corresponding residues. In particular, sequences representing the catalytic site of the synthase can be aligned. For example, the region of Sequence ID No. 1, extending from residue 296 to residue 428, can be aligned with the sequence of another PylRS to produce the corresponding mutation. Further information regarding the transfer of mutations from one PylRS backchain to the other is provided in International Publication No. 2013 / 171485A1 (incorporated herein by reference). An example alignment is provided in Figure 35. 【0082】 Therefore, in one embodiment, an acyl-tRNA synthase is provided that can specifically acylate tRNA using beta amino acids, comprising an amino acid sequence including any set of mutations disclosed in connection with the first embodiment and optionally a wild-type residue, or any set of mutations disclosed in connection with the first embodiment and optionally a wild-type residue, wherein the acyl-tRNA synthase backbone is PylRS. 【0083】 The mutation can be applied to PylRS derived from Nitrososphaeria archaeon (Nitra). This sequence is provided below. MSKIRFTRGQIHRLIELGAEPTELERDFETEAERDKEFNKIAENLARKNLKNIKDFLEQRRKPLVRVIEEKLRTTALRLGFSEVVTPIIIPRLFIKRMGIDEGDPLWKQVMLIDDKRALRPMLAPNLYVLMAKLSNIVRPVKIFEIGPCFRRETGGRYHLEEFTMFNMVELAPEGDPKERLLDYIDTIMRDIGLNYTISVEPSNVYGETLDVVVNGIEVASAAIGPKPIDANWGVREPWIGVGFGVERLAMLVGGYNSIARIAKSLSYLDGSTLSVIKLRW (Sequence ID 3) 【0084】 To assist in alignment, a version of the Nitra PylRS sequence is provided below, where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each of the mutations of the present invention can be applied. Further corresponding positions can be identified in the same manner. MSKIRFTRGQIHRLIELGAEPTELERDFETEAERDKEFNKIAENLARKNLKNIKDFLEQRRKPLVRVIEEKLRTTALRLGFSEVVTPIIIPRLFIKRMGIDEGDPLWKQVMLIDDKRALRPXLXPNXXVLXAKLSNIVRPVKIFEIGPCFRRETGGRYHLEEFTMFXMXELAPEGDPKERLLDYIDTIMRDIGLNYTISVEPSNVXGETLDVVVNGIEVASAXIGPKPIDANWGVREPWIGVGFGVERLAMLVGGYNSIARIAKSLSYLDGSTLSVIKLRW (Sequence ID 4) 【0085】 The mutation can be applied to PylRS derived from bacteria of the order Clostridiales (Clos). This sequence is provided below. MENFTITQTERLKQLNCENDVLELEFEDSEARNSKFREIEIGRVKKGKENIKNLLKEKHITISDEVGNKLSDWLMSKDYTKVLTPTIISKDQLKAMTIDEENHLFSQVFWIDNNKCLRPMLAPNLYIVMRELKRITNEPVKIFEIGSCFRKESQGARHMNEFTMLNMVELASVEDGKQLDTLKALAHEAMESLGVESYELVIEESAVYGSTLDIEIDGIEVASGSYGPHELDANWDIFDTWVGIGFGIERLAMAINGGSTIKKYGRSINFIDGETMKL(Sequence ID 5) 【0086】 To assist in alignment, a version of the Clos PylRS sequence is provided below, where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each of the mutations of the present invention can be applied. Further corresponding positions can be identified in the same manner. MENFTITQTERLKQLNCENDVLELEFEDSEARNSKFREIEIGRVKKGKENIKNLLKEKHITISDEVGNKLSDWLMSKDYTKVLTPTIISKDQLKAMTIDEENHLFSQVFWIDNNKCLRPXLXPNXXIVXRELKRITNEPVKIFEIGSCFRKESQGARHMNEFTMLXMXELASVEDGKQLDTLKALAHEAMESLGVESYELVIEESAVXGSTLDIEIDGIEVASGXYGPHELDANWDIFDTWVGIGFGIERLAMAINGGSTIKKYGRSINFIDGETMKL(Sequence ID 6) 【0087】 The mutation can be applied to PylRS derived from the Methanomethylophilus sp. 1R26 (1R26). This sequence is provided below. MAEHFTDAQIQRLREYGNGTYKDMEFADVSAREKAFTKLMSDASRDNESALKGMIAHPARQGLSRLMNDIADALVADGFIEVRTPIIISKDALAKMTITPDKPLFKQVFWIDDKRALRPMLAPSLYTVMRSLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPAGDATESLKKYIGIVMKAAGLPDYQLVHEESDVYKETIDVEINGQEVCSAAVGPHYLDAAHDVHEPWAGAGFGLERLLTIRQGYSTVMKGGASTTYLNGAKMD(Sequence ID 7) 【0088】 To assist in alignment, a version of the 1R26 PylRS sequence is provided below, where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each of the mutations of the present invention can be applied. Furthermore, corresponding positions can be identified in the same manner. MAEHFTDAQIQRLREYGNGTYKDMEFADVSAREKAFTKLMSDASRDNESALKGMIAHPARQGLSRLMNDIADALVADGFIEVRTPIIISKDALAKMTITPDKPLFKQVFWIDDKRALRPXMXAPXXYTXMRSLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTMLXLXDMGPAGDATESLKKYIGIVMKAAGLPDYQLVHEESDVXKETIDVEINGQEVCSAXVGPHYLDAAHDVHEPWAGAGFGLERLLTIRQGYSTVMKGGASTTYLNGAKMD(Sequence ID 8) 【0089】 The mutation can be applied to PylRS derived from Methanomassiliicoccus luminyensis 1 (Lum1). This sequence is provided below. MDTRLTPAQAQRIREMGGTVDPSLAFSSEAERESAFQRISADLQGANLAKIRRCAEAPERHPIGSLENTLACALAAKGFIEVKTPMMIPADGLVKMGIDESHPLWNQVFWVGPKKALRPMLAPNLYFLMRHLRRSVPAPLLLFEIGPCFRKERGSNHLEEFTMLNLVELAPQADATERLKEHIATVMNAVGLPYELVVEGSEVYGTTIDVEVDGVELASGAVGPLPMDPHGITEPWAGVGFGLERIALMRTKEQNIKKVGRSLVYVNGARIDI (Sequence ID 9) 【0090】 To assist in alignment, a version of the Lum1 PylRS sequence is provided below, where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each of the mutations of the present invention can be applied. Furthermore, corresponding positions can be identified in the same manner. MDTRLTPAQAQRIREMGGTVDPSLAFSSEAERESAFQRISADLQGANLAKIRRCAEAPERHPIGSLENTLACALAAKGFIEVKTPMMIPADGLVKMGIDESHPLWNQVFWVGPKKALRPXLXPNXXFLXRHLRRSVPAPLLLFEIGPCFRKERGSNHLEEFTMLXLXELAPQADATERLKEHIATVMNAVGLPYELVVEGSEVXGTTIDVEVDGVELASGXVGPLPMDPHGITEPWAGVGFGLERIALMRTKEQNIKKVGRSLVYVNGARIDI (Sequence ID 10) 【0091】 Other main chains to which the mutations of the present invention can be applied include Mb PylRS (SEQ ID NO: 11), Lum1 PylRS (SEQ ID NO: 12), Tron PylRS (SEQ ID NO: 13), Gemm PylRS (SEQ ID NO: 14), PG48 PylRS (SEQ ID NO: 15), I2 PylRS (SEQ ID NO: 16), D121 PylRS (SEQ ID NO: 17), and D416 PylRS (SEQ ID NO: 18). 【0092】 Accordingly, in one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18, wherein the amino acid sequence comprises either the mutations disclosed in connection with the first embodiment and optionally wild-type residues, or a set of mutations and optionally wild-type residues. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18. In one embodiment, the amino acid sequence may be identical to any one of SEQ ID NOs: 1 to 18, apart from the described mutations. 【0093】 In certain embodiments, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 10, wherein the amino acid sequence comprises either the mutations disclosed in connection with the first embodiment and optionally wild-type residues, or a set of mutations and optionally wild-type residues. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 10. In some embodiments, the amino acid sequence may be identical to any one of SEQ ID NOs: 1 to 10, apart from the described mutations. 【0094】 Therefore, the acyl-tRNA synthetase may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to any one of the amino acid sequences of SEQ ID NOs: 1-18, and may include an amino acid sequence containing mutations corresponding to the M300D, A302H, A302Y, or A302C mutations in SEQ ID NO: 1, and the N346G, N346A, or N346S mutations (indicated by the relevant "X" in SEQ ID NOs: 2, 4, 6, 8, or 10). The amino acid sequence may also contain mutations corresponding to any one of the mutations in Table 1: 12_1, 12_2, 12_3, 12_4, 12_A, 12_B, 12_C, or 12_D. The amino acid sequence may include residues corresponding to any one of the mutations 12_1, 12_2, 12_3, 12_4, 12_A, 12_B, 12_C, or 12_D in Table 1, as well as wild-type residues. The amino acid sequence may include residues corresponding to any one set of mutations in Tables 1-6. The amino acid sequence may include residues corresponding to any one set of mutations in Tables 1-6, as well as wild-type residues. The amino acid sequence may include residues corresponding to A1_1, A1_2, A1_3, A1_4, A1_5, A1_6, A1_7, A1_8, A1_9, A1_10, A1_11, A1_12, A2_1, A2_2, A2_3, A2_5, A2_6, A2_7, A2_8, A2_9, A2_10, A2_11, A2_12, A3_1, A3_2, The amino acid sequence may further include mutations corresponding to sets of mutations, or sets of mutations and wild-type residues, labeled as A3_3, A3_4, A3_5, A3_7, A3_8, A3_9, A3_10, A3_11, A4_1, A4_3, A4_6, A4_7, A5_1, A5_2, A5_3, A5_4, A5_5, A5_6, or A5_8. The amino acid sequence may further include mutations corresponding to mutations listed as sets a), b), c), d), e), or f) herein. 【0095】 In a preferred embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using beta amino acids, and comprises an amino acid sequence containing mutations corresponding to M300D, A302H, L305H, N307K, N346G, C348F, F366L, and V401C relative to SEQ ID NO: 1. The acyl-tRNA synthetase may also comprise an amino acid sequence containing residues corresponding to M300D, A302H, L305H, Y306Y, N307K, L309L, M344M, N346G, C348F, F366L, Y384Y, S399S, and V401C relative to SEQ ID NO: 1. The amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18. The amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of sequence numbers 1 to 10. 【0096】 The synthase of the first embodiment may include sequence changes relative to the wild-type sequence in addition to the mutations described in detail herein. Specifically, the synthase may include sequence changes at sites that do not significantly impair the function or operation of the synthase described herein. To verify that the function of the synthase is not inactivated and has not been significantly altered, its function can be tested by manipulating the synthase as described in the Examples section, etc. Therefore, provided that the synthase retains its function and can be tested as described herein, sequence changes relative to the wild-type reference sequence can be produced in the synthase. 【0097】 Figure 35 provides sequence alignments, where "*" indicates residues found in all aligned aaRS sequences, ":" indicates residues with strong similarity between aligned sequences, and "." indicates residues with some similarity between aligned sequences. Synthases may be at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any one of sequence numbers 1-18, and variations are found only in i) "*"; ii) "*" or ":", or iii) residues not marked with "*", ":", or ".", apart from the specifically described mutations discussed herein. 【0098】 For example, conservative substitutions can be made according to the following table. The amino acids shown in the same block in the second column and preferably in the same row in the third column can be substituted for each other: [Table 7] 【0099】 The acyl-tRNA synthetase of the first embodiment can be isolated or purified. The acyl-tRNA synthetase of the first embodiment may be a non-natural acyl-tRNA synthetase. 【0100】 In a second embodiment, the use of acyl-tRNA synthetase in a method for producing a polymer containing beta amino acids is provided. 【0101】 In one embodiment, a method for producing a polymer containing beta amino acids, i) Use of acyl-tRNA synthetase for acylation of tRNA using beta amino acids, wherein the acyl-tRNA synthetase includes a mutation that enables acylation of tRNA using beta amino acids, and ii) Incorporation of beta amino acids into polymers A method is provided that includes this. 【0102】 In one embodiment, the use of a first-mode acyl-tRNA synthetase in a method for producing a polymer containing beta amino acids is provided. 【0103】 In one embodiment, a method for producing a polymer containing beta amino acids, i) Use of acyl-tRNA synthetase in a first aspect for acylating tRNA using beta amino acids, and ii) Incorporation of beta amino acids into polymers A method is provided that includes this. 【0104】 The beta-amino acid may also be an analog of S-beta-3-phenylalanine. In particular, the beta-amino acid may be (S)-3-amino-3-(3-bromophenyl)propanoic acid, (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β 3 MDF), (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β 3 pBrF), (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β 3 pmFF), (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β 3 oBrF), or (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β 3 (mCF3F) is also acceptable. 【0105】 The polymer may contain one or more standard amino acids or one or more naturally occurring amino acids. The polymer may also contain one or more unnatural amino acids and / or hydroxy acids. The unnatural amino acids may be α-amino acids, and / or the hydroxy acids may be α-hydroxy acids. 【0106】 Polymers can be formed by the genetic incorporation of monomers using ribosomes. Polymers can also be formed by the translation of nucleic acid sequences by ribosomes. Translation may include the binding of tRNA charged with beta-amino acids to ribosomes and the formation of bonds between the beta-amino acids and pre- and / or post-monomers. Therefore, the method may include providing a nucleic acid sequence encoding a polymer and using charged tRNA and ribosomes to translate the sequence and form a polymer. 【0107】 The use or method can be carried out in cells, such as prokaryotic cells, bacterial cells, or E. coli cells. 【0108】 In a third embodiment, a nucleic acid encoding the acyl-tRNA synthetase of the first embodiment is provided. The nucleic acid may be DNA. The vector may contain the nucleic acid. 【0109】 In a fourth embodiment, cells are provided that include an acyl-tRNA synthetase according to the first embodiment, a nucleic acid according to the third embodiment, or a vector comprising the nucleic acid according to the third embodiment. 【0110】 In certain embodiments, cells contain or express acyl-tRNA synthetase of the first embodiment, and the acyl-tRNA synthetase is orthogonal to endogenous tRNA. Therefore, the acyl-tRNA synthetase does not acylate endogenous tRNA to the extent that it renders the cell unviable. In some embodiments, for example, cell viability, as measured by proliferation, decreases by less than 50%, less than 25%, less than 10%, or less than 5%, or does not decrease if acyl-tRNA synthetase is expressed. 【0111】 α,α-disubstituted amino acid-acyl-tRNA synthetase The present inventors have identified an acyl-tRNA synthetase capable of acylating tRNA using α,α-disubstituted amino acids, utilizing the methods and means disclosed herein. 【0112】 Therefore, in the fifth embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using α,α-disubstituted amino acids, A302C, A302G, A302H, or A302S for Sequence ID No. 1; and N346A, N346C, N346E, N346G, N346T, or N346V for Sequence ID No. 1 An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0113】 The definition of "specifically" acylation is provided in the section relating to the first aspect. 【0114】 In certain embodiments, the amino acid sequence includes A302G or A302H and / or N346A, N346C, or N346G. 【0115】 The amino acid sequence may contain a mutation at the position corresponding to M300 in SEQ ID NO: 1. The mutation may be M300A, M300C, M300D, M300E, M300S, or M300T. In some embodiments, the residue is wild-type (M300M). The most common mutation in screening was M300D. In certain embodiments, the mutation is M300D. 【0116】 The amino acid sequence may contain a mutation at the position corresponding to L305 in SEQ ID NO: 1. The mutation may be L305C, L305F, L305H, L305I, L305N, or L305S. More specifically, the mutation may be L305C, L305F, or L305S. In some embodiments, the residue is wild-type (L305L). The most common mutation in screening was L305C. 【0117】 The amino acid sequence may contain a mutation at the position corresponding to Y306 in SEQ ID NO: 1. The mutation may be Y306D, Y306F, Y306L, or Y306N. In some embodiments, the residue is wild-type (Y306Y). In certain embodiments, the amino acid sequence includes Y306F or Y306Y. 【0118】 The amino acid sequence may contain a mutation at the position corresponding to L309 in SEQ ID NO: 1. The mutation may be L309C, L309F, L309G, L309H, L309N, or L309V. In some embodiments, the residue is wild-type (L309L). In certain embodiments, the amino acid sequence includes L309F or L309L. 【0119】 The amino acid sequence may contain a mutation at the position corresponding to M344 in SEQ ID NO: 1. The mutation may be M344H or M344Q. In some embodiments, the residue is wild-type (M344M), and the wild-type residue was the most common in screening. 【0120】 The amino acid sequence may contain a mutation at the position corresponding to C348 in SEQ ID NO: 1. The mutation may be C348F, C348G, C348H, C348I, C348L, C348S, or C348V. In some embodiments, the residue is wild-type (C348C). In certain embodiments, the amino acid sequence contains C348G or C348C. The most common mutation in screening was C348G. 【0121】 The amino acid sequence may include a wild-type residue at the position corresponding to Y384 in SEQ ID NO: 1. This can be referred to as containing Y384Y. 【0122】 The amino acid sequence may include a wild-type residue at the position corresponding to S399 in SEQ ID NO: 1. This can be referred to as containing S399S. 【0123】 The amino acid sequence may contain a mutation at the position corresponding to V401 in SEQ ID NO: 1. In the example, the mutation is V401A, V401C, V401K, V401L, or a conserved substitution of the residue. More specifically, the mutation may be V401C or V401L. In some embodiments, the residue is wild-type (V401V). In certain embodiments, the amino acid sequence includes V401C, V401L, or V401V. 【0124】 In one embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302C, A302G, A302H, or A302S; or more specifically, A302G or A302H; and N346A, N346C, N346E, N346G, N346T, or N346V; or more specifically, N346A, N346C, N346G An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0125】 In a particular embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302G or A302H; and N346A, N346C, or N346G An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0126】 An acyl-tRNA synthetase capable of specifically acylating tRNA using α,α-disubstituted amino acids, M300D; A302G or A302H; L305 mutation and N346 mutation (for either residue) An acyl-tRNA synthetase comprising an amino acid sequence containing the corresponding residue is provided herein. 【0127】 In a particular embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302G or A302H; L305 mutation, Y306 mutation, L309 mutation, N346 mutation, and C348 mutation; and optionally V401 mutation An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0128】 In a particular embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302H; N346A or N346G; and C348G An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0129】 In a particular embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302G or A302H; L305C, L305F, or L305S; N346A, N346C, or N346G; and C348C or C348G An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0130】 In a particular embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302G or A302H; L305C, L305F, or L305S; Y306F or Y306Y; L309F or L309L; N346A, N346C, or N346G; C348C or C348G; and V401C, V401L, or V401V An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0131】 In a particular embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302G or A302H; L305C; Y306F; L309F N346A or N346G; and C348G An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0132】 In a particular embodiment, an acyl-tRNA synthetase capable of specifically acylating tRNA using an α,α-disubstituted amino acid, M300D; A302G or A302H; L305C, L305F, or L305S; Y306F or Y306Y; L309F or L309L; M344M; N346A, N346C, or N346G; C348C or C348G; Y384Y; S399S; and V401C, V401L, or V401V An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0133】 The α,α-disubstituted amino acid may also be (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S)α-Me-pIF). 【0134】 The inventors provide specific embodiments in Table 7. These embodiments are defined for Sequence ID No. 1. For example, A6_2 includes M300D, A302H, L305S, etc. [Table 8] 【0135】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations in Table 7. For example, acyl-tRNA synthetase may contain an amino acid sequence that includes residues corresponding to M300D, A302H, L305S, L309F, N346G, and C348G. Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations and wild-type residues in Table 7. For example, acyl-tRNA synthetase may contain an amino acid sequence that includes residues corresponding to M300D, A302H, L305S, Y306Y, L309F, M344M, N346G, C348G, Y384Y, S399S, and V401V. 【0136】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations labeled as A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7 (for example, for A6_2, M300D, A302H, L305S, L309F, N346G, and C348G). Acyl-tRNA synthetase may contain any of the sets of mutant and wild-type residues labeled as A6_1, A6_2, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7 (for example, for A6_2, M300D, A302H, L305S, Y306Y, L309F, M344M, N346G, C348G, Y384Y, S399S, and V401V). 【0137】 The fifth embodiment of the mutation is the sequence of pyrrolidyl tRNA synthetase (Mm PylRS) of wild-type metanosarsinina mazei, provided herein with reference to Sequence ID No. 1. To aid in alignment, a version of the Mm PylRS sequence in which "X" is marked at positions 300, 302, 305, 306, 309, 346, 348, 384, and 401 is provided as Sequence ID No. 2. 【0138】 Accordingly, in one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using α,α-disubstituted amino acids, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid sequence comprises any of the mutations or sets of mutations disclosed in connection with the fifth embodiment. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the amino acid sequence may be identical to SEQ ID NO: 1 or SEQ ID NO: 2, apart from the described mutations. 【0139】 The acyl-tRNA synthetase has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may include an amino acid sequence comprising M300D;A302G or A302H; and N346A, N346C, or N346G. Acyl-tRNA synthetase may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may include an amino acid sequence comprising M300D;A302G or A302H;L305C, L305F, or L305S;Y306F or Y306Y;L309F or L309L;M344M;N346A, N346C, or N346G;C348C or C348G;Y384Y;S399S; and V401C, V401L, V401V. The amino acid sequence may also include a mutation corresponding to any one of the mutation sets in Table 7. The amino acid sequence may include a mutation corresponding to one of the mutations in the set labeled A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7. The amino acid sequence may also include a mutation corresponding to one of the mutations in the set labeled A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7 and a residue corresponding to the wild-type residue. 【0140】 One skilled in the art can also apply the identified mutations to other PylRS backbones that contain a backbone not disclosed herein. This is despite the fact that other PylRSs may have low sequence identity to Mm PylRS. To apply the mutations described with reference to Mm PylRS to other synthetases, the sequences should be aligned to identify the corresponding residues. In particular, the sequences representing the catalytic sites of the synthetases can be aligned. Thus, the region of SEQ ID NO: 1 that extends from residue 296 to residue 428 can be aligned with the sequence of another PylRS to create the corresponding mutations. Further information regarding the transfer of mutations from one PylRS backbone to another is provided in WO 2013 / 171485 A1 (incorporated herein by reference). An exemplary alignment is provided in Figure 35. 【0141】 Thus, in one embodiment, there is provided an aminoacyl-tRNA synthetase that can specifically aminoacylate tRNA with an α,α-disubstituted amino acid, comprising an amino acid sequence comprising any of the mutations and optionally wild-type residues disclosed in connection with the fifth aspect, or any set of the mutations and optionally wild-type residues disclosed in connection with the fifth aspect, wherein the aminoacyl-tRNA synthetase backbone is PylRS. 【0142】 The mutations can be applied to PylRS from Nitospora archias (Nitra). This sequence is provided herein as SEQ ID NO: 3. To assist with the alignment, a version of the Nitra PylRS sequence is provided herein (SEQ ID NO: 4), with "X" marked at positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, 401 in the Mm PylRS sequence. These are the positions at which the respective mutations of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0143】 The mutation can be applied to PylRS derived from bacteria of the order Clostridiales (Clos). This sequence is provided as Sequence ID No. 5. To aid in alignment, a version of the Clos PylRS sequence is provided herein (Sequence ID No. 6), where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each mutation of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0144】 The mutation can be applied to PylRS derived from the Metanomethylophilus species 1R26 (1R26). This sequence is provided herein (SEQ ID NO: 7). To aid in alignment, a version of the 1R26 PylRS sequence is provided herein (SEQ ID NO: 8), where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each mutation of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0145】 The mutation can be applied to PylRS derived from Metanomassillicoccus luminuensis 1 (Lum1). This sequence is provided herein (SEQ ID NO: 9). To aid in alignment, a version of the Lum1 PylRS sequence is provided herein (SEQ ID NO: 10), where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each mutation of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0146】 Other main chains to which the mutations of the present invention can be applied include Mb PylRS (SEQ ID NO: 11), Lum1 PylRS (SEQ ID NO: 12), Tron PylRS (SEQ ID NO: 13), Gemm PylRS (SEQ ID NO: 14), PG48 PylRS (SEQ ID NO: 15), I2 PylRS (SEQ ID NO: 16), D121 PylRS (SEQ ID NO: 17), and D416 PylRS (SEQ ID NO: 18). 【0147】 Accordingly, in one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using α,α-disubstituted amino acids, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18, wherein the amino acid sequence comprises either the mutations disclosed in connection with the fifth embodiment and optionally wild-type residues, or a set of mutations and optionally wild-type residues. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18. In one embodiment, the amino acid sequence may be identical to any one of SEQ ID NOs: 1 to 18, apart from the described mutations. 【0148】 In certain embodiments, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using α,α-disubstituted amino acids, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 10, wherein the amino acid sequence comprises either a mutation and optionally a wild-type residue, or a set of a mutation and optionally a wild-type residue, as disclosed in connection with the fifth embodiment. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 10. In some embodiments, the amino acid sequence may be identical to any one of SEQ ID NOs: 1 to 10, apart from the described mutation. 【0149】 Therefore, in the example, the acyl-tRNA synthetase may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to any one amino acid sequence of sequence numbers 1-18, and may include an amino acid sequence containing any one set of mutations in Table 7 or any one set of mutations labeled as A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7. The amino acid sequence may include any one set of mutations in Table 7 or any one set of mutations labeled as A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7, and residues corresponding to wild-type residues. 【0150】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using α,α-disubstituted amino acids, and comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18, wherein the amino acid sequence comprises M300D for SEQ ID NO: 1; A302C, A302G, A302H, or A302S for SEQ ID NO: 1; or more specifically, A302G or A302H; and N346A, N346C, N346E, N346G, N346T, or N346V for SEQ ID NO: 1; or more specifically, N346A, N346C, or N346G. In one embodiment, the amino acid sequence includes M300D;A302G or A302H;L305C, L305F, or L305S;Y306F, or Y306Y;L309F, or L309L;M344M;N346A, N346C, or N346G;C348C or C348G;Y384Y;S399S; and V401C, V401L, or V401V. 【0151】 The synthase of the fifth embodiment may include sequence changes relative to the wild-type sequence in addition to the mutations described in detail herein. Specifically, the synthase may include sequence changes at sites that do not significantly impair the function or operation of the synthase described herein. To verify that the function of the synthase is not inactivated and has not been significantly altered, its function can be tested by manipulating the synthase as described in the Examples section, etc. Therefore, provided that the synthase retains its function and can be tested as described herein, sequence changes relative to the wild-type reference sequence can be produced in the synthase. 【0152】 As will be discussed in relation to the first aspect, Figure 35 provides sequence alignments. The synthase may be at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any one of sequence numbers 1-18, and changes are found only in residues not marked with i) "*"; ii) "*" or ":", or iii) "*", ":", or ".", apart from the mutations specifically described herein. 【0153】 For example, conservative substitutions can be made according to the table disclosed in connection with the first embodiment. 【0154】 The acyl-tRNA synthetase of the fifth embodiment can be isolated or purified. The acyl-tRNA synthetase of the fifth embodiment may be a non-natural acyl-tRNA synthetase. 【0155】 A sixth aspect provides the use of acyl-tRNA synthetase in a method for producing polymers containing α,α-disubstituted amino acids. 【0156】 In one embodiment, a method for producing a polymer containing an α,α-disubstituted amino acid, i) Use of acyl-tRNA synthetase that acylates tRNA using α,α-disubstituted amino acids, wherein the acyl-tRNA synthetase includes a mutation that enables acylation of tRNA using α,α-disubstituted amino acids, and ii) Incorporation of α,α-disubstituted amino acids into polymers A method is provided that includes this. 【0157】 In one embodiment, a fifth aspect of the use of acyl-tRNA synthetase in a method for producing a polymer containing an α,α-disubstituted amino acid is provided. 【0158】 In one embodiment, a method for producing a polymer containing an α,α-disubstituted amino acid, i) Use of a fifth embodiment of acyl-tRNA synthetase for acylation of tRNA using α,α-disubstituted amino acids, and ii) Incorporation of α,α-disubstituted amino acids into polymers A method is provided that includes this. 【0159】 The α,α-disubstituted amino acid may also be (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S)α-Me-pIF). 【0160】 The polymer may contain one or more standard amino acids or one or more naturally occurring amino acids. The polymer may also contain one or more unnatural amino acids and / or hydroxy acids. The unnatural amino acids may be α-amino acids, and / or the hydroxy acids may be α-hydroxy acids. 【0161】 A polymer can be formed by genetic incorporation of monomers using ribosomes. A polymer can be formed by translation of a nucleic acid sequence by ribosomes. Translation may include the binding of a tRNA charged with an α,α-disubstituted amino acid to a ribosome, and the formation of a bond between the α,α-disubstituted amino acid and the preceding and / or following monomers. Thus, the method may include providing a nucleic acid sequence encoding a polymer, and using charged tRNA and ribosomes to translate said sequence to form a polymer. 【0162】 Thus, there is provided a method for producing a polymer comprising genetic incorporation of at least one α,α-disubstituted amino acid, which may be (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S)α-Me-pIF). 【0163】 The use or method can be carried out in a cell. For example, a prokaryotic cell, a bacterial cell, or an Escherichia coli cell. 【0164】 In a seventh aspect, there is provided a nucleic acid encoding the acyl-tRNA synthetase of the fifth aspect. The nucleic acid may be DNA. The vector may contain the nucleic acid. 【0165】 In an eighth aspect, there is provided a cell comprising the acyl-tRNA synthetase of the fifth aspect, the nucleic acid of the seventh aspect, or a vector containing the nucleic acid of the seventh aspect. 【0166】 In certain embodiments, the cell contains or expresses the acyl-tRNA synthetase of the fifth aspect, and the acyl-tRNA synthetase is orthogonal to the endogenous tRNA. Thus, the acyl-tRNA synthetase does not acylate the endogenous tRNA to an extent that renders the cell non-viable. In certain embodiments, for example, the fitness of the cell, measured by growth, is reduced by less than 50%, less than 25%, less than 10%, less than 5%, or not reduced when the acyl-tRNA synthetase is expressed. 【0167】 Beta-hydroxy acid-acyl tRNA synthetase The present inventors have identified acyl-tRNA synthetases capable of acylating tRNA with beta-hydroxy acids using the methods and means disclosed herein. 【0168】 Therefore, in the ninth aspect, an acyl-tRNA synthetase capable of specifically acyling tRNA using a beta-hydroxy acid, M300A, M300D, M300M, M300N, or M300S for Sequence ID No. 1; and A302D, A302G, A302H, or A302N for Sequence ID No. 1 An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0169】 The definition of "specifically" acylation is provided in the section relating to the first aspect. 【0170】 In particular, acyl-tRNA synthetase may contain residues corresponding to M300N or M300S. M300N was the most common in screening. Acyl-tRNA synthetase may also contain residues corresponding to A302H. A302H was the most common in screening. Acyl-tRNA synthetase may contain residues corresponding to both M300N and A302H. 【0171】 The amino acid sequence may contain a mutation at the position corresponding to N346 in SEQ ID NO: 1. The mutation may be N346A, N346C, N346E, N346G, N346S, or N346V. In some embodiments, the residue is wild-type (N346N). In certain embodiments, the mutation is N346A, N346C, or N346G. 【0172】 The amino acid sequence may contain a mutation at the position corresponding to L305 in SEQ ID NO: 1. The mutation may be L305C, L305N, L305S, or L305V. In some embodiments, the residue is wild-type (L305L). In embodiments, the amino acid sequence may contain L305L or L305V. The most common residue in screening was wild-type. 【0173】 The amino acid sequence may contain a mutation at the position corresponding to Y306 in SEQ ID NO: 1. The mutation may be Y306D, Y306F, Y306I, Y306N, Y306R, or Y306S. In some embodiments, the residue is wild-type (Y306Y). While the wild-type residue was the most common in screening, particularly high selectivity was achieved by variants containing Y306N. 【0174】 The amino acid sequence may contain a mutation at the position corresponding to L309 in SEQ ID NO: 1. The mutation may be L309D, L309H, L309I, L309R, or L309S. More specifically, the mutation may be L309I or L309S. In some embodiments, the residue is wild-type (L309L). The most common residue in screening was wild-type, but particularly high selectivity was achieved by variants containing L309I. 【0175】 The amino acid sequence may contain a mutation at the position corresponding to M344 in SEQ ID NO: 1. The mutation may be M344E or M344Q. In some embodiments, the residue is wild-type (M344M), and the wild-type residue was the most common in screening. 【0176】 The amino acid sequence may contain a mutation at the position corresponding to C348 in SEQ ID NO: 1. The mutation may be C348F, C348I, C348L, C348T, or C348V. More specifically, the mutation may be C348I, C348L, C348T, or C348V. In some embodiments, the residue is wild-type (C348C). This residue was mutated in most variants during screening. 【0177】 The amino acid sequence may include a wild-type residue at the position corresponding to Y384 in SEQ ID NO: 1. This can be referred to as containing Y384Y. 【0178】 The amino acid sequence may include a wild-type residue at the position corresponding to S399 in SEQ ID NO: 1. This can be referred to as containing S399S. 【0179】 The amino acid sequence may contain a mutation at the position corresponding to V401 in SEQ ID NO: 1. In the example, the mutation is V401A, V401C, V401K, V401L, V401S, or V401T. More specifically, the mutation may be V401C, V401K, or V401L. In some embodiments, the residue is wild-type (V401V). 【0180】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using a beta-hydroxy acid, and comprises an amino acid sequence including residues corresponding to M300N and A300H, and optionally mutations at positions N346 and / or C348. 【0181】 In one embodiment, an acyl-tRNA synthetase capable of specifically acyling tRNA using a beta-hydroxy acid, M300A, M300D, M300M, M300N, or M300S; more specifically, M300N or M300S; A302D, A302G, A302H, or A302N; more specifically, A302H; and N346A, N346C, N346E, N346G, N346S, or N346V; more specifically, N346A, N346C, or N346G An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0182】 In one embodiment, an acyl-tRNA synthetase capable of specifically acyling tRNA using a beta-hydroxy acid, M300N or M300S; more specifically, M300N; A302H; and N346A, N346C, or N346G; more specifically, N346G An acyl-tRNA synthetase is provided, which includes an amino acid sequence containing the corresponding residue. 【0183】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using a beta-hydroxy acid, and comprises an amino acid sequence that includes residues corresponding to M300N, A302H, and N346G, and may further include a C348 mutation and / or a V401 mutation. 【0184】 In one embodiment, an acyl-tRNA synthetase capable of specifically acyling tRNA using a beta-hydroxy acid, M300N or M300S; A302H; The residue corresponding to N346A, N346C, or N346G; and C348 mutation and / or V401 mutation An acyl-tRNA synthetase containing an amino acid sequence including is provided. 【0185】 In one embodiment, an acyl-tRNA synthetase capable of specifically acyling tRNA using a beta-hydroxy acid, M300N or M300S; A302H; The residue corresponding to N346A, N346C, or N346G; and A C348 mutation that is arbitrarily C348I, C348L, C348T, or C348V. An acyl-tRNA synthetase containing an amino acid sequence including is provided. 【0186】 In one embodiment, an acyl-tRNA synthetase capable of specifically acyling tRNA using a beta-hydroxy acid, M300N; A302H; and The residue corresponding to N346G; Furthermore, the C348 mutation and / or the V401 mutation It includes an amino acid sequence containing; optionally, The C348 mutation is C348I, C348L, C348T, or C348V; and / or Acyl-tRNA synthetases are provided in which the V401 mutation is V401C, V401K, V401L, or V401V. 【0187】 In one embodiment, an acyl-tRNA synthetase capable of specifically acyling tRNA using a beta-hydroxy acid, M300N or M300S; A302H; N346A, N346C, or N346G; and C348I, C348L, C348T, or C348V; and An amino acid sequence and acyl-tRNA synthetase are provided, which optionally include residues corresponding to V401C, V401K, V401L, or V401V. 【0188】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using a beta-hydroxy acid, and comprises an amino acid sequence including residues corresponding to M300N;A302H;N346A, N346C, or N346G; and C348I, C348L, or C348V. 【0189】 Beta-hydroxy acids include (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S)β 3 mClF) may also be used. 【0190】 The inventors provide specific embodiments in Table 8. These embodiments are defined for Sequence ID No. 1. For example, A7_2 includes M300N, A302H, L305L, etc. [Table 9] 【0191】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations in Table 8. For example, acyl-tRNA synthetase may contain an amino acid sequence that includes residues corresponding to M300N, A302H, Y306N, N346G, C348L, and V401L. Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations and wild-type residues in Table 8. For example, acyl-tRNA synthetase may contain an amino acid sequence that includes residues corresponding to M300N, A302H, L305L, Y306N, L309L, M344M, N346G, C348L, Y384Y, S399S, and V401L. 【0192】 Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations labeled A7_1, A7_2, A7_3, A7_4, A7_5, or A7_7 in Table 8. Acyl-tRNA synthetase may contain an amino acid sequence that includes any of the sets of mutations labeled A7_1, A7_2, A7_3, A7_4, A7_5, or A7_7 in Table 8 and wild-type residues. 【0193】 The mutation of the ninth aspect is the sequence of pyrrolidyl tRNA synthetase (Mm PylRS) of wild-type metanosarcina mazei, provided herein with reference to Sequence ID No. 1. To aid in alignment, a version of the sequence of Mm PylRS is provided as Sequence ID No. 2, in which "X" is marked at positions 300, 302, 305, 306, 309, 346, 348, 384, and 401. 【0194】 Accordingly, in one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using a beta-hydroxy acid, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid sequence comprises any of the mutations or sets of mutations disclosed in connection with the ninth embodiment. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the amino acid sequence may be identical to SEQ ID NO: 1 or SEQ ID NO: 2, apart from the described mutations. 【0195】 Acyl-tRNA synthetase has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may include amino acid sequences comprising M300A, M300D, M300M, M300N, or M300S; more specifically, M300N, or M300S; A302D, A302G, A302H, or A302N; more specifically, A302H; and N346A, N346C, N346E, N346G, N346S, or N346V; more specifically, N346A, N346C, or N346G. Acyl-tRNA synthetase has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and may include an amino acid sequence containing M300N or M300S; more specifically, M300N;A302H; and N346A, N346C, or N346G; more specifically, N346G. The amino acid sequence may include a mutation corresponding to any one of the mutations in the set of mutations in Table 8. The amino acid sequence may include a mutation corresponding to any one of the mutations labeled A7_1, A7_2, A7_3, A7_4, A7_5, and A7_7 in Table 8. The amino acid sequence may include a mutation corresponding to any one of the mutations in A7_1, A7_2, A7_3, A7_4, A7_5, and A7_7 in Table 8 and a residue corresponding to the wild-type residue. 【0196】 Those skilled in the art can also apply the identified mutations to other PylRS backchains, including those not disclosed herein. This is not limited to the fact that other PylRS may have low sequence identity with respect to Mm PylRS. To apply the mutations described with reference to Mm PylRS to other synthases, the sequences should be aligned to identify the corresponding residues. In particular, sequences representing the catalytic site of the synthase can be aligned. For example, the region of Sequence ID No. 1, extending from residue 296 to residue 428, can be aligned with the sequence of another PylRS to produce the corresponding mutation. Further information regarding the transfer of mutations from one PylRS backchain to the other is provided in International Publication No. 2013 / 171485A1 (incorporated herein by reference). An example alignment is provided in Figure 35. 【0197】 Therefore, in one embodiment, an acyl-tRNA synthase is provided that can specifically acylate tRNA using a beta-hydroxy acid, comprising an amino acid sequence including any set of mutations disclosed in connection with the ninth embodiment and optionally a wild-type residue, or any set of mutations disclosed in connection with the ninth embodiment and optionally a wild-type residue, wherein the acyl-tRNA synthase backbone is PylRS. 【0198】 Mutations can be applied to PylRS derived from Nitrosophaeria archaeon (Nitra). This sequence is provided herein as Sequence ID No. 3. To aid in alignment, a version of the Nitra PylRS sequence is provided herein (Sequence ID No. 4), where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each of the mutations of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0199】 The mutations can be applied to PylRS derived from Clostridial bacteria (Clos). This sequence is provided as Sequence ID No. 5. To aid in alignment, a version of the Clos PylRS sequence is provided herein (Sequence ID No. 6), where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each of the mutations of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0200】 The mutation can be applied to PylRS derived from the Metanomethylophilus species 1R26 (1R26). This sequence is provided herein (SEQ ID NO: 7). To aid in alignment, a version of the 1R26 PylRS sequence is provided herein (SEQ ID NO: 8), where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each mutation of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0201】 The mutation can be applied to PylRS derived from Metanomassillicoccus luminuensis 1 (Lum1). This sequence is provided herein (SEQ ID NO: 9). To aid in alignment, a version of the Lum1 PylRS sequence is provided herein (SEQ ID NO: 10), where "X" marks the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the Mm PylRS sequence. These are the positions to which each mutation of the present invention can be applied. Further corresponding positions can be identified in the same manner. 【0202】 Other main chains to which the mutations of the present invention can be applied include Mb PylRS (SEQ ID NO: 11), Lum1 PylRS (SEQ ID NO: 12), Tron PylRS (SEQ ID NO: 13), Gemm PylRS (SEQ ID NO: 14), PG48 PylRS (SEQ ID NO: 15), I2 PylRS (SEQ ID NO: 16), D121 PylRS (SEQ ID NO: 17), and D416 PylRS (SEQ ID NO: 18). 【0203】 Accordingly, in one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using a beta-hydroxy acid, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18, wherein the amino acid sequence comprises either the mutations and optionally wild-type residues disclosed in connection with the ninth embodiment, or a set of mutations and optionally wild-type residues. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18. In one embodiment, the amino acid sequence may be identical to any one of SEQ ID NOs: 1 to 18, apart from the described mutations. 【0204】 In certain embodiments, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using a beta-hydroxy acid, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 10, wherein the amino acid sequence comprises either the mutations disclosed in connection with the ninth embodiment and optionally wild-type residues, or a set of mutations and optionally wild-type residues. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 10. In some embodiments, the amino acid sequence may be identical to any one of SEQ ID NOs: 1 to 10, apart from the described mutations. 【0205】 Therefore, in the example, the acyl-tRNA synthetase may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to any one of the amino acid sequences of SEQ ID NOs: 1-18, and may include an amino acid sequence containing any one of the sets of mutations in Table 8 or any one of the sets of mutations labeled A7_1, A7_2, A7_3, A7_4, A7_5, and A7_7 in Table 8. The amino acid sequence may include any one of the sets in Table 8 or any one of the sets of mutations labeled A7_1, A7_2, A7_3, A7_4, A7_5, and A7_7 in Table 8, and residues corresponding to wild-type residues. 【0206】 In one embodiment, an acyl-tRNA synthetase is provided that can specifically acylate tRNA using a beta-hydroxy acid, comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18, wherein the amino acid sequence comprises mutations corresponding to M300A, M300D, M300M, M300N, or M300S; more specifically, M300N, or M300S; A302D, A302G, A302H, or A302N; more specifically, A302H; and N346A, N346C, N346E, N346G, N346S, or N346V; more specifically, N346A, N346C, or N346G. The amino acid sequence may include mutations corresponding to M300N or M300S; more specifically, M300N;A302H; and N346A, N346C, or N346G; more specifically, N346G. The amino acid sequence may also include M300N, A302H, and optionally N346 and / or C348 mutations. 【0207】 The synthase of the ninth embodiment may include sequence changes relative to the wild-type sequence in addition to the mutations described in detail herein. Specifically, the synthase may include sequence changes at sites that do not significantly impair the function or operation of the synthase described herein. To verify that the function of the synthase is not inactivated and has not been significantly altered, its function can be tested by manipulating the synthase as described in the Examples section, etc. Therefore, provided that the synthase retains its function and can be tested as described herein, sequence changes relative to the wild-type reference sequence can be produced in the synthase. 【0208】 As will be discussed in relation to the first aspect, Figure 35 provides sequence alignments. The synthase may be at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any one of sequence numbers 1-18, and changes are found only in residues not marked with i) "*"; ii) "*" or ":", or iii) "*", ":", or ".", apart from the mutations specifically described herein. 【0209】 For example, conservative substitutions can be made according to the table disclosed in connection with the first embodiment. 【0210】 The acyl-tRNA synthetase of the ninth embodiment can be isolated or purified. The acyl-tRNA synthetase of the ninth embodiment may be a non-natural acyl-tRNA synthetase. 【0211】 In a tenth aspect, the use of acyl-tRNA synthetase in a method for producing a polymer containing a beta-hydroxy acid is provided. 【0212】 In one embodiment, a method for producing a polymer containing a beta-hydroxy acid, i) Use of acyl-tRNA synthetase that acylates tRNA using a beta-hydroxy acid, wherein the acyl-tRNA synthetase includes a mutation that enables acylation of tRNA using a beta-hydroxy acid, and ii) Incorporation of beta-hydroxy acids into polymers A method is provided that includes this. 【0213】 In one embodiment, the use of acyl-tRNA synthetase according to a ninth aspect of a method for producing a polymer containing a beta-hydroxy acid is provided. 【0214】 In one embodiment, a method for producing a polymer containing a beta-hydroxy acid, i) Use of acyl-tRNA synthetase of the ninth aspect for acylation of tRNA using a beta-hydroxy acid, and ii) Incorporation of beta-hydroxy acids into polymers A method is provided that includes this. 【0215】 Beta-hydroxy acids include (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S)β 3 mClF) may also be used. 【0216】 The polymer may contain one or more standard amino acids or one or more naturally occurring amino acids. The polymer may also contain one or more unnatural amino acids and / or additional hydroxy acids. The unnatural amino acids may be α-amino acids, and / or additional hydroxy acids may be α-hydroxy acids. 【0217】 Polymers can be formed by the genetic incorporation of monomers using ribosomes. Polymers can also be formed by the translation of nucleic acid sequences by ribosomes. Translation may include the binding of tRNA charged with a beta-hydroxy acid to ribosomes and the formation of bonds between the beta-hydroxy acid and the preceding and / or succeeding monomers. Therefore, the method may include providing a nucleic acid sequence encoding a polymer and using charged tRNA and ribosomes to translate the sequence and form a polymer. 【0218】 Therefore, (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S)β 3 A method is provided for producing a polymer that includes the genetic incorporation of at least one beta-hydroxy acid, which may be mClF. 【0219】 The use or method can be carried out in cells, such as prokaryotic cells, bacterial cells, or E. coli cells. 【0220】 In the eleventh aspect, a nucleic acid encoding the acyl-tRNA synthetase of the ninth aspect is provided. 【0221】 The nucleic acid may be DNA. The vector may contain nucleic acid. 【0222】 In the twelfth aspect, a cell is provided that includes an acyl-tRNA synthetase according to the ninth aspect, a nucleic acid according to the eleventh aspect, or a vector comprising the nucleic acid according to the eleventh aspect. 【0223】 In certain embodiments, cells contain or express acyl-tRNA synthetase of the ninth embodiment, and the acyl-tRNA synthetase is orthogonal to endogenous tRNA. Therefore, the acyl-tRNA synthetase does not acylate endogenous tRNA to the extent that it renders the cell unviable. In some embodiments, for example, cell viability, as measured by proliferation, decreases by less than 50%, less than 25%, less than 10%, or less than 5%, or does not decrease if acyl-tRNA synthetase is expressed. 【0224】 Sequence comparisons can be performed using readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences. 【0225】 Those skilled in the art will understand how to calculate the percentage of identity between two nucleic acid sequences. To calculate the percentage of identity between two nucleic acid sequences or two amino acid sequences, it is necessary to first prepare an alignment of the two sequences and then calculate the value of sequence identity. The percentage of identity between two sequences may take different values ​​depending on (i) the method used to align the sequences, e.g., the Needleman-Wunsch algorithm (e.g., applied by Needle(EMBOSS) or Stretcher(EMBOSS)), the Smith-Waterman algorithm (e.g., applied by Water(EMBOSS)), or the LALIGN application (e.g., applied by Matcher(EMBOSS)); and (ii) the parameters used by the alignment method, e.g., partial-to-whole alignment, the matrix used, and the parameters applied to the gaps. In certain embodiments, sequence identity as disclosed herein can be calculated based on the overall alignment of relevant features, for example, a comparison of the full length of the sequence encoding the PylRS catalytic site, the full length of the sequence encoding the PylRS C-terminal domain, or the full length of the sequence encoding PylRS. In particular, proteins defined herein by reference to the degree of identity with respect to the sequence number may have that degree of identity in comparison to the full length of the sequence number. 【0226】 Once an alignment is created, there are many different methods for calculating the identity percentage between two sequences. For example, the number of identities can be divided by (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (iv) the number of equivalent positions excluding protrusions. Furthermore, it is understood that the identity percentage is strongly length-dependent. Therefore, the shorter the pair of sequences, the higher the sequence identity that can be expected to occur by chance. 【0227】 Next, the percentage of identity between the two nucleic acid sequences is calculated (N / T). * Such alignments can be calculated as 100 (wherein N is the number of positions in which the sequences share identical residues, and T is the total number of compared positions, including gaps but excluding protrusions). 【0228】 Sequence alignment may be pairwise sequence alignment. Suitable services include Needle(EMBOSS), Stretcher(EMBOSS), Water(EMBOSS), Matcher(EMBOSS), LALIGN, or GeneWise. In one example, the identity between two amino acid sequences can be calculated using the Needle(EMBOSS) service with default parameters set to, for example, matrix (BLOSUM62), gap open (10), gap elongation (0.5), end gap penalty (false), end gap open (10), and end gap elongation (0.5). In another example, the identity between two amino acid sequences can be calculated using the Matcher(EMBOSS) service with default parameters set to, for example, matrix (BLOSUM62), gap open (14), gap elongation (4), and alternative match (1). In one example, the identity between two nucleic acid sequences can be calculated using the Needle (EMBOSS) service with default parameters set to, for example, matrix (DNAfull), gap open (10), gap extension (0.5), end gap penalty (false), end gap open (10), and end gap extension (0.5). In another example, the identity between two nucleic acid sequences can be calculated using the Matcher (EMBOSS) service with default parameters set to, for example, matrix (DNAfull), gap open (16), gap extension (4), and alternative match (1). 【0229】 All of the features described herein (including the appended claims, abstract and drawings), and / or all of the steps of any method or process so disclosed herein, may be combined with any of the above embodiments in any combination, except for any combination in which at least part of such features and / or steps are mutually exclusive. 【0230】 For a better understanding of the present invention and to illustrate how its embodiments can be carried out, examples are referenced herein that are not intended to limit the invention in any sense. [Examples] 【0231】 Summary of Examples The genetic code of living cells has been reprogrammed to enable site-specific incorporation of hundreds of non-standard amino acids (ncAAs) into proteins, as well as the encoded synthesis of non-standard polymers, macrocyclic peptides, and depsipeptides. Current methods for modifying orthogonal (O)-aminoacyl-tRNA synthetases to acylate novel monomers rely on translational readouts and therefore require the monomer to be a ribosomal substrate. O-synthetases cannot evolve to acylate O-tRNA using non-standard monomers (ncMs) that are weak ribosomal substrates (and ribosomes cannot evolve to polymerize ncMs that cannot acylate on O-tRNA); this codependency creates an evolutionary deadlock that limits the scope of translation in living cells to alpha-L-amino acids and closely related hydroxy acids. Here, we break this deadlock by developing direct selection of O-synthetases that acylate congeneral O-tRNAs using ncMs, regardless of whether the ncMs are ribosomal substrates. The inventors have developed a split tmRNA consisting of a non-covalent assembly between the 5' half of an O-tRNA, the 3' half of an O-tRNA, and an mRNA encoding a congenerase; this couples the synthase genotype with split tmRNA acylation in cells. The inventors have also developed a method for specifically isolating and enriching acylated split tmRNA. The inventors combine these advantages in a tRNA display to enable direct, rapid, and scalable selection of O-synthetases that acylate their congenerase O-tRNA; this method uses 1 / 50th of the ncM of translation-based selection, and the inventors parallelize the tRNA display to efficiently select synthetases for eight ncAAs. Using the tRNA display, the inventors directly select O-synthetases that acylate congenerase O-tRNA using β-amino acids. Based on this advance, the inventors demonstrate the genetically encoded, site-specific cellular integration of β-amino acids into proteins, thereby expanding the chemical range of the E. coli gene code. 【0232】 Introduction Here, the inventors have developed a derivative of tREX that isolates specific acylated tRNAs from cells and labels them with dNTP analogs by primer extension; this enables fluorescence imaging (fluoro-tREX) or capture (bio-tREX) of acylated tRNAs. The inventors have also developed a cyclically substituted metanosarcina mazeipyrrolidine tRNA by joining the 5' and 3' ends via an intervening sequence and by creating new 5' and 3' ends with an anticodon. CUA (MmtRNA Pyl CUA , hereafter tRNA Pyl CUA (The inventors have identified a gene called cissplit(s)tRNA) Pyl It produces a gene. The intercalating sequence is processed from the transcript and acylated by Mmpyrrolidyl-tRNA synthetase (hereafter referred to as PylRS), a homologous aminoacyl-tRNA synthetase of the parent tRNA, to produce a split stRNA consisting of a 5' half and a 3' half. 【0233】 The inventors have identified the PylRS gene as cis-stRNA Pyl By fusing it with the gene, cis-stmRNA Pyl By creating this, the genotype responsible for acylation is associated with acylation itself. The inventors demonstrate that they can selectively enrich (by more than 300 times) stmRNA encoding an active PylRS variant with respect to a variant of attenuated activity using bio-mREX (a modification of bio-tREX applied to stmRNA). The inventors increase the dynamic range of PylRS enrichment by maximizing stmRNA transcription while minimizing translation of the PylRS mRNA encoded within the stmRNA. 【0234】 The inventors generate stmRNA libraries containing combinations of mutations in the PylRS gene and rapidly and scalably define active and selective PylRS variants by a process they call tRNA display, using parallel bio-mREX-based enrichment, reverse transcription, and NGS, both in the presence and absence of ncAA. The inventors have used tRNA display to identify carboxylic acids and weak ribosomal substrates that are generally considered to be unable to function in translation (Katoh, T. & Suga, H. In vitro genetic code reprogramming for the expansion of usable noncanonical amino acids. Annual Review of Biochemistry 91, 221-243 (2022), Melo Czekster, C., Robertson, WE, Walker, AS, Soll, D. & Schepartz, A. In Vivo Biosynthesis of a beta-Amino Acid-Containing Protein. J Am Chem Soc 138, 5194-5197, doi:10.1021 / jacs.6b01023 (2016), Tan, Z., Forster, AC, Blacklow, SC & Cornish, VW Amino Acid Backbone Specificity of the Escherichia coli Translation Machinery. Journal of the American Chemical Society 126). 12752-12753 (2004), Pavlov, MY et al. Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proceedings of the National Academy of Sciences 106, 50-54 (2009), Katoh, T., Tajima, K. & Suga, H.Consecutive Elongation of D-Amino Acids in Translation. Cell Chem Biol 24, 46-54, doi:10.1016 / j.chembiol.2016.11.012 (2017), Katoh, T. & Suga, H. Ribosomal Incorporation of Consecutive beta-Amino Acids. J Am Chem Soc 140, 12159-12167, doi:10.1021 / jacs.8b07247 (2018), Dedkova, LM et al. beta-Puromycin selection of modified ribosomes for in vitro incorporation of beta-amino acids. Biochemistry 51, 401-415, doi:10.1021 / bi2016124 (2012)) We select an orthogonal aminoacyl-tRNA synthetase (aaRS) of the ncM class that specifically acylates congeneral orthogonal tRNAs using β-amino acids. Furthermore, using β-amino acid pairs, we demonstrate site-specific co-translational incorporation of β-amino acids into recombinant proteins. [Example 1] 【0235】 High-sensitivity detection and efficient isolation of acylated tRNAs The inventors have demonstrated that the aminoacylation state of specific tRNAs isolated from cells can be determined by selective, primer-mediated extension of non-oxidized tRNAs using periodate oxidation followed by a nucleotide derivative supporting a fluorophore (Cy5) or biotin. These experiments demonstrate that the inventors' method, which they have named fluorescent tREX (fluoro-tREX), can track tRNA acylation by generating a fluorescent signal. 【0236】 Next, the inventors created biotin-tREX (bio-tREX) by replacing Cy5-dCTP with biotinylated dCTP (bio-dCTP) in the fluoro-tREX extension step. The inventors selectively captured the biotinylated extension product (obtained from a tRNA molecule protected from periodate oxidation by its aminoacylation) on magnetic streptavidin beads, washed the beads, and eluted the bound tRNA extension product. tRNA in the eluate Pyl CUA The presence of the elongation product depended on the presence of PylRS and BocK(1) in the cell, and the addition of bio-dCTP to the elongation reaction. We conclude that biotin-tREX enabled the selective capture of tRNA elongation products from aminoacylated tRNA. 【0237】 Further information is provided in the priority documents (European Patent Application No. 2306393.6, filed on 28 April 2023, and European Patent Application No. 2400299.0, filed on 9 January 2024, which are incorporated herein by reference). [Example 2] 【0238】 Split tRNA: In vivo assembly, maturation, and acylation Next, the present inventors used the anticodon to form tRNA Pyl CUA By splitting the gene, split tRNA is produced. Pyl (stRNA Pyl The inventors sought to determine whether it was possible to create such a system. They hypothesized the creation of a system in which the 5' and 3' halves of a split tRNA are transcribed, assembled in vivo by non-covalent interactions (including base pairing), matured by cellular tRNA processing mechanisms, recognized by PylRS, and efficiently acylated. 【0239】 The inventors initially designed a series of constructs that split a tRNA gene into two halves using an anticodon. Some of these constructs resulted in robust aminoacylation, which depended on the presence of both tRNA halves, PylRS, and BocK. To the inventors' knowledge, this data represents the first example of split tRNAs that assemble, mature, and acylate in cells. Further information is provided in the priority literature. 【0240】 Next, the inventors ensure (1) equimolar stoichiometry of half of both tRNAs, and (2) spatial proximity, thereby facilitating the assembly of half of both tRNAs into an acylated tRNA body. Pyl We focused on designing the optimal expression system for tRNA. To achieve this, we used tRNA Pyl CUA We demonstrated that the split tRNA could be split and expressed cis-tRNA from a single transcript, and that the split tRNA functioned as an efficient substrate for acylation by PylRS. Further information is provided in the priority literature. [Example 3] 【0241】 Covalent linkage between acylation phenotype and genotype Next, the inventors used the PylRS coding sequence and linker sequence as cis-stRNA. Pyl The 3' half of the expression cassette is fused to the 5' end, and the stmRNA is attached. PylA cassette was created. After conducting experiments, the inventors concluded that their stmRNA construct is functional. This construct is transcribed and processed to produce a split tRNA in which the 3' half is fused to the synthase mRNA. The synthase gene is transcribed, and the resulting protein catalyzes the acylation of the 3' end of the 3' half of the stmRNA. In fluoro-mREX, acylation is converted into a fluorescent "phenotype" by the addition of a fluorescent nucleotide to the RNA chain containing the synthase mRNA. This creates a physical link between the synthase mRNA sequence, its genotype, and the fluorescent "phenotype," which are produced as a result of synthase activity. Further information is provided in the priority literature. [Example 4] 【0242】 Efficient acylation-specific enrichment of the PylRS genotype Next, the inventors aimed to selectively isolate acylated stmRNAs from non-acylated stmRNAs and directly obtain the cDNA of the PylRS gene responsible for acylation by reverse transcribing the isolated PylRS mRNA within the stmRNAs. The inventors then assumed that the obtained cDNA could be used as a direct readout in quantitative (q)PCR to be converted into DNA for further selection and directed evolution, or sequenced. To selectively isolate acylated stmRNAs from non-acylated stmRNAs, the inventors created bio-mREX, which is an adaptation of bio-tREX to stmRNAs. Experimentally testing this method, the inventors concluded that bio-mREX enables efficient and selective recovery of stmRNAs and the genes of synthases that acylate the split tRNAs within them. Further information is provided in the priority literature. 【0243】 In conclusion, stmRNA vol2 By combining the construct with bio-mREX, we have developed a pulldown that can selectively isolate cDNA of active PylRS variants over a wide dynamic range, beyond inactive variants. [Example 5] 【0244】 Direct selection for cellular acylation by tRNA display Next, the inventors devised a novel method for directly selecting synthases based on their tRNA acylation activity. The inventors hypothesized that active and selective PylRS variants could be efficiently detected by performing selection based on parallel bio-mREX coupled with next-generation sequencing (NGS) deep sequencing (Figure 2a). In such experiments, a PylRS active site library was compared with stmRNA vol2 During construction, the selected monomers were cloned and transformed into E. coli cells. The cultures were then diluted in culture medium in the presence (positive sample) and absence (negative sample) of the selected monomers overnight, and bio-mREX was performed. After performing the experiment in multiple replicates, the cDNAs of the positive and negative samples, as well as the cDNA reverse-transcribed from the input library, were barcoded and sequenced by NGS (Figure 2a). The inventors referred to this selection method as tRNA display. 【0245】 NGS data from sample selection allows for the calculation of two key parameters: (1) enrichment, defined as the average abundance of the same sequence in positive samples compared to the abundance of a particular sequence in the input RNA; and (2) selectivity, defined as the ratio of the abundance of the same sequence in positive samples compared to the abundance of a given sequence in negative samples. Desired PylRS variants correspond to sequences that are both highly enriched and highly selective. Plotting the natural logarithm of enrichment against the natural logarithm of selectivity yields a spindle-shaped distribution. We anticipate that all desirable sequences lie in the upper right quadrant of the spindle plot (Figure 2a) (highly enriched and highly selective). 【0246】 To test tRNA display, the inventors used stmRNA vol2As a construct, we generated a small PylRS library in which we expect many sequences to be active. The library contains mutations previously identified that enable efficient incorporation of CbzK(2) (Yanagisawa, T. et al. Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase. Cell Chem Biol 26, 936-949 e913, doi:10.1016 / j.chembiol.2019.03.008 (2019), Dunkelmann, DL, Willis, JCW, Beattie, AT & Chin, JW Engineered triply orthogonal pyrrolysyl-tRNA synthetase / tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids. Nat Chem 12, 535-544, doi:10.1038 / s41557-020-0472-x). (2020), the Y306, L309, and N346 positions of PylRS were targeted (Figures 2b and 6). The inventors transformed E. coli cells with this library and performed one round of tRNA display. The inventors observed a large population of highly enriched and selective variant PylRS sequences in spindle plots derived from sequencing of this experiment (Figure 2c). 【0247】 To evaluate the predictive power of tRNA display, the inventors identified tRNAs that are considered potential hits based on their position on the spindle plot. Pyl CUA and GFP(150TAG) in the presence of 65 PylRS variants His6The in vivo production of GFP(150CbzK) from was measured (Figure 7). The inventors observed a positive correlation (Figure 2d) between the enrichment derived from NGS data and the translation activity derived from GFP production for these hits, and the majority of these hits were selective (Figure 7). 【0248】 The inventors concluded that tRNA display enables the direct, translation-independent identification of active and selective PylRS enzymes from a library of PylRS sequences. [Example 6] 【0249】 High-throughput selection of ncAAs by tRNA display To further validate the utility of tRNA display, the inventors performed parallel selections (Figure 8) using six independent, highly diverse, PylRS active-site libraries (Figure 6) and ten ncAAs, 2 - 11 (Figure 2b). After two rounds of selection, the inventors analyzed the spindle plots derived from the NGS data and identified individual PylRS variants for the enriched and selective ncAAs 2, 3, 4, 7, 8, 9, 10, 11; the enriched and selective variants for each of these ncAAs showed convergent sequence motifs (Figures 8 - 18). 【0250】 The inventors used tRNA Pyl CUAThe incorporation of ncAAs 3, 4, 7, 8, 9, 10, and 11 in response to the amber codon in GFP(150TAG)His6 in cells containing the corresponding PylRS mutants identified by tRNA display was demonstrated. GFP production was ncAA-dependent, and ESI-MS confirmed the incorporation of each ncAA in GFP (Figures 2e-i, 9-18). The inventors note that their results include aminoacyl-tRNA synthetase for 7, enabling the first incorporation of this thiophene-containing ncAA into the protein. Of the 30 characterized variants having selectivity scores higher than or equal to 10 and enrichment scores higher than or equal to 5, 27 were active in protein expression for their homologous ncAAs (20 variants had at least 50% of the activity of PylRS with BocK(1)). All 27 variants selectively incorporated their respective ncAA substrates, as determined by mass spectrometry (Figures 2e-i, 9-18). 【0251】 In summary, the inventors demonstrated parallel, scalable, and rapid selection of PylRS variants using diverse libraries and ncAAs via tRNA display. The inventors note that the amount of ncAA used in each tRNA display selection is 1 / 100 to 1 / 50 of the amount used in current methods for synthase selection. [Example 7] 【0252】 Searching for β-amino acid-specific PylRS variants using tRNA display Next, the inventors attempted tRNA display to search for synthases for a class of ncMs known to be untranslatable or weak ribosomal substrates from in vitro testing (Figure 3) (Katoh, T. & Suga, H. In vitro genetic code reprogramming for the expansion of usable noncanonical amino acids. Annual Review of Biochemistry 91, 221-243 (2022), Melo Czekster, C., Robertson, WE, Walker, AS, Soll, D. & Schepartz, A. In Vivo Biosynthesis of a beta-Amino Acid-Containing Protein. J Am Chem Soc 138, 5194-5197, doi:10.1021 / jacs.6b01023 (2016), Tan, Z., Forster, AC, Blacklow, SC & Cornish, VW Amino Acid Backbone Specificity of the Escherichia coli Translation Machinery. Journal of the American Chemical Society 126, 12752-12753 (2004), Pavlov, MY et al. Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proceedings of the National Academy of Sciences 106, 50-54 (2009), Katoh, T., Tajima, K. & Suga, H. Consecutive Elongation of D-Amino Acids in Translation. Cell Chem Biol 24, 46-54, doi:10.1016 / j.chembiol.2016.11.012 (2017), Katoh, T.Suga, H. Ribosomal Incorporation of Consecutive beta-Amino Acids. J Am Chem Soc 140, 12159-12167, doi:10.1021 / jacs.8b07247 (2018), Dedkova, LM et al. beta-Puromycin selection of modified ribosomes for in vitro incorporation of beta-amino acids. Biochemistry 51, 401-415, doi:10.1021 / bi2016124 (2012). The inventors focused on nine ncMs (12-20, Figure 3a), but these monomers exhibit β-stoichiometry that fluctuates. 2 and β 3 It contains β-hydroxy acids and carboxylic acids, which are amino acids. 6-((tert-butoxycarbonyl)amino)hexanoic acid (BocAhx, 15) is a known substrate for wt PylRS (Kobayashi, T., Yanagisawa, T., Sakamoto, K. & Yokoyama, S. Recognition of non-alpha-amino substrates by pyrrolysyl-tRNA synthetase. J Mol Biol 385, 1352-1360, doi:10.1016 / j.jmb.2008.11.059 (2009)) and provided a control for the inventors' ncM selection. 【0253】 To select PylRS variants for these monomers, we performed parallel tRNA display selection (Figure 19) using highly diverse libraries (Figure 6) with mutations in residues 300, 302, 305, 306, 309, 344, 346, 348, and 401 in the PylRS active site, and their respective ncMs. We identified enriched and selective PylRS variants by analyzing spindle plots derived from NGS data for each ncM (Figures 20-28). For 12 and 15, we identified enriched and selective sequences, and the most selective hits converged to different sequence patterns for each of these ncMs. 【0254】 The inventors identified sequences similar to the wild-type PylRS sequence for ncM 15,6-((tert-butoxycarbonyl)amino)hexanoic acid (BocAhx), indicating that the selection converged on the active synthase for this monomer. Four of the most selective and active hits from the selection, PylRS(15_1)~PylRS(15_4), differed from the wild-type sequence by a point mutation at V401; the inventors demonstrated that all of these selected sequences were active and selective for 15 using fluoro-tREX (Figure 3b, Figure 23). Since 15 is expected to be more difficult to cleave from tRNA than other monomers containing β-amino acids (Figure 4), this result provided confidence that tRNA display would enable synthase selection for a wide range of other monomers containing β-amino acids. 【0255】 Notably, two PylRS variants, PylRS(12_1) and PylRS(12_2), identified by tRNA display selection, were determined by fluoro-tREX. Pyl CUA (S)-3-amino-3-(3-bromophenyl)propanoic acid (β 3-mBrF directed acylations that were 12-dependent (Figure 3c, Figure 20). To verify the identity of the monomers bound to tRNA by PylRS(12_1) and PylRS(12_2), we Pyl CUA captured acylated tRNA Pyl CUA onto streptavidin beads with a biotinylated probe for the tRNA Pyl CUA washed the beads, and eluted ncM by heating under alkaline conditions; then we derivatized the released ncM and analyzed the sample by LC-MS (Figure 29). Using this approach, we confirmed that both PylRS variants charge tRNA Pyl CUA with ncM12 (Figure 3e, f). To our knowledge, PylRS(12_1) / tRNA Pyl CUA and PylRS(12_2) / tRNA 【0256】 are the first β-amino acid-specific orthogonal aminoacyl-tRNA synthetase / tRNA pairs described to date. evol1~3 Next, we increased the activities of PylRS(12_1) and PylRS(12_2) by random mutagenesis of the active site region of the PylRS gene within the stmRNA construct, followed by selection based on tRNA display (Figure 30, 31). From the resulting spinner plots, we identified sequences carrying 1-3 additional mutations relative to the parental clones, which were enriched and selective. The three most enriched and selective hits, PylRS(12_1 Pyl CUA ) were derivatives of PylRS(12_1). We confirmed the specificity of PylRS(12_1 evol1~3 ) for acylating tRNA evol1~3 ) with 12 by both fluoro-tREX and our LC-MS-based assay (Figure 3d, f, g, Figure 29 and 32). PylRS(12_1 evol1~3 ) is more specific for tRNA using 12 than PylRS(12_1)Pyl CUA It showed significantly higher activity during acylation (Figures 3c, d, e, f). 【0257】 Next, the inventors of the present invention have found that PylRS(12_1) or PylRS(12_1) in cells evol1 ) to tRNA Pyl CUA and GFP150TAG His6It is possible that β-link fragments can be synthesized and isolated (Katoh, T. & Suga, H. In vitro genetic Annual Review of Biochemistry 91 , 221–243 ( 2022 ) Melo Czekster , C. , Robertson , WE , Walker , AS , Soll , D. & Schepartz , A. In Vivo Biosynthesis of a Beta-Amino Acid-Containing Protein Soc 138, 5194–5197, doi:10.1021 / jacs.6b01023 (2016);Tan, Z., Forster, AC, Blacklow, SC & Cornish, VW Amino Acid Backbone Specificity of the Escherichia coli Translation Machinery. Proceedings of the National Academy of Sciences 106, 50-54 (2009) Pavlov, MY et al., Consecutive Elongation of D-Amino Acids in Translation Cell Chem Biol 24, 46–54;Ribosomal Incorporation of Consecutive beta-Amino Acids. J Am Chem Soc 140, 12159-12167, doi:10.1021 / jacs.8b07247 (2018), Dedkova, LM et al. beta-Puromycin selection of modified ribosomes for in vitro incorporation of beta-amino acids. Biochemistry 51, 401-415, doi:10.1021 / bi2016124 (2012)), the inventors found that GFP production depends on the addition of 12 to cells. GFP fluorescence is more pronounced than PylRS(12_1). evol1 ) is three times higher, and PylRS(12_1 evol1 This was consistent with the higher acylation activity of PylRS(12_1) or PylRS(12_1) by ESI-MS and MS / MS. evol1 GFP purified from cells containing ) His6We confirmed the incorporation of 12 at position 150. In the absence of 12, we observed some GFP production resulting from Phe incorporation. However, in the presence of 12 (Figure 3g, h, Figure 33), we observed more GFP production, and we detected only the incorporation of 12 by intact MS and MS / MS. We conclude that in the presence of 12, the background incorporation of Phe observed in the absence of 12 is effectively overwhelmed. Similar observations have been made previously regarding efficient and selective ncAA integration systems (Wang, K., Neumann, H., Peak-Chew, SY & Chin, JW Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat Biotechnol 25, 770-777, doi:10.1038 / nbt1314 (2007)), and it is also known that the fidelity of the natural code relies on competition (Fredens, J. et al. Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514-518, doi:10.1038 / s41586-019-1192-5 (2019)). The inventors of this invention have developed GFP3TAG His6In experiments using this method, we did not observe the incorporation of 12 at the 3-position of GFP (Figure 33), indicating that β-amino acids are weak ribosomal substrates (Katoh, T. & Suga, H. In vitro genetic code reprogramming for the expansion of usable noncanonical amino acids. Annual Review of Biochemistry 91, 221-243 (2022), Melo Czekster, C., Robertson, WE, Walker, AS, Soll, D. & Schepartz, A. In Vivo Biosynthesis of a beta-Amino Acid-Containing Protein. J Am Chem Soc 138, 5194-5197, doi:10.1021 / jacs.6b01023 (2016), Tan, Z., Forster, AC, Blacklow, SC & Cornish, VW Amino Acid Backbone Specificity of the Escherichia coli Translation Machinery. Journal of the American Chemical Society). 126, 12752-12753 (2004), Pavlov, MY et al. Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proceedings of the National Academy of Sciences 106, 50-54 (2009), Katoh, T., Tajima, K. & Suga, H. Consecutive Elongation of D-Amino Acids in Translation. Cell Chem Biol 24, 46-54, doi:10.1016 / j.chembiol.2016.11.012 (2017), Katoh, T. & Suga, H.Note that this finding was consistent with "Ribosomal Incorporation of Consecutive beta-Amino Acids. J Am Chem Soc 140, 12159-12167, doi:10.1021 / jacs.8b07247 (2018)" and "Dedkova, LM et al. beta-Puromycin selection of modified ribosomes for in vitro incorporation of beta-amino acids. Biochemistry 51, 401-415, doi:10.1021 / bi2016124 (2012)," and note that β-amino acids are not permissible at all positions in proteins. 【0258】 Next, the inventors performed two rounds of selection using lib14 and ncM A1, A2, A3, A4, A5 (β-amino acids with altered side chains), A6 (α,α-disubstituted amino acid), and A7 (β-hydroxy acid) (Figures 36a and 38). From the resulting spindle plots (Figures 39-45), the inventors identified enriched and selective sequences, and the most selective hits converged to different sequence patterns for each ncM. The inventors note that the selected synthases for all six β-amino acids differ in sequence but contain common mutations M300D and A302H (Figures 20, 39-43). The sequence pattern observed for β-hydroxy acid A7 is similar to that observed for the β-amino acids. However, the residue at position 300, which may be directly adjacent to the amine / hydroxy group, is changed from aspartic acid to asparagine (Figure 45). PylRS variants identified by tRNA display selection, when determined by fluoro-tREX and our LC-MS-based assays, are associated with tRNAs composed of their homologous monomers. Pyl CUAThe specific acylation of was instructed (Figures 36d-q). The inventors quantified the acylation fraction as a function of ncM concentration (Figure 46). To the best of the inventors' knowledge, they have searched for the first specific aminoacyl-tRNA synthetase / tRNA pairs for three different classes of ncM: β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids. [Example 8] 【0259】 ncM coding (enoding) in proteins To investigate the incorporation of β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids into proteins, the inventors searched for orthogonal synthase / orthogonal tRNA pairs in cells for eight types of ncM, 12, A1, A2, A3, A4, A5, A6, and A7, and used GFP150TAG. His6 In combination with the above, a congeneral ncM was added. The inventors observed ncM-dependent GFP production for 12, A2, A5, and A6, with isolated yields ranging from 3 to 35 mg per liter of culture. Mass spectrometry confirmed the incorporation of these β-amino acids and α,α-disubstituted amino acids into GFP (Figures 3h, 33, 36r, 36s, 47, and 48). In the absence of 12, the inventors observed some GFP production resulting from the incorporation of native amino acids (Figure 36r). However, in the presence of 12, the inventors produced more GFP, and detected only the incorporation of 12 by intact MS and MS / MS (Figures 3h, 33, 47). The inventors concluded that in the presence of 12, background incorporation observed in the absence of 12 was effectively overridden; the inventors made similar observations for A2, A5, and A6 (Figures 3h, 36s, 33, 47). Similar observations have been previously made for efficient and selective ncAA incorporation systems, and it is also known that the fidelity of natural codes relies on competition. The inventors conclude that 12, A2, A5, and A6 are incorporated site-specifically with high fidelity. The inventors also found that GFP at position 3 (GFP3TAG) His6We did not observe the integration of ncM 12, A2, A5, or A6 (from the source) (Figure 48), indicating that these ncMs are not permissible at all locations in the protein; similar site-dependent integration efficiencies have been previously observed for ncAA. 【0260】 The present inventors have found that when cells are provided with A1, A3, A4, or A7 and their homologous orthogonal synthase / orthogonal tRNA pairs, GFP150TAG is produced using these ncMs. His6 or GFP3TAG His6 It should be noted that we did not observe an ncM-dependent increase in GFP production from (Figures 36r, 48). These observations are consistent with these ncMs being weak substrates for ribosome polymerization. For A4 and A7, we observed a decrease in GFP production upon addition of the ncM (Figure 36r); this is consistent with these ncMs inhibiting amber codon readthrough once acylated on orthogonal tRNA. Searching for orthogonal synthases specific to these ncMs provides a starting point for selecting ribosomes to polymerize them. [Example 9] 【0261】 Structure of β-amino acid proteins To further characterize the 12 incorporations at position 150 of GFP, the inventors performed X-ray crystallography to determine GFP(150(S)β) at 1.5 Å. 3 mBrF His6 The structure of (protein PylRS(12_1) and tRNA was elucidated.) Pyl CUA (Purified from cells carrying the protein) (Figure 3i). The electron density indicates two consecutive carbon atoms (C2 and C3) in the protein backbone at position 150; the metabromophenyl substituent is bonded to C3 of the β-amino acid, and the stereochemistry at C3 corresponds to the expected (S) stereoisomer. Our structure is the expected (S)β to the protein. 3 Confirm site-specific integration of mBrF amino acids. (S)β 3The introduction of mBrF results in a significant twist in the beta barrel of GFP compared to the wild-type protein (Figure 34). Interestingly, the hydrogen bonding network of the residues immediately before and after the β-amino acid in the polypeptide chain remains essentially unperturbed; this is because this beta barrel is β at this position. 3 - This demonstrates the ability to accommodate amino acids (Figure 34). To the best of our knowledge, this is the first structure of an in vivo produced protein containing β-amino acids. Taken together, our data demonstrate site-specific incorporation of β-amino acids in proteins produced in cells. [Example 10] 【0262】 Consideration In vivo incorporation of main-chain modified monomers has long been an unresolved challenge in expanding the range of encoded cellular polymer synthesis beyond α-L amino acids and their close analogues (De La Torre, D. & Chin, JW Reprogramming the genetic code. Nature Reviews Genetics 22, 169-184, doi:10.1038 / s41576-020-00307-7 (2021), Arranz-Gibert, P., Vanderschuren, K. & Isaacs, FJ Next-generation genetic code expansion. Current Opinion in Chemical Biology 46, 203-211 (2018)).tRNA acylation (Santoro, SW, Wang, L., Herberich, B., King, DS & Schultz, PG An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nature biotechnology 20, 1044-1048 (2002), Chin, JW, Martin, AB, King, DS, Wang, L. & Schultz, PG Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proceedings of the National Academy of Sciences 99, 11020-11024 (2002)) or tRNA acylation and ribosome polymerization in translational readouts commonly used for selection for ribosome polymerization (Schmied, WH et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444-448, The codependency of doi:10.1038 / s41586-018-0773-z (2018)) creates an evolutionary deadlock; this deadlock has so far limited the range of monomers that can be used to acylate specific orthogonal tRNAs in vivo. 【0263】 tRNA display breaks this deadlock based on codependency. This is achieved by creating a selectable acylation phenotype and coupling this phenotype to the sequence of the corresponding synthase gene. This allows for the direct selection of orthogonal aminoacyl-tRNA synthetases that acylate their congeneral orthogonal tRNAs using ncM, without the ncM needing to be a ribosomal substrate or function in translation. Using tRNA display, the inventors rapidly and scalably selected efficient aaRS systems for eight ncAAs. These selections consume orders of magnitude fewer compounds than previous methods. Furthermore, the inventors used tRNA display to select and improve tRNA acylation systems for ncMs. These systems could not be discovered using previous methods. 【0264】 tRNA Pyl CUASince it has been established that ncM12 is acylated in cells, the inventors were able to identify a site in GFP where this incorporation of ncM is permissible to achieve genetically encoded incorporation of a β-amino acid in the protein. In future research, the inventors will utilize cellular acylation of tRNA using ncM to enable translation-based selection for orthogonal ribosomes that can polymerize ncM over a wider range of sites (Wang, K., Neumann, H., Peak-Chew, SY & Chin, JW Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat Biotechnol 25, 770-777, doi:10.1038 / nbt1314 (2007), Schmied, WH et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444-448, doi:10.1038 / s41586-018-0773-z (2018), Rackham, O. & Chin, JW A network of orthogonal ribosome x mRNA pairs. Nat Chem Biol 1, 159-166, doi:10.1038 / nchembio719 (2005), Neumann, H., Wang, K., Davis, L., Garcia-Alai, M. & Chin, JW Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441-444, doi:10.1038 / nature08817 (2010)).Translation-based selection (Schmied, WH et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444-448, doi:10.1038 / s41586-018-0773-z (2018)) can also generate orthogonal ribosomes that facilitate the cellular synthesis of polymers composed of a wider variety of ncMs.The inventors have developed a method for directly encoding ncM, and a clear method for increasing the chemical range of peptides and proteins through post-translational modification and protein ligation (Morinaka, BI et al. Natural noncanonical protein splicing yields products with diverse β-amino acid residues. Science 359, 779-782 (2018), Lakis, E., Magyari, S. & Piel, J. In Vivo Production of Diverse β-Amino Acid-Containing Proteins. Angewandte Chemie 134, e202202695 (2022), Camarero, JA & Muir, TW Native chemical ligation of polypeptides. Current Protocols in Protein Science 15, 18.14. 11-18.14. 21 (1999), Niquille, DL et al. Nonribosomal biosynthesis of backbone-modified peptides. Nature We anticipate that combining this with Chemistry 10, 282-287 (2018) and Arnison, PG et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Natural Product Reports 30, 108-160 (2013) will further expand the repertoire of ncMs that can be used to construct modified proteins and non-standard polymers. 【0265】 Gene coding of β-amino acids can enable the creation of protease-resistant proteins in cells (Seebach, D. et al. β-Peptides: Synthesis by Arndt-Eistert homologation with concomitant peptide coupling. Structure determination by NMR and CD spectroscopy and by X-ray crystallography. Helical secondary structure of a β-hexapeptide in solution and its stability towards pepsin. Helvetica Chimica Acta 79, 913-941 (1996), Hintermann, T. & Seebach, D. The Biological Stability of β-Peptides: No Interactions between β-and β-Peptidic Structures? Chimia 51, 244-244 (1997), Seebach, D. et al. Biological and pharmacokinetic studies with β-peptides. Chimia 52, 734-734 (1998)).Future developments will involve strategies for encoding non-standard polymers in cells (Robertson, WE et al. Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021)) and foldamers composed of β-amino acids and other ncMs that can form secondary, tertiary, and quaternary structures (Horne, WS, Price, JL & Gellman, SH Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly. Proceedings of the National Academy of Sciences 105, 9151-9156 (2008), Wang, PS & Schepartz, A. β-Peptide bundles: Design. Build. Analyze. Biosynthesize. Chemical Communications 52, 7420-7432 (2016)), such as foldamers (Gellman, SH Foldamers: a manifesto. Accounts of chemical research 31, This may be combined with an expansion of the range of monomers that can be encoded to enable encoded biosynthesis (173-180 (1998)). The inventors anticipate that the design and directed evolution of genetically encoded foldomers can complement and enhance the standard functions of cells. 【0266】 In addition to ncM12, using tRNA display, the inventors searched for orthogonal synthase / orthogonal tRNA pairs that are selective for eight novel ncMs, including β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids, thereby directly facilitating gene coding and site-specific incorporation of β-amino acids and α,α-disubstituted amino acids into proteins in living organisms. [Example 9] 【0267】 method buffer solution Resuspension buffer (RB): 100mM NaOAc, 50mM NaCl, 0.1mM EDTA, pH5.0 Deacylation buffer (DB): 50mM Bicine pH9.6, 1mM EDTA Buffer D(D): 50 NaOAc pH5, 150 NaCl, 10mM Mg2Cl, 0.1mM EDTA Hybridization buffer (HB): 10 mM Tris-HCl, 25 mM NaCl, pH 7.4 Kreno-Exo(-) Master Mix with Cy5-11-dCTP (KMM-Cy5): 17 μL water, 5 μL 2.0 10x NE buffer, 1 μL Kreno-Exo(-), 1 μL dNTPS-dCTP (10 mM), 1 μL Cy5-11-dCTP (20 μM) Orange loading pigment (OLD): 8M urea, Orange G Cy5-11-dCTP Mini with Creno-(Exo-) Master Mix (KMM-Cy5-Mini): 1.1 μL water, 1.2 μL 10x NE Buffer 2.0, 2 μL Creno-Exo(-), 0.5 μL dNTPS-dCTP (5 mM), 1 μL Cy5-11-dCTP (5 μM) Creno-Exo(-) Master Mix (KMM-Bio) with Bio-11-dCTP: 17 μL water, 5 μL 10x NE buffer 2.0, 1 μL Creno-Exo(-), 1 μL dNTPS-dCTP (10 mM), 1 μL Bio-11-dCTP (20 μM) Wash buffer (WB): 10mM Tris-HCl, 150mM LiCl, 1mM EDTA, 0.05%v / v Tween20, pH7.5 Binding buffer (BB): 20mM Tris-HCl, 1M LiCl, 2mM EDTA, 0.05%v / v Tween20, pH7.5 Formamide loading buffer (FMB): 90% formamide SDS lysis buffer (SLB): 100mM NaOAc, 50mM NaCl, 0.1mM EDTA, pH5.0, 1% (m / w) SDS Alkaline washing buffer (AWB): 25 mM NaOH, 4 mM EDTA, 0.05% Tween20 Reverse transcription hybridization mix (RHM): 1 μL of DNA primer (2 μM), 1 μL of 10 mM dNTPs, 1 μL of 10xHB, 10 μL of water RT Master Mix (RMM): 4 μL SSIV buffer, 1 μL RNAse Out, 1 μL SSIV RT, 1 μL 0.1 M dithiothreitol (DTT) Acidic washing buffer 1 (aWB1): 100 mM NaOAc pH 5 Acidic wash buffer 1+Tween20 (aWB1-T): 100mM NaOAc pH5, 0.01% (v / v) Tween-20 Acidic washing buffer 2 (aWB2): 20 mM NaOAc pH 5 【0268】 Culture medium SOC: Super Optimal Broth + Glucose Yeast extract tryptone supplemented with 2xYT-s: 75 μg / mL spectinomycin. 2xYT-st: Tryptone yeast extract supplemented with 75 μg / mL spectinomycin and 10 μg / mL tetracycline. 2xYT-s-ap: Tryptone yeast extract supplemented with 75 μg / mL of spectinomycin and 50 μg / mL of apramycin. Yeast extract tryptone supplemented with 2xYT-am: 75 μg / mL ampicillin. 【0269】 chemicals NcM1 and 2 were purchased from Bachem. NcM4 was purchased from Fluorochem. NcM5 was purchased from Ambeed. NcM6, 8, 13, 17, and 18 were purchased from Enamine. NcM7 was purchased from aaBlocks. NcM9, 10, 15, and 16 were purchased from Merck. NcM12 and 20 were purchased from BLD. NcM14 was purchased from Advanced ChemBlock. NcM19 was purchased from AstaTech. NcM3 was synthesized as previously described (Spinck, M. et al. Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles. Nature Chemistry 15, 61-69 (2023)), and ncM11 was custom synthesized as previously described (Tang, S. et al. Nature 602, 701-707 (2022)). NcM13 and 18 were deprotected with Boc in concentrated HCl in dioxane. 【0270】 Cloning of DNA constructs Standard cloning was performed by Gibson assembly using NEBuilder® HiFi DNA Assembly Master Mix (NEB) in accordance with the manufacturer's guidelines. As previously described, libraries were generated by enzymatic inverse PCR. Briefly, the template plasmid was amplified by PCR using two primers (see primer list) containing degenerate codons at the desired mutagenesis site and BsaI cleavage site. In the case of custom mixes, primers containing different codons were manually mixed and used for the PCR reaction. The PCR products were gel-purified and digested with BsaI and DpnI. Subsequently, the samples were purified, ligated using T4 ligase, and transformed into electrocompetent E. coli DH10β cells. 9The minimum transformation efficiency was ensured. Individual colonies (more than 10) were evaluated using Sanger sequencing for quality control of library assembly. Total plasmid DNA was prepared from the resulting culture, sequenced in bulk using Sanger sequencing, and used for subsequent experiments. 【0271】 General protocol NGS Data Analysis NGS was performed on a MiSeq system (for test evolutions using Library 1 and Substrate 1) or a NextSeq2000 system (for all other cases). cDNA obtained from tRNA display was amplified by PCR using oligo-NGS_A(1-8) and NGS_B(1-8) containing different combinations of Nextera sequencing barcodes. Samples were purified, quantified, and combined in equimolar amounts. Paired-end reads were initially paired using PEAR (Ellman, JA, Mendel, D. & Schultz, PG Site-specific incorporation of novel backbone structures into proteins. Science 255, 197-200 (1992)) and aligned with the MmPylRS reference sequence using Bowtie2 (Mendel, D., Cornish, VW & Schultz, PG Site-directed mutagenesis with an expanded genetic code. Annual review of biophysics and biomolecular structure 24, 435-462 (1995)). Relevant library locations were extracted, translated into amino acids, and the resulting variants were counted using an R script. The frequency of each variant in each library was used for subsequent operations, and the count value was calculated by dividing by the total count in that library. Enrichment and selectivity scores were calculated for all variants using an R script as follows: Firstly, variants present only in all positive repeats were considered (tables were merged using the AND operator). Assuming that highly enriched sequences could not potentially be included in the negative and input samples, but could be desirable, negative and naive repeats were merged into the positive table using the OR operator. If a replica did not contain a particular variant, a placeholder value of 0.95 was used.Using the obtained dataset, the mean enrichment in the presence and absence of each substrate was calculated and expressed as a quotient of the mean frequencies under one condition and the input condition. Using the obtained positive and negative enrichments, the selectivity value (equal to the quotient of positive and negative frequencies) for each variant was calculated. For further analysis, the variants were filtered using a threshold empirically determined for the normalized standard deviation of the positive frequency (plus the variance error under the substrate condition). 【0272】 Identification of ncMs by tRNA pulldown and LC-MS tRNA was isolated from 8 mL of cells according to general protocol B, which omits oxidation with NaIO4. The RNA pellet was resuspended in 90 μL of buffer D, and the RNA concentration was adjusted to match the lowest concentration in the samples being compared. 0.5 μL of biotinylated DNA probe (100 μM) was added to the RNA, and the DNA probe was hybridized at 65°C for 5 minutes. 40 μL of Streptavidin Dynabeads MyOne C1 (Invitrogen) was washed twice with buffer DT and added to 10 μL of buffer D. The beads were added to the hybridization reaction, and the probe was bound to the beads at 4°C for a minimum of 30 minutes while inverting and mixing. The samples were washed three times with 200 μL of acW1-T, twice with 200 μL of acW1, three times with 200 μL of acW2, and once with 200 μL of water, all on a magnetic stand. 24 μL of DB and beads were added and incubated at 42°C for 1 hour. 12 μL of the deacylation mix was added to 3 μL of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (3 mg / mL in AQC-acetonitrile), and the reaction was incubated at 55°C for 15 minutes. The samples were analyzed on an Agilent Technologies 6130 Quadrupole LC / MS using single-ion monitoring. 【0273】 GFP (150X) His6 and GFP(3X) His6 (In the formula, X represents any ncM) Activity assay GFP150TAG His6 or GFP3TAG His6 Chemically competent DH10β cells carrying a p15A plasmid encoding (two versions of the p15A plasmid containing tetracycline or apramycin-resistant cassettes, which yield similar results, were used interchangeably) were rescued in MmPylRS or its variants, and MmtRNA rescued in SOC and grown overnight in 2xYTs-t or 2xYTs-ap. Pyl CUA Cells were transformed with the pMB1 plasmid encoding .20 μL of overnight culture was diluted in 480 μL of 2xYTs-t or 2xYTs-ap containing 0.2% L-arabinose in the presence and absence of 2–4 mM of each ncM in a 96-well plate. Cells were grown at 700 r.pm for 16–20 hours at 37°C. The plates were centrifuged at 4200 rcf for 12 minutes at 4°C, and the cells were resuspended in 150 μL of PBS. 100 μL of the resuspended cells were transferred to a Costar 96-well flat-bottom plate and OD 600 GFP fluorescence was measured using a PHERAstar FS plate reader. 【0274】 GFP (150X) for MS analysis His6 Isolation The three repeats of the protein produced as described above were mixed in a 1.5 mL Eppendorf tube, centrifuged at 4200 rcf for 3 minutes, frozen at -20°C, thawed, and resuspended in 150 μL of BugBuster (Millipore). The cells were lysed for 1 hour with inversion mixing. The lysed cells were centrifuged at 20000 rcf at 4°C for 20 minutes, and the lysate was added to 20 μL of NiNTA beads. GFP(150) Hi6 The protein was allowed to bind to the beads at room temperature for 20 minutes while being mixed by inversion. The beads were washed six times with 60 μL of 30 mM imidazole in PBS, and the protein was eluted five times with 30 μL of 300 mM imidazole in PBS. 【0275】 For the low-activity mutant of ncM12, 5–15 mL of cell culture was used for protein production. The volume of Bug Buster was adjusted proportionally, while all other volumes were kept constant. 【0276】 mass spectrometry ESI-MS as well as MS / MS were acquired as previously described (Dunkelmann, DL, Oehm, SB, Beattie, AT & Chin, JW A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat Chem, doi:10.1038 / s41557-021-00764-5 (2021), Dunkelmann, DL, Willis, JCW, Beattie, AT & Chin, JW Engineered triply orthogonal pyrrolysyl-tRNA synthetase / tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids. Nat Chem 12, 535-544, doi:10.1038 / s41557-020-0472-x (2020)). 【0277】 Protein expression, purification, and crystallization GFP150TAG His6 The p15A plasmid encoding MmPylRS(12_1) and MmtRNA Pyl Chemically competent DH10β cells carrying the pMB1 plasmid encoding the pMB1 plasmid were transformed, rescued in SOC, and grown overnight in 2xYTs-ap. 10 mL of the overnight culture was diluted in 1 L of 2xYTs-ap containing 0.2% L-arabinose in the presence of 5 mM 12. 【0278】 GFP150(S)β3 A bacterial pellet of mBrF-His6 expression culture (1 L) was lysed by sonication, centrifuged at 142,000 rcf for 30 minutes, and the supernatant was conjugated to Ni-NTA beads (Qiagen). After washing the beads three times, the protein was eluted and further purified by gel filtration using a Superdex 75 HiLoad 26 / 60 pg column (GE Healthcare) in 25 mM Tris pH 7.4, 200 mM NaCl, and 0.06% NaN3. The purified protein was concentrated to 6 mg / mL using Vivaspin 20, 10,000 MWCO (Sartorius). The sample was trypsin-digested with Sequencing Grade Modified Trypsin (Promega) in a 50:1 ratio. The sample was incubated at 37°C for 1 hour, centrifuged at 21,000 rcf for 10 minutes, and then seeded into a crystal tray. Crystallization tests using multiple commercially available crystallization kits were performed at 18°C ​​in a 96-well sitting drop vapor diffusion plate (Molecular Dimensions) and set up using a Mosquito HTS robot (TTP Labtech). The drop ratio of 0.2 μL protein solution + 0.2 μL reservoir solution was used for screening. The only useful dataset was collected from crystals taken from a Fusion screen (Molecular Dimensions) with the following composition: 37.5% PEG3350 / PEG 1K / MPD (1:1:1), 0.1 M bicine / trisma pH 8.5, 0.8% (w / v) Morpheus III alkaloids, and 0.12 M Morpheus alcohol. Crystals were collected and briefly frozen in liquid nitrogen. 【0279】 Diffraction data collection, processing, and structural analysis. Diffraction data was collected using ESRF on beamline ID23-2 at an energy of 14.2 keV. The data was processed using XDS via pipeline autoProc (Global Phasing ltd.). The structure was elucidated by molecular substitution with MolRep using the homolog model PDB 2B3P. Interaction construction was performed using Coot, refinement using REFMAC5, and verification using Molprobity. Structural drawings were prepared using PyMOL (PyMOL Molecular Graphics System, Schrodinger, LLC). 【0280】 ncAA selection Selection was performed as shown in Figure 8. RNA was isolated and oxidized as described in General Procedure A. Bio-mREX was performed as specified in General Procedure. After the first selection, a new library was assembled from the amplified cDNA described above. After the second selection, an NGS sample was prepared from the isolated cDNA described above, and NGS was performed using a P2 600-cycle cartridge, and the data was analyzed as specified above. 【0281】 ncM selection Selection was performed as shown in Figure 19. RNA was isolated and oxidized as described in General Procedure A. Bio-mREX was performed as specified in General Procedure. NGS samples were prepared from the isolated cDNA described above, and NGS was performed using a P1 600-cycle cartridge, with data analyzed as specified above. 【0282】 Selection of substrate 12 using a random mutagenesis library The concentrations of the pMB1 plasmid encoding PylRS hits 12_1 and 12_2 were measured using a Qubit 2 fluorometer (Life Technologies) and a Qubit 1x dsDNA HS assay kit (Invitrogen), and the plasmids were mixed in equimolar amounts. The combined plasmids were used for mutagenic PCR of the PylRS active site using golden gate primers and the GeneMorph II kit (Agilent) under conditions that yield the maximum number of random mutations. The amplicon was cloned into a new pColE1 backbone by a two-piece Golden Gate assembly according to NEB (New England Biolabs) guidelines. Selection was performed as schematic in Figure 30. RNA was isolated and oxidized as described in General Procedure A. Bio-mREX was performed as specified in General Procedure. NGS samples were prepared from the isolated cDNA described above, NGS was performed using a P2 600 cycle cartridge, and the data were analyzed as specified above. 【0283】 Code availability The code for analyzing tRNA display NGS data will be available at https: / / github.com / JWChin-Lab / tRNA_display upon release. 【0284】 References 1 Dumas, A., Lercher, L., Spicer, CD & Davis, BG Designing logical codon reassignment - Expanding the chemistry in biology. 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Nat Biotechnol 25, 770-777, doi:10.1038 / nbt1314 (2007). 29 Fredens, J. et al. Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514-518, doi:10.1038 / s41586-019-1192-5 (2019). 30 Arranz-Gibert, P., Vanderschuren, K. & Isaacs, F. J. Next-generation genetic code expansion. Current Opinion in Chemical Biology 46, 203-211 (2018). 31 Schmied, W. H. et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444-448, doi:10.1038 / s41586-018-0773-z (2018). 32 Beattie, A. T., Dunkelmann, D. L., Chin, J. W. Quintuply orthogonal pyrrolysyl-tRNA synthetase / tRNAPyl pairs. Nature Chemistry In press (accepted) (2023). 33 Rackham, O. & Chin, J. W. A network of orthogonal ribosome x mRNA pairs. Nat Chem Biol 1, 159-166, doi:10.1038 / nchembio719 (2005). 34 Neumann, H., Wang, K., Davis, L., Garcia-Alai, M. & Chin, J. W. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. 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Claims

[Claim 1] An acyl-tRNA synthetase that can specifically acylate tRNA using beta amino acids, M300A, M300C, M300D, M300M, or M300S for Sequence ID No. 1; A302A, A302C, A302D, A302G, A302H, A302L, A302N, or A302Y for Sequence ID No. 1; and N346A, N346C, N346G, N346S, N346T, or N346V for Sequence ID No. 1 The acyl-tRNA synthetase comprising an amino acid sequence containing the corresponding residue. [Claim 2] The acyl-tRNA synthetase according to claim 1, comprising M300A, M300C, M300D, or M300S; A302C, A302D, A302G, A302H, A302L, A302N, or A302Y; and N346A, N346C, N346G, N346S, N346T, or N346V. [Claim 3] The acyl-tRNA synthetase according to claim 1, comprising M300D; A302H; and optionally N346A, N346G, or N346S. [Claim 4] The acyl-tRNA synthetase according to claim 3, comprising N346G. [Claim 5] An acyl-tRNA synthetase capable of specifically acylating tRNA using a beta-amino acid, wherein the beta-amino acid may be (S)-3-amino-3-(3-bromophenyl)propanoic acid, and the acyl-tRNA synthetase is M300D mutation for sequence number 1, A302H, A302Y, or A302C mutations in Sequence ID No. 1, and N346G, N346A, or N346S mutations in Sequence ID No. 1 Acyl-tRNA synthetase containing an amino acid sequence that includes the corresponding mutation. [Claim 6] The acyl-tRNA synthetase according to claim 5, wherein the A302 mutation is A302H and the N346 mutation is N346G. [Claim 7] The acyl-tRNA synthetase according to any one of claims 1 to 6, wherein the amino acid sequence includes one or any combination of mutations at the positions corresponding to the following positions in SEQ ID NO: F295, L305, Y306, N307, L309, M344, N346, C348, T364, F366, K375, T387, V401, D414, and G421. [Claim 8] The amino acid sequence is L305C, L305F, L305G, L305H, L305I, L305L, L305N, or L305V; Y306C, Y306F, Y306H, Y306I, Y306L, Y306N, Y306R, Y306V, or Y306Y; L309C, L309F, L309G, L309H, L309I, L309L, L309N, L309S, L309V, or L309Y; M344E, M344G, M344M, or M3 44Q; C348A, C348C, C348F, C348G, C348H, C348I, C348L, C348M, C348N, C348R, C348S, C348T, C348V, or C348Y; Y384Y; S399S; and one, any combination, or all of V401A, V401C, V401F, V401K, V401L, V401S, V401T, or V401V, according to claim 7. [Claim 9] The acyl-tRNA synthetase according to claim 7, wherein the beta-amino acid may be (S)-3-amino-3-(3-bromophenyl)propanoic acid, and the amino acid sequence comprises one or a combination of any of F295L or F295I;L305H or L305C;Y306L, Y306F, Y306R, or Y306C;N307K;L309V or L309C;C348I, C348L, C348V, or C348F;T364A;F366L;K375R;T387I or T387S;V401X, V401C, V401S, V401L, V401K, or V401A;D414V; and G421A. [Claim 10] The beta-amino acid may be (S)-3-amino-3-(3-bromophenyl)propanoic acid, and the amino acid sequence may be one of the following sets of mutations at the positions corresponding to the positions in SEQ ID NO: i) A302H, L305H, N346G, C348F, and V401C; ii) A302H, Y306L, N346G, C348L, and V401S; iii) A302H, Y306L, N346G, C348L, and V401L; iv) A302C, L305C, Y306F, L309V, N346G, C348L, and V401C; v) A302Y, Y306R, N346G, and C348I; vi) A302H, Y306R, N346G, and C348L; vii) A302Y, Y306C, L309C, N346G, and C348I; viii) A302H, L305C, Y306F, N346G, and V401K; or ix) A302H, L305C, Y306F, N346G, C348L, and V401A The acyl-tRNA synthetase according to any one of claims 1 to 9, comprising: [Claim 11] The beta amino acid is ((S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β ３ The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence may be MDF, and the amino acid sequence comprises the following residues at positions corresponding to the positions in SEQ ID NO: M300D; A302H; optionally L305C, L305I, or L305L; optionally Y306H, Y306I, Y306L, or Y306Y; optionally L309C, L309F, L309H, L309L, or L309N; optionally M344E, M344M, or M344Q; N346G or N346A; C348 mutation, optionally C348G, C348N, C348S, or C348V; optionally Y384Y; optionally S399S; and optionally V401C, V401L, or V401V. [Claim 12] The beta amino acid is ((S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β ３ The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence comprises the following residues at positions corresponding to the positions in SEQ ID NO: M300D; A302H; optionally L305F, L305I, L305L, or L305N; optionally Y306I or Y306Y; optionally L309C, L309F, L309L, or L309N; optionally M344Q or M344M; N346A or N346G; optionally C348C, C348G, C348I, or C348S; optionally Y384Y; optionally S399S; and optionally V401C, V401L, or V401V. [Claim 13] The beta amino acid is ((S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β ３ The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence may be pmFF) and the amino acid sequence comprises the following residues at positions corresponding to the positions in Sequence ID No. 1: M300D; A302H; optionally L305C, L305I, L305L, or L305V; optionally Y306I, Y306L, or Y306Y; optionally L309G, L309H, L309L, L309N, L309S, or L309V; optionally M344Q or M344M; N346G; C348 mutation, optionally C348F, C348L, C348S, or C348V; optionally Y384Y; optionally S399S; and optionally V401C, V401L, or V401V. [Claim 14] The beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β ３ The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence includes, at positions corresponding to the positions in SEQ ID NO: M300D or M300M; A302D or A302H, optionally L305L, optionally Y306H, Y306L, Y306R, or Y306Y; optionally L309G, L309H, or L309L; optionally M344Q; N346G or N346S; optionally C348C, C348I, C348S, or C348Y; optionally Y384Y; optionally S399S; and optionally V401C or V401V. [Claim 15] The beta amino acid is ((S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β ３ The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence includes, at positions corresponding to the positions in Sequence ID No. 1, the residues: M300D; A302H or A302Y; optionally L305F, L305I, L305L, or L305V; optionally Y306F, Y306L, or Y306Y; optionally L309C, L309L, L309N, or L309V; optionally M344Q or M344M; N346G; optionally C348F, C348H, or C348V; optionally Y384Y; optionally S399S; and optionally V401S, V401T, or V401V. [Claim 16] The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence includes one of the sets of mutations from any one of Tables 1 to 6 at a position corresponding to the position in SEQ ID NO:

1. [Claim 17] The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence includes, at the position corresponding to the position in SEQ ID NO: 1, either a mutation from any one of Tables 1 to 6 or a set of wild-type residues. [Claim 18] The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence includes, at a position corresponding to the position in SEQ ID NO: 1, one of a set of mutations associated with an identifier (e.g., 12_1) in any one of Tables 1 to 6. [Claim 19] The acyl-tRNA synthetase according to any one of claims 1 to 9, wherein the amino acid sequence includes, at the position corresponding to the position in SEQ ID NO: 1, either a mutation associated with an identifier (e.g., 12_1) from any one of Tables 1 to 6 or a set of wild-type residues. [Claim 20] The amino acid sequence has one of the following sets of mutations at the position corresponding to the position in SEQ ID NO: a) N307K and F366L; b) F295I and T387I; c) G421A; d) T364A and T387I; e) T387S and D414V; or f) F295L and K375R The acyl-tRNA synthetase according to any one of claims 1 to 19, comprising: [Claim 21] The acyl-tRNA synthetase according to any one of claims 1 to 20, wherein the beta-amino acid may be (S)-3-amino-3-(3-bromophenyl)propanoic acid, and the amino acid sequence includes mutations corresponding to A302H, L305H, N307K, N346G, C348F, F366L, and V401C relative to SEQ ID NO:

1. [Claim 22] The acyl-tRNA synthetase according to claim 5, wherein the amino acid sequence includes mutations corresponding to A302H, L305H, Y306Y, N307K, L309L, M344M, N346G, C348F, F366L, Y384Y, S399S, and V401C relative to SEQ ID NO:

1. [Claim 23] The acyl-tRNA synthetase according to any one of claims 1 to 22, wherein the amino acid sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% similarity or identity with the amino acid sequence of SEQ ID NO:

1. [Claim 24] The acyl-tRNA synthetase according to any one of claims 1 to 23, wherein the amino acid sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% similarity or identity with any one of the amino acid sequences of sequence numbers 1 to 18. [Claim 25] The acyl-tRNA synthetase according to any one of claims 1 to 24, wherein the amino acid sequence is a PylRS amino acid sequence. [Claim 26] Use of acyl-tRNA synthetase according to any one of claims 1 to 25 in a method for producing a polymer containing beta amino acids. [Claim 27] A method for producing a polymer containing beta amino acids, i) Use of acyl-tRNA synthetase for acylation of tRNA using beta amino acids, and ii) Incorporation of the beta amino acid into the polymer The method, including the method described above. [Claim 28] A method for producing a polymer containing beta amino acids, i) Use of acyl-tRNA synthetase according to any one of claims 1 to 25 for acylation of tRNA using beta amino acids, and ii) Incorporation of the beta amino acid into the polymer chain The method, including the method described above. [Claim 29] The acyl-tRNA synthetase according to any one of claims 1 to 25, wherein the beta-amino acid is an analog of S-beta-3-phenylalanine, the use according to claim 26, or the method according to claim 27 or 28. [Claim 30] The beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)β ３ MDF), (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)β ３ pBrF), (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)β ３ pmFF), (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)β ３ oBrF), or (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β ３ mCF3F), the acyl-tRNA synthetase according to any one of claims 1 to 25, the use according to claim 26, or the method according to claim 27 or 28. [Claim 31] A nucleic acid encoding an acyl-tRNA synthetase according to any one of claims 1 to 25. [Claim 32] A host cell comprising an acyl-tRNA synthetase according to any one of claims 1 to 25 or a nucleic acid according to claim 31. [Claim 33] The host cell according to claim 32, wherein the acyl-tRNA synthetase is orthogonal to endogenous tRNA. [Claim 34] A host cell according to claim 32 or 33 that is alive. [Claim 35] An acyl-tRNA synthetase capable of specifically acylating tRNA using α,α-disubstituted amino acids, A302C, A302G, A302H, or A302S for Sequence ID No. 1; and N346A, N346C, N346E, N346G, N346T, or N346V for Sequence ID No. 1 The acyl-tRNA synthetase comprising an amino acid sequence containing the corresponding residue. [Claim 36] The acyl-tRNA synthetase according to claim 35, wherein the amino acid sequence includes residues corresponding to M300A, M300C, M300D, M300E, M300M, M300S, or M300T for SEQ ID NO:

1. [Claim 37] The acyl-tRNA synthetase according to claim 35, wherein the amino acid sequence includes a residue corresponding to M300D for SEQ ID NO:

1. [Claim 38] The amino acid sequence is M300D; A302G or A302H; and N346A, N346C, or N346G The acyl-tRNA synthetase according to claim 35, comprising a residue corresponding to the following: [Claim 39] The acyl-tRNA synthetase according to any one of claims 35 to 38, wherein the amino acid sequence includes one or any combination of mutations at the positions corresponding to the following positions in SEQ ID NO: F295, M300, L305, Y306, N307, L309, M344, N346, C348, T364, F366, K375, T387, V401, D414, and G421. [Claim 40] The amino acid sequence is L305C, L305F, L305H, L305I, L305L, L305N, or L305S; Y306D, Y306F, Y306L, Y306N, or Y306Y; L309C, L309F, L309G, L309H, L309L, L309N, or L309V; M344H, M344M, or M3 The acyl-tRNA synthetase according to claim 39, comprising 44Q; C348C, C348F, C348G, C348H, C348I, C348L, C348S, or C348V; Y384Y; S399S; and one, any combination, or all of V401A, V401C, V401K, V401L, or V401V. [Claim 41] The amino acid sequence is M300D; A302G or A302H; L305C, L305F, or L305S; N346A, N346C, or N346G; and C348C or C348G An acyl-tRNA synthetase according to any one of claims 35 to 40, comprising a residue corresponding to the specified residue. [Claim 42] The amino acid sequence is M300D; A302G or A302H; L305C, L305F, or L305S; Y306F or Y306Y; L309F or L309L; N346A, N346C, or N346G; C348C or C348G; and V401C, V401L, or V401V An acyl-tRNA synthetase according to any one of claims 35 to 40, comprising a residue corresponding to the specified residue. [Claim 43] The amino acid sequence is M300D; A302G or A302H; L305C, L305F, or L305S; Y306F or Y306Y; L309F or L309L; M344M; N346A, N346C, or N346G; C348C or C348G; Y384Y; S399S; and V401C, V401L, or V401V An acyl-tRNA synthetase according to any one of claims 35 to 40, comprising a residue corresponding to the specified residue. [Claim 44] The acyl-tRNA synthetase according to claim 35, wherein the amino acid sequence includes one of the sets of mutations in Table 7 at a position corresponding to the position in SEQ ID NO:

1. [Claim 45] The acyl-tRNA synthetase according to claim 35, wherein the amino acid sequence includes one of the mutant and wild-type residue sets in Table 7 at the position corresponding to the position in SEQ ID NO:

1. [Claim 46] The acyl-tRNA synthetase according to claim 35, wherein the amino acid sequence includes, at a position corresponding to the position in SEQ ID NO: 1, one of the sets of mutations associated with the identifier (e.g., A6_2) in Table 7. [Claim 47] The acyl-tRNA synthetase according to claim 35, wherein the amino acid sequence includes, at the position corresponding to the position in SEQ ID NO: 1, either a mutation associated with an identifier in Table 7 (e.g., A6_2) or a set of wild-type residues. [Claim 48] The amino acid sequence has one of the following sets of mutations at the position corresponding to the position in SEQ ID NO: a) N307K and F366L; b) F295I and T387I; c) G421A; d) T364A and T387I; e) T387S and D414V; or f) F295L and K375R An acyl-tRNA synthetase according to any one of claims 35 to 47, comprising: [Claim 49] The acyl-tRNA synthetase according to any one of claims 35 to 48, wherein the amino acid sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% similarity or identity with the amino acid sequence of SEQ ID NO:

1. [Claim 50] The acyl-tRNA synthetase according to any one of claims 35 to 49, wherein the amino acid sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18. [Claim 51] The acyl-tRNA synthetase according to any one of claims 35 to 50, wherein the amino acid sequence is a PylRS amino acid sequence. [Claim 52] Use of acyl-tRNA synthetase according to any one of claims 35 to 51 in a method for producing a polymer containing an α,α-disubstituted amino acid. [Claim 53] A method for producing polymers containing α,α-disubstituted amino acids, i) Use of acyl-tRNA synthetase for acylation of tRNA using α,α-disubstituted amino acids, and ii) Incorporation of the α,α-disubstituted amino acids into the polymer The method, including the method described above. [Claim 54] A method for producing polymers containing α,α-disubstituted amino acids, i) Use of acyl-tRNA synthetase according to any one of claims 35 to 51 for acylation of tRNA using α,α-disubstituted amino acids, and ii) Incorporation of the α,α-disubstituted amino acids into the polymer chain The method, including the method described above. [Claim 55] The acyl-tRNA synthetase according to any one of claims 35 to 51, wherein the α,α-disubstituted amino acid is (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S)α-Me-pIF), the use according to claim 52, or the method according to claim 53 or 54. [Claim 56] A nucleic acid encoding an acyl-tRNA synthetase according to any one of claims 35 to 51. [Claim 57] A host cell comprising an acyl-tRNA synthetase according to any one of claims 35 to 51 or a nucleic acid according to claim 56. [Claim 58] The host cell according to claim 57, wherein the acyl-tRNA synthetase is orthogonal to endogenous tRNA. [Claim 59] A host cell according to claim 57 or 58 that is alive. [Claim 60] An acyl-tRNA synthetase capable of specifically acylating tRNA using a beta-hydroxy acid, M300A, M300D, M300M, M300N, or M300S for Sequence ID No. 1; and A302D, A302G, A302H, or A302N for Sequence ID No. 1 The acyl-tRNA synthetase comprising an amino acid sequence containing the corresponding residue. [Claim 61] The acyl-tRNA synthetase according to claim 60, wherein the amino acid sequence includes residues corresponding to M300D, M300N, or M300S. [Claim 62] The acyl-tRNA synthetase according to claim 60 or 61, wherein the amino acid sequence includes residues corresponding to N346A, N346C, N346E, N346G, N346N, N346S, or N346V. [Claim 63] The acyl-tRNA synthetase according to claim 62, wherein the amino acid sequence includes residues corresponding to N346A, N346C, or N346G. [Claim 64] The acyl-tRNA synthetase according to any one of claims 60 to 63, wherein the amino acid sequence includes residues corresponding to M300N and A302H for SEQ ID NO:

1. [Claim 65] The acyl-tRNA synthetase according to claim 64, wherein the amino acid sequence comprises mutations at N346 and / or C348 relative to SEQ ID NO:

1. [Claim 66] The amino acid sequence is M300N or M300S; A302H; and N346A, N346C, or N346G The acyl-tRNA synthetase according to claim 60, comprising a residue corresponding to the following: [Claim 67] The acyl-tRNA synthetase according to claim 60, wherein the amino acid sequence includes residues corresponding to M300N, A302H; and N346G. [Claim 68] The acyl-tRNA synthetase according to any one of claims 60 to 67, wherein the amino acid sequence includes one or any combination of mutations at the positions corresponding to the following positions in SEQ ID NO: F295, L305, Y306, N307, L309, M344, N346, C348, T364, F366, K375, T387, V401, D414, and G421. [Claim 69] The amino acid sequence is L305C, L305L, L305N, L305S, or L305V; Y306D, Y306F, Y306I, Y306N, Y306R, Y306S, or Y306Y; L309D, L309H, L309I, L309L, L309R, or L309S; M344E, M344M, or M344 Q; C348C, C348F, C348I, C348L, C348T, or C348V; Y384Y; S399S; and one of V401A, V401C, V401K, V401L, V401S, V401T, V401V, any combination thereof, or all of them, according to claim 68. [Claim 70] The amino acid sequence is M300N or M300S; A302H; N346A, N346C, or N346G; and A C348 mutation that is arbitrarily C348I, C348L, C348T, or C348V. An acyl-tRNA synthetase according to any one of claims 60 to 69, comprising a residue corresponding to the following: [Claim 71] The amino acid sequence is M300N or M300S; A302H; L305L or L305V; Y306N or Y306Y L309I, L309L, or L309S; M344E, M344M, or M344Q; N346A, N346C, or N346G; C348I, C348L, C348T, or C348V; and V401C, V401K, V401L, or V401V An acyl-tRNA synthetase according to any one of claims 60 to 69, comprising a residue corresponding to the following: [Claim 72] The amino acid sequence is M300N or M300S; A302H; L305L or L305V; Y306N or Y306Y; L309I, L309L, or L309S; M344E, M344M, or M344Q; N346A, N346C, or N346G; C348I, C348L, C348T, or C348V; Y384Y; S399S; and V401C, V401K, V401L, or V401V An acyl-tRNA synthetase according to any one of claims 60 to 69, comprising a residue corresponding to the following: [Claim 73] The acyl-tRNA synthetase according to claim 60, wherein the amino acid sequence includes one of the sets of mutations in Table 8 at a position corresponding to the position in SEQ ID NO:

1. [Claim 74] The acyl-tRNA synthetase according to claim 60, wherein the amino acid sequence includes one of the mutant and wild-type residue sets in Table 8 at the position corresponding to the position in SEQ ID NO:

1. [Claim 75] The acyl-tRNA synthetase according to claim 60, wherein the amino acid sequence includes, at a position corresponding to the position in SEQ ID NO: 1, any of the sets of mutations associated with the identifier (e.g., A7_2) in Table 8. [Claim 76] The acyl-tRNA synthetase according to claim 60, wherein the amino acid sequence includes, at the position corresponding to the position in SEQ ID NO: 1, either a mutation associated with an identifier in Table 8 (e.g., A7_2) or a set of wild-type residues. [Claim 77] The amino acid sequence has one of the following sets of mutations at the position corresponding to the position in SEQ ID NO: a) N307K and F366L; b) F295I and T387I; c) G421A; d) T364A and T387I; e) T387S and D414V; or f) F295L and K375R An acyl-tRNA synthetase according to any one of claims 60 to 76, comprising: [Claim 78] The acyl-tRNA synthetase according to any one of claims 60 to 77, wherein the amino acid sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% similarity or identity with the amino acid sequence of SEQ ID NO:

1. [Claim 79] The acyl-tRNA synthetase according to any one of claims 60 to 78, wherein the amino acid sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% similarity or identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 18. [Claim 80] The acyl-tRNA synthetase according to any one of claims 60 to 79, wherein the amino acid sequence is the PylRS amino acid sequence. [Claim 81] Use of acyl-tRNA synthetase according to any one of claims 60 to 80 in a method for producing a polymer containing a beta-hydroxy acid. [Claim 82] A method for producing a polymer containing a beta-hydroxy acid, i) Use of acyl-tRNA synthetase for acylation of tRNA using beta-hydroxy acids, and ii) Incorporation of the beta-hydroxy acid into the polymer The method, including the method described above. [Claim 83] A method for producing a polymer containing a beta-hydroxy acid, i) Use of acyl-tRNA synthetase according to any one of claims 60 to 80 for acylation of tRNA using a beta-hydroxy acid, and ii) The method comprising incorporating the beta-hydroxy acid into a polymer chain. [Claim 84] Beta-hydroxy acids include (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S)β ３ The acyl-tRNA synthetase according to any one of claims 60 to 80, which is mClF, the use according to claim 81, or the method according to claim 82 or 83. [Claim 85] A nucleic acid encoding an acyl-tRNA synthetase according to any one of claims 60 to 80. [Claim 86] A host cell comprising an acyl-tRNA synthetase according to any one of claims 60 to 80 or a nucleic acid according to claim 85. [Claim 87] The host cell according to claim 86, wherein the acyl-tRNA synthetase is orthogonal to endogenous tRNA. [Claim 88] A host cell according to claim 86 or 87 that is alive.

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