Acyl-trna synthetases

Description

ACYL-tRNA SYNTHETASES FIELD OF THE INVENTION

[001] The invention relates to acyl-tRNA synthetases with altered activities. For example, beta-amino acid-tRNA synthetases, nucleic acids encoding said synthetases, cells expressing synthetases, and their uses and methods. The invention also relates to beta-hydroxy-tRNA synthetases, nucleic acids encoding said synthetases, cells expressing said synthetases, and their uses and methods. Furthermore, the invention relates to α,α-amino acid-acyltRNA synthetases, nucleic acids encoding said synthetases, cells expressing synthetases, and their uses and methods. The invention also relates to methods for producing polymers comprising the genetic incorporation of disubstituted α,α-amino acids. BACKGROUND OF THE INVENTION

[002] The genetic code of living cells has been reprogrammed to allow site-specific incorporation of hundreds of non-canonical amino acids (ncAAs) into proteins1,2, and the encoded synthesis of non-canonical polymers, macrocyclic peptides, and depsipeptides3-6. Despite remarkable progress, the monomers that can be site-specifically incorporated into cellular proteins are essentially limited to α-L amino acids with variant side chains and closely related hydroxy acids. Although a wider range of monomers has been incorporated in in vitro translation reactions7-10—primarily short peptides—these in vitro approaches cannot be extended to living cells. (S)e3-para Bromohomophenylalanine ((S)e3-p BrhF) was incorporated at very low levels in competition with phenylalanine (Phe) at Phe codons in E. coli, using forced Phe deprivation conditions11; this approach leads to a mixture of amino acids at all Phe codons and not Petition 870250088848, dated 09 / 30 / 2025, page 16 / 242 2 / 141 allows site-specific incorporation of (S)e3-p BrhF at a single position in response to a single codon, as needed to reprogram the genetic code.

[003] The encoded and site-specific incorporation of a non-canonical monomer (ncM) via cellular translation requires both the acylation of a tRNA orthogonal to the ncM by an orthogonal synthetase and the ribosomal polymerization of the ncM into a polymeric chain (Fig. 1). Current methods for modifying aminoacyl-tRNA synthetases that acylate new monomers rely on translational reads12'13e, therefore requiring the monomers to be ribosomal substrates for incorporation, often at specific sites on the proteins. As many ncMs of interest are poor ribosomal substrates10'11'14-18, this creates an evolutionary impasse in cells; An orthogonal synthetase cannot be developed to acylate an orthogonal tRNA with ncMs that are poor ribosomal substrates, and ribosomes cannot be developed to polymerize ncMs that cannot be acylated into orthogonal tRNAs.

[004] The inventors previously described tRNA extension (tREX), a rapid and scalable method for determining the aminoacylation status of user-defined tRNAs from cells19. In this approach, total tRNA is isolated from cells and the 2',3' diol on the ribose at the 3' end of unacylated tRNAs is selectively oxidized to a dialdehyde, while acylated tRNAs are protected from diol oxidation. A DNA probe containing a fluorophore is then annealed to the 3' end of the tRNA of interest, under conditions that facilitate deacylation of acylated tRNAs, to reveal the free diol at their 3' ends. This allows polymerase-mediated extension of unoxidized (that have been acylated) tRNAs. The resulting mass difference between oxidized, unextended tRNAs and unoxidized, extended tRNAs is resolved by gel electrophoresis, allowing for distinction. Petition 870250088848, dated 09 / 30 / 2025, page 17 / 242 3 / 141 between acylated and free tRNAs.

[005] Another earlier method is described in Saito et al. (EMBO Journal, Vol. 20, No. 7, pp. 1797-1806, 2001). This method is based on a biotinylated substrate and is performed in vitro. SUMMARY OF THE INVENTION

[006] In one aspect, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: M300A, M300C, M300D, M300M or M300S in relation to SEQ ID NO: 1; A302A, A302C, A302D, A302G, A302H, A302L, A302N or A302Y in relation to SEQ ID NO: 1; and N346A, N346C, N346G, N346S, N346T or N346V in relation to SEQ ID NO: 1.

[007] In one embodiment, the acyl-tRNA synthetase comprises an M300D mutation relative to SEQ ID NO: 1, an A302H, A302Y or A302C mutation relative to SEQ ID NO: 1 and an N346G, N346A or N346S mutation relative to SEQ ID NO: 1.

[008] In a second aspect, the use of an acyl-tRNA synthetase is provided in a method for generating a polymer comprising a beta amino acid. In one embodiment, the use of an acyl-tRNA synthetase, as described herein, is provided in a method for generating a polymer comprising a beta amino acid.

[009] A method is also provided for preparing a polymer comprising a beta amino acid, wherein the method comprises: i) using an acyl-tRNA synthetase, as described herein, to acylate a tRNA with the beta amino acid, and ii) incorporating the beta amino acid into a polymer chain.

[0010] In a third aspect, a nucleic acid is provided that Petition 870250088848, dated 09 / 30 / 2025, page 18 / 242 4 / 141 encodes a first-aspect acyl-tRNA synthetase.

[0011] In a fourth aspect, a cell comprising an acyl-tRNA synthetase from the first aspect, a nucleic acid from the third aspect, or a vector comprising a nucleic acid from the third aspect is provided.

[0012] In a fifth aspect, an acyl-tRNA synthetase is provided that is capable of specific acylation of a tRNA with a disubstituted α,α-amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: A302C, A302G, A302H or A302S with respect to SEQ ID NO: 1; and N346A, N346C, N346E, N346G, N346T or N346V with respect to SEQ ID NO: 1.

[0013] In a sixth aspect, the use of an acyl-tRNA synthetase is provided in a method for generating a polymer comprising a disubstituted α,α-amino acid.

[0014] A method is also provided for producing a polymer comprising a disubstituted α,α-amino acid, wherein the method comprises: i) using an acyl-tRNA synthetase, as described in this document, to acylate a tRNA containing the disubstituted α,α-amino acid and ii) incorporating the disubstituted α,α-amino acid into a polymer chain.

[0015] In a seventh aspect, a nucleic acid encoding an acyl-tRNA synthetase from the fifth aspect is provided.

[0016] In an eighth aspect, a cell comprising an acyl-tRNA synthetase from the fifth aspect, a nucleic acid from the seventh aspect, or a vector comprising a nucleic acid from the seventh aspect is provided.

[0017] In a ninth aspect, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence. Petition 870250088848, dated 09 / 30 / 2025, page 19 / 242 5 / 141 acids comprising residues corresponding to: M300A, M300D, M300M, M300N or M300S in relation to SEQ ID NO: 1; and A302D, A302G, A302H or A302N in relation to SEQ ID NO: 1.

[0018] In a tenth aspect, the use of an acyltRNA synthetase in a method for generating a polymer comprising a beta-hydroxy acid is provided.

[0019] A method is also provided for producing a polymer comprising a beta-hydroxy acid, wherein the method comprises: i) using an acyl-tRNA synthetase, as described herein, to acylate a tRNA with the beta-hydroxy acid, and ii) incorporating the beta-hydroxy acid into a polymer chain.

[0020] In an eleventh aspect, a nucleic acid is provided that encodes an acyl-tRNA synthetase from the ninth aspect.

[0021] In a twelfth aspect, a cell comprising an acyl-tRNA synthetase of the ninth aspect, a nucleic acid of the eleventh aspect, or a vector comprising a nucleic acid of the eleventh aspect is provided.

[0022] The use of an acyl-tRNA synthetase, as described herein, in a method of genetically incorporating a monomer into a polymer is also provided.

[0023] A method for producing a polymer is also provided, wherein the method comprises: i) using an acyl-tRNA synthetase, as described in this document, to acylate a tRNA with a monomer and ii) incorporating the monomer into a polymer chain. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Fig. 1: Cellular-encoded incorporation of non-canonical monomers into proteins and non-canonical polymers requires both tRNA acylation and ribosomal polymerization. Petition 870250088848, dated 09 / 30 / 2025, page 20 / 242 6 / 141 mica. The encoded, site-specific incorporation of a non-canonical monomer (ncM, yellow star) via cellular translation requires both the acylation of a tRNA orthogonal to the ncM by an orthogonal synthetase and the ribosomal polymerization of the ncM into a polymer chain. Current methods for modifying aminoacyl-tRNA synthetases that acylate new monomers rely on translational reads and therefore require the monomers to be ribosomal substrates. For ncMs that are poor ribosomal substrates, this codependency creates an evolutionary impasse in cells; an orthogonal synthetase cannot be developed to acylate a tRNA orthogonal to ncMs that are poor ribosomal substrates, and ribosomes cannot be developed to polymerize ncMs that cannot be acylated into orthogonal tRNAs.To overcome this impasse, we developed direct selections for orthogonal synthetases to aminoacylate their orthogonal tRNAs cognate with ncMs, regardless of whether the ncMs are ribosomal substrates. Fig. 2. The tRNA display allows direct selection of orthogonal aminoacyltRNA synthetases that aminoacylate their orthogonal tRNAs cognate with ncAAs.

[0025] a, Schematic representation of tRNA display, a translation-independent strategy for selecting aaRS enzymes that aminoacylate a specific tRNA with a desired monomer. In tRNA display, a library of stmRNAs is transformed into cells and cultured in the presence and absence of non-canonical monomers of interest (represented by the yellow star) in multiple replicates. The library contains PylRS variants that are active and selective for the ncM (yellow), PylRS variants that are not active with the ncMs or canonical amino acids in the cell (light blue), and PylRS variants that are active with one or more canonical amino acids in the cell and are not selective for the ncM (dark blue). Bio-mREX Petition 870250088848, dated 09 / 30 / 2025, page 21 / 242 7 / 141 is performed for each replicate and the cDNA is subjected to next-generation sequencing (NGS). The results are analyzed by plotting the selectivity (the ratio of the relative abundance of a specific sequence in the positive samples (+ncM) divided by the relative abundance in the negative sample (-ncM)) against the enrichment (the ratio of the same sequence in the positive samples divided by the relative abundance in the input library) of all observed sequences; this results in a spindle-shaped plot (hereinafter referred to as a spindle plot). Active and selective PylRS variants are expected to be highly selective and enriched (yellow dot - upper right quadrant), active but non-selective variants are expected to be non-selective but highly enriched (dark blue dot), and inactive PylRS variants are expected to be non-selective and not enriched (light blue dot). b, The structures of non-canonical α-alpha amino acids used in this study.IN ((benzyloxy)carbonyl)-L-lysine (CbzK) (2), N6-((prop-2-yn-1-yloxy)carbonyl)L-lysine (AlkyneK) (3), N6-benzoyl -L-lysine (BenzK) (4), 3-([2,2'bipyridin]-5-yl)-2-aminopropanoic acid (BiPyA) (5), Ντ-methyl -L-histidine (NTmH) (6), (S)-2-amino-3-(thiophen-3-yl)propanoic acid (3-ThiA) (7)), (S)-2-amino-3-(pyridin-3-yl)propanoic acid (PyA) (8), (S)-2amino-3-(4-iodophenyl)propanoic acid (pIF) (9), Acid (S)-2-amino-3-(4-bromothiophen-2-yl)propanoic acid (BrThiA) (10), (2S)-2-amino-3-(((2((1-(6-nitrobenzo[d][1,3]dioxol5yl)ethyl)thio)ethoxy)carbonyl)amino)propanoic acid (pcDAP) (11). c, tRNA display with PylRS library (stmRNAvol2-lib1), this library mutates at three positions for all other canonical amino acids (Y306X, L309X and N346X). The spindle plot shows the result of tRNA display selection against CbzK (2) using a parallel selection step, as described in (a).The samples were run in triplicate and the data processed as described in Petition 870250088848, dated 09 / 30 / 2025, page 22 / 242. 8 / 141 all. Red dots indicate 65 clones that were subsequently characterized. d, ln(Enrichment, +2) of tRNA-derived PylRS mutants display (red dots in (c)) vs. GFP fluorescence measured in cells containing the corresponding PylRS / tRNAPylCUA mutant pair, GFP(150TAG)hís6 and CbzK (2). The dashed line represents the linear regression for the displayed data points; R-squared = 0.6611, p < 0.0001. ej, (left) white bar: GFP fluorescence from cells containing GFP(150TAG)hís6, the PylRS / tRNAPylCUA variant pair indicated and the ncAA indicated. Gray bar: the wtPylRS / tRNAPylCUA pair with the same ncAA. Fluorescence is shown as a fraction of the fluorescence generated by the wt PylRS / tRNAPylCUA pair with 2 mM BocK (1) and GFP(150TAG)hís6. (right) ESI-MS of GFP(150X)hís6, where X is the indicated ncAA.f, mass found: 27922.0 Da, expected mass 27923.3 Da; g, mass found: 27944.8 Da, expected mass 27945.5 Da; h, mass found: 27867.6 Da, expected mass 27866.4 Da; i, mass found:. 27862.0 Da, expected mass; 27861.4 Da; j, mass found: 27986.4 Da, expected mass; 27986.2 Da; k, mass found: 27945.6 Da, expected mass: 27944.3 Da. Fig. 3. The tRNA display allows the selection of orthogonal aminoacyl-tRNA synthetases that carry non-canonical monomers.

[0026] a, Structures of the non-canonical monomers used in this study. (S)-3-amino-3-(3-bromophenyl)propanoic acid ((S^mBrF) (12), (S)-3-amino-6-(((benzyloxy)carbonyl)amino)hexanoic acid ((S)β3CbzK) (13), (S)-6-acetamido-3-aminohexanoic acid ((Sj^AcK) (14), BocAhx (15), acid 6-(((benzyloxy)carbonyl)amino)hexanoic acid (CbzAhx) (16), 3-amino-2-((1-ethyl-1H-imidazol-5yl)methyl)propanoic acid ^2NeH (17)), 3-amino-4-(4bromophenyl)butanoic acid ^3pBrhF) (18), 2-benzyl-3hydroxypropanoic acid ^2OH-F) (19), acid 3-amino-3-phenylpropanoic acid ^3F) Petition 870250088848, dated 09 / 30 / 2025, page 23 / 242 9 / 141 (20). b, Fluoro-tREX for the indicated PylRS variants. The experiments were performed in triplicate, using tRNAs extracted from cells containing a pMB1 plasmid encoding each PylRS variant and TRNAPylcuA in the presence and absence of 4 mM of 15. cd, Fluoro-tREX for the indicated PylRS variants from primary selection (panel c) and after further evolution (panel d). The experiments were performed in triplicate, using tRNA extracted from cells containing a pMB1 plasmid encoding each variant of PylRS and RNAPylCUA in the presence and absence of 4 mM of 12. Selected variants of PylRS acylate tRNAPylCUA with 12. Traces of LC-MS (ion scanning mode in the AQC adduct of substrate 12) in AQC-derivative eluates of tRNA pulldowns from cells expressing the indicated PylRS variants.Cells harboring a pMB1 plasmid encoding the corresponding PylRS variant and tRNAPylcua (or just tRNA (-) as a control) were cultured in the presence of substrate 12, and tRNA pulldowns were performed using a biotinylated probe against tRNAPylcuA. f, Quantification of the relative acylation of tRNAPylCUA by selected PylRS variants. The integrated area under the peak for LC-MS traces shown in, e.g., GFP fluorescence from cells containing GFP(150TAG)hís6, PylRS(12_1) or PylRS(12_1evQl1) and tRNAPylCUA, and cultured in the presence or absence of substrate 12. Fluorescence is shown as a fraction of the fluorescence generated by the PylRS / tRNAPylcuA pair wt with 2 mM BocK (1) and GFP(150TAG)hís6. h, intact ESI-MS GFP(150(S)β3mBrF)His6 purified from cells containing PylRS(12_1evQl1), tRNAPylcUA and GFP(150TAG)His6 cultured in the presence of 4 mM of 12. Mass found: 27,939.0 Da, predicted mass: 27,938.2 Da.i, Close-up of residue 150 of GFP(150(S)β3mBrF)His6 from a crystal structure determined at 1.5 Å. The 2Fo-Fc map is shown at the sigma = 2 boundary level (PDB code 8OVY). The electron density (blue) confirms. Petition 870250088848, dated 09 / 30 / 2025, page 24 / 242 10 / 141 the incorporation of 12 at position 150, clearly demonstrating the extension of the peptide structure by a methylene group and the stereochemistry of the β-amino acid in the protein.

[0027] Fig. 4. After oxidation, deacylation of tRNAs under alkaline conditions increases the acylation signal in fluro-tREX and therefore allows robust detection of acylation by hydroxy acids and carboxylic acids. a, Chemical structure of N6-(tert-butoxycarbonyl)-L-lysine (BocK) 1, (S)-6-((tert-butoxycarbonyl)amino)-2-hydroxyhexanoic acid (OH-BocK) 21, as well as 6-((tert-butoxycarbonyl)amino)hexanoic acid (BocAhx) 15. Mm PylRS is highly active with BocK 1, OH-BocK121 and shows acylation activity with BocAhx 152. b, The free acid of 1, 21 and 15 spans a range of the estimated pKas. The rate constant for the alkaline hydrolysis of esters, to produce a fixed alcohol and a variable carboxylic acid, increases as the pKa of the resulting carboxylic acid decreases.Therefore, we expect the hydrolysis rate of acylated tRNAs to be slower when the acylating monomers are α-hydroxy acids, simple carboxylic acids (and β-amino acids) than when the acylating monomers are α-amino acids. c, Mm PylRS acylates Mm tRNAPyl in cells with BocK 1, OH-BocK 21, and BocAhx 15, respectively. Northern blot of MmtRNAPyl from tRNAs isolated from cells containing a pMB1 plasmid encoding the Mm PylRS / Mm tRNAPyl pair in the presence and absence of BocK, OH-BocK, or BocAhx. The experiments were performed in three biological replicates, yielding similar results. d, After oxidation, a deacylation step under alkaline conditions is required to robustly detect the acylation activity of MmPylRS by fluoro-tREX with non-alpha amino acid substrates. Fluoro-tREX was performed with the tRNA samples described in panel c, with and without incubation of the tRNAs for 45 minutes with 50 mM bicin at pH 9.6 after oxidation. The OH-BocK acylation signals... Petition 870250088848, dated 09 / 30 / 2025, page 25 / 242 The results of the 11 / 141 experiment, as well as those of BocAhx, were dependent on the deacylation of tRNAs prior to the Klenow extension (exo) step of the protocol. The experiments were performed on three biological replicates, producing similar results.

[0028] Fig. 5. Relationship between the acylation signal measured, by bio-mREX, for stmRNAs and the GFP fluorescence signal measured for intact and translationally competent tRNAs. For stmRNAs, active aminoacyl-tRNA synthetases (aaRS) lead to the acylation of their coding stmRNAs, which, by bio-mREX, are extended, separated, and finally reverse transcribed. This results in the cDNA of the active synthetase, which can be quantified by qPCR. In the case of an inactive aaRS, the stmRNAs are not acylated and no cDNA is produced in bio-mREX experiments. Therefore, the activity of a synthetase in bio-mREX correlates with the number of cDNA molecules measured by qPCR. In canonical translation, an active aaRS enzyme leads to a cognate, intact, and acylated tRNAoUA, which is used in protein translation. Inactive aaRS enzymes lead to unacylated tRNAs, which are not used in protein translation.The production of the GFP protein from GFP150TAGhí6, measured by GFP fluorescence, reports the acylation of tRNAoUA, as well as the other steps in protein production.

[0029] Fig. 6. PylRS libraries used in this work. a, Overview of the seven libraries designed and created. These libraries target a total of 11 amino acid residues in the PylRS active site and employ several types of degenerate codons. NNK codons are represented in dark red, DBK codons (+ lysine codon) in blue, NDT codons in dark green, and NRT codons in yellow. For certain sites, custom residue mixtures covering the most commonly observed mutations were used (mixtures 1 to 7, represented as gray spheres). Petition 870250088848, dated 09 / 30 / 2025, page 26 / 242 12 / 141 All libraries were created with 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 mixtures are described in the methods. b, The eleven amino acid residues targeted for mutagenesis in the PylRS active site are shown in red. The image was rendered using Pymol, based on the 2ZIN structure from the PDB.

[0030] Fig. 7. tRNA display identifies active and selective orthogonal variants of aaRS from an stmRNA library. a, Spindle plot from tRNA display selection using stmRNA library 1 and ncAA 1. b, Identifying the spindle plot region enriched in active and selective clones. We expect the upper right quadrant of the spindle plot to be enriched in active and selective clones. Since selectivity is derived from the ratio between +ncAA and -ncAA sequence counts, clones enriched with negative selectivity values ​​would correspond to the specific enrichment of a clone in the -ncAA condition relative to the +ncAA condition. We postulate that most apparent enrichments of this type were spurious and therefore that the spindle plot regions where positive selectivity values ​​were mirrored by negative selectivity values ​​of the same magnitude may contain substantial noise.Based on this postulate, we expected that active and selective ncAA clones would be most enriched in the region of the graph where, for a given positive enrichment value, selectivity becomes asymmetrical. To enrich this asymmetrical population, we grouped the average selectivity values: 5,350 points on the spindle graph (Fig. 2c) were divided into 500 equal compartments along the enrichment+1 dimension (163 compartments contained data). The average selectivity of each compartment was plotted against the natural logarithm of enrichment+1. From this, a threshold of approximately 7.4 (corresponding to a logarithmic score value) was obtained. Petition 870250088848, dated 09 / 30 / 2025, p. 27 / 242 13 / 141 rhythmic of 2) was used to define the enrichment value at which the spindle plot is asymmetric along the selectivity axis. c, Experimental fluorescence values ​​of GFP for 100 clones plotted against the natural logarithm of the enrichment score in the presence of 1. GFP was expressed from GFP150TAGhís6 in the presence of the variant clone Mm PylRS, the cognate Mm tRNAPylCUA and ncAA (1). Points above the symmetry threshold are colored red (65 points), points below are colored blue (21 points). There is a strong and significant positive correlation between the tRNA display sequence data and the experimental expression data for the red dots (R-squared value = 0.6611, p-value < 0.0001), but no significant correlation for the blue dots (R2 value = 0.0392, p-value = 0.397), which is consistent with the postulate. The red dots are also shown in Fig. 2c.In subsequent selections, based on this analysis, we focused primarily on identifying clones on the right side of the spindle plot where, for a given enrichment value, the magnitude of the positive selectivity value for a clone is greater than the negative selectivity values ​​for clones with the same enrichment value. In panel d, the tRNA display identifies PylRS variants specific to ncAA. The graph shows experimental selectivity versus spindle plot selectivity for the clones shown in panel b, color-coded as in panel b. Experimental selectivity is derived from GFP expression experiments, as in panel b, but + / - ncAA.

[0031] Fig. 8. Schematic representation of the tRNA display-based strategy for selecting PylRS variants that direct the incorporation of ncAAs into proteins. In the first cycle, the 2, 13, 14, 3D, 4D, and 5D libraries of naive stmRNAvol2 were transformed into BL21 cells and cultured overnight. The 3D and 4D libraries were combined to generate 3D4D libraries. The five libraries Petition 870250088848, dated 09 / 30 / 2025, page 28 / 242 14 / 141 thecae were cultured to an OD600 of 0.3-0.4, and 2.6 mL of cell culture from each library was added to a stock solution of each ncAA; this resulted in 50 samples (five libraries x ten ncAAs). Cells were cultured for 40 min, stmRNAs were induced, and cells were cultured for a further 20 min. Bio-mREX was performed on the isolated RNA for each of the 50 samples. For each reaction, cDNA was amplified with primers suitable for Golden Gate assembly. Then, all amplicons from the selected libraries for the same ncAA were combined in equimolar ratios (resulting in ten combined libraries in total) and cloned into a new ColE1 vector main strand. This created ten pre-selected libraries. The ten pre-selected libraries were transformed into BL21 cells and cultured overnight. The pre-selected libraries for ncAAs 2 through 6 were combined to create a single cluster library.Similarly, the pre-selected libraries for ncAAs 7 to 11 were combined to create a second cluster library. 2.6 mL of the first cluster library were added to solutions of ncAAs 2 to 6. 2.6 mL of the first cluster library were also added to a sample without ncAAs, as a control. Cells were cultured for 40 min, stmRNAs were induced, and cells were cultured for a further 20 min, and RNA was isolated. Three RNA samples were converted to cDNA as input controls for bio-mREX. Bio-mREX was performed on the isolated RNA. This generated seven samples (bio-mREX input, control -ncAA for bio-mREX, and five bio-mREX samples for ncAAs 2 to 6). The experiment was performed in triplicate, generating 21 samples. The cDNA of each sample was sequenced by NGS and analyzed to generate spindle plots and sequence tables. The second cluster was treated analogously to the first cluster, using ncAAs 7 to 11 instead of 2 to 6. Petition 870250088848, dated 09 / 30 / 2025, p. 29 / 242 15 / 141

[0032] Fig. 9. Selection of PylRS variants for CbzK2 by tRNA display. a, Chemical structure of CbzK2. b, Spindle plot resulting from tRNA display selection, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+2) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in 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 the positive samples.c, 25 main sequences ordered by selectivity resulting from selection for substrate 2 with selectivities > 5 and enrichments > 5.

[0033] Fig. 10. Selection of PylRS variants for N6-((prop-2-yn-1-yloxy)carbonyl)-L-lysine (AlkyneK)3 by tRNA visualization. a, Chemical structure of AlkyneK3. b, Spindle diagram resulting from tRNA visualization selection, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+3) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only Petition 870250088848, dated 09 / 30 / 2025, p. 30 / 242 16 / 141 in the positive samples. c, Top 25 sequences, ordered by selectivity, resulting from selection for substrate 3 with selectivities > 5 and enrichments > 5. d, Amber suppressor activity data for selected PylRS variants, measured by the production of GFP150AlkyneKHis6 from GFP150TAGhis6 from cells containing a pMB1 plasmid encoding wild-type (wt) MmPylRS or the indicated MmPylRS variants, and a p15A plasmid encoding GFP150TAGhis6 in the presence and absence of 4 mM 3. Fluorescence is shown relative to cells harboring wt MmPylRS, Mm tRNAPylcuA expressing GFP from GFP150TAGhis6 in the presence of 2 mM substrate 1. e, Crude mass spectrum for purified GFP150AlkyneKHis6 produced with PylRS 3_1. f, Deconvolved mass spectrum resulting from the primary spectrum shown in panel e. Predicted mass: 27,923.3 Da, found mass: 27,922.0 Da. The smaller peak, labeled with -met, corresponds to the cleavage of the N-terminal methionine residue.gh, same as eef, except for GFP150AlkyneKHis6 produced with PylRS 3_2. Predicted mass: 27.923.3 Da, found mass: 27.923.6 Da. ij, same as eef, except for GFP150AlkyneKHis6 produced with PylRS 3_3. Predicted mass: 27.923.3 Da, found mass: 27.923.2 Da.

[0034] Fig. 11. Selection of PylRS variants for N-benzoyl-Llysine (BenzK) 4 by tRNA display. a, Chemical structure of BenzK 4. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+ 4) divided by the relative abundance in the control sample (ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are ob sequences. Petition 870250088848, dated 09 / 30 / 2025, p. 31 / 242 17 / 141 observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples. c, 25 main sequences, ordered by selectivity, resulting from selection for substrate 4 with selectivities > 5 and enrichments > 5. d, Amber suppressor activity data for selected PylRS variants, measured by GFP150BenzKHis6 production from GFP150TAG hís6 in cells harboring a pMB1 plasmid encoding wild-type (wt) Mm PylRS or the indicated Mm PylRS variants, and a p15A plasmid encoding GFP150TAG hís6 in the presence and absence of 4 mM 4. Fluorescence is shown relative to cells harboring Mm PylRS wt, Mm tRNAPylcuA expressing GFP from GFP150TAG hís6 in the presence of 2 mM substrate 1. and, raw mass spectrum for purified GFP150BenzKHis6 produced with PylRS 4_1.f, Deconvolved mass spectrum resulting from the primary spectrum shown in panel e. Predicted mass 27945.5 Da, found mass 27944.8 Da. The smaller peak labeled -met corresponds to the cleavage of the N-terminal methionine residue. gh, same as eef, but for GFP150BenzKHis6 produced with PylRS 4_2. Predicted mass 27945.5 Da, found mass 27944.8 Da. ij, same as eef, but for GFP150BenzKHis6 produced with PylRS 4_3. Predicted mass: 27945.5 Da, found mass: 27944.4 Da. kl, same as eef, except for GFP150BenzKHis6 produced with PylRS 4_4. Predicted mass: 27945.5 Da, found mass: 27944.4 Da.

[0035] Fig. 12. Selection of PylRS variants for 3-([2,2'-bipyridin]-5-yl)-2-aminopropanoic acid (BiPyA) 5 by tRNA display. a, Chemical structure of BiPyA 5. b, Spindle diagram resulting from selection by tRNA display, as described in Fig. 8. Selectivity is defined as the ratio between relative abundance Petition 870250088848, dated 09 / 30 / 2025, p. 32 / 242 18 / 141 of a specific sequence in the positive samples (+ 5), divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples, divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples. c, 25 main sequences, ordered by selectivity, resulting from selection for substrate 5 with selectivities > 5 and enrichments > 5.d, Data on amber suppressor activity for selected PylRS variants, measured by the production of GFP150BiPyA hís6 from GFP150TAGhís6 from cells containing a pMB1 plasmid encoding wild-type (wt) Mm PylRS or the indicated Mm PylRS variants, and a p15A plasmid encoding GFP150TAGhís6 in the presence and absence of 4 mM of 5. Fluorescence is shown relative to cells containing MmPylRS wt, MmtRNAPylcuA expressing GFP from GFP150TAGhís6 in the presence of 2 mM of substrate 1.

[0036] Fig. 13. Selection of PylRS variants for NT-methyl-Lhistidine (NTmH) 6 by tRNA display. a, Chemical structure of NTmH 6. b, Spindle diagram resulting from tRNA display selection as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+ 6) divided by the relative abundance in the control sample (ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are ob sequences. Petition 870250088848, dated 09 / 30 / 2025, page 33 / 242 19 / 141 observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples. c, The 16 sequences, ordered by selectivity, resulting from selection for substrate 6 with selectivities > 5 and enrichments > 5. d, Amber suppressor activity data for selected PylRS variants, measured by the production of GFP150NTmHHis6 from GFP150TAGhís6 from cells containing a pMB1 plasmid encoding wild-type (wt) Mm PylRS or the indicated Mm PylRS variants, and a p15A plasmid encoding GFP150TAGhís6 in the presence and absence of 4 mM of 6. Fluorescence is shown relative to cells containing Mm PylRS wt, Mm tRNAPylcuA expressing GFP from GFP150TAGhís6 in the presence of 2 mM of substrate 1.

[0037] Fig. 14. Selection of PylRS variants for (S)-2-amino-3-(thiophen-3-yl)propanoic acid (3-ThiA) 7 by tRNA display. a, Chemical structure of 3-ThiA 7. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+7) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, 25 main sequences, ordered by selectivity, resulting from selection for substrate 7 with selectivities >. Petition 870250088848, dated 09 / 30 / 2025, p. 34 / 242 20 / 141 and enrichments > 5. d, 7. Fluorescence is shown relative to cells harboring Mm PylRS wt, Mm tRNAPylcuA expressing GFP from GFP150TAGhís6 in the presence of 2 mM of substrate 1. e, Crude mass spectrum for purified GFP150-3-ThiA Khís6 produced with PylRS 7_1. f, Deconvolved mass spectrum resulting from the primary spectrum shown in panel e. Predicted mass 27866.4 Da, found mass 27867.2 Da. The smaller peak labeled -met corresponds to the cleavage of the N-terminal methionine residue. gh, same as eef, but for GFP150-3-ThiAHis6 produced with PylRS 7_2. Predicted mass 27866.4 Da, found mass 27866.8 Da. ij, same as eef, but for GFP150-3-ThiAHis6 produced with PylRS 7_3. Predicted mass 27866.4 Da, found mass 27866.8 Da. kl, same as eef, but for GFP150-3-ThiAHis6 produced with PylRS 7_4. Predicted mass 27866.4 Da, found mass 27866.4 Da. mn, same as eef, but for GFP150-3-ThiAHis6 produced with PylRS 7_5.Predicted mass 27866.4 Da, found mass 27867.6 Da. op, same as eef, but for GFP150-3-ThiAHis6 produced with PylRS 7_6. Predicted mass 27866.4 Da, found mass 27867.6 Da. qr, same as eef, but for GFP150-3-ThiAHis6 produced with PylRS 7_7. Predicted mass: 27866.4 Da, found mass: 27867.6 Da. st, same as eef, but for GFP150-3-ThiA His6 produced with PylRS 7_8. Predicted mass: 27866.4 Da, found mass: 27868.0 Da.

[0038] Fig. 15. Selection of PylRS variants for (S)-2amino-3-(pyridin-3-yl)propanoic acid (PyA) 8 by means of tRNA display. a, Chemical structure of PyA 8. b, Spindle diagram resulting from the selection of tRNA display, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+ 8), divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples, divided by Petition 870250088848, dated 09 / 30 / 2025, page 35 / 242 21 / 141 relative abundance in the input library. The delimited region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples. c, The five sequences, ordered by selectivity, resulting from selection for substrate 8 with selectivities of > 5 and enrichments of > 5. d, Amber suppressor activity data for selected PylRS variants measured by the production of GFP150PyAHis6 from GFP150TAGhís6 from cells harboring a pMB1 plasmid encoding wild-type (wt) MmPylRS or the indicated MmPylRS variants, and a p15A plasmid encoding GFP150TAGhís6 in the presence and absence of 8 4 mM.Fluorescence is shown relative to cells harboring Mm PylRS wt, Mm tRNAPylcuA expressing GFP from GFP150TAGhis6 in the presence of 2 mM substrate 1. e, Crude mass spectrum for purified GFP150PyAHis6 produced with PylRS 8_1. f, Deconvolved mass spectrum resulting from the primary spectrum shown in panel e. Predicted mass: 27,861.4 Da, found mass: 27,860.4 Da. The smaller peak, labeled -met, corresponds to the cleavage of the N-terminal methionine residue. gh, the same as eef, except for GFP150PyAHis6 produced with PylRS 8_2. Predicted mass: 27,861.4 Da, found mass: 27,862.0 Da. ij, same as eef, except for GFP150PyAHis6 produced with PylRS 8_3. Predicted mass: 27,861.4 Da, found mass: 27,859.6 Da. kl, same as eef, except for GFP150PyAHis6 produced with PylRS 8_4. Predicted mass: 27,861.4 Da, found mass: 27,860.8 Da.

[0039] Fig. 16. Selection of PylRS variants for (S)-2amino-3-(4-iodophenyl)propanoic acid (pIF) 9 by tRNA display. a, Structure Petition 870250088848, dated 09 / 30 / 2025, p. 36 / 242 22 / 141 Chemical structure of pIF 9. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+ 9) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples. c, 25 main sequences, ordered by selectivity, resulting from selection for substrate 9 with selectivities > 5 and enrichments > 5.d, Amber suppressor activity data for selected PylRS variants measured by producing GFP150PIFhís6 from GFP150TAGhís6 from cells harboring a pMB1 plasmid encoding wild-type (wt) MmPylRS or the indicated MmPylRS variants, and a p15A plasmid encoding GFP150TAGhís6 in the presence and absence of 9 4 mM. Fluorescence is shown relative to cells harboring MmPylRS wt., Mm tRNAPylcuA expressing GFP from GFP150TAGhís6 in the presence of 2 mM of substrate 1. e, Crude mass spectrum for purified GFP150pIFKHis6 produced with PylRS 9_1. f, Deconvolved mass spectrum resulting from the primary spectrum shown in panel e. Predicted mass 27986.2 Da, found mass 27987.6 Da. The smaller peak marked met corresponds to the cleavage of the N-terminal methionine residue. gh, the same as eef, but for GFP150pIFKHis6 produced with PylRS 9_2. Predicted mass 27986.2 Da, found mass 27986.0 Da.ij, the same as eef, but for GFP150pIFKHis6 produced with PylRS. Petition 870250088848, dated 09 / 30 / 2025, p. 37 / 242 23 / 141 9_3. Predicted mass: 27986.2 Da, found mass: 27986.4 Da. kl, same as eef, but for GFP150pIFKHis6 produced with PylRS 9_4. Predicted mass: 27986.2 Da, found mass: 27986.4 Da. mn, same as eef, but for GFP150pIFKHis6 produced with PylRS 9_5. Predicted mass: 27986.2 Da, found mass: 27986.4 Da. op, same as eef, but for GFP150pIFKHis6 produced with PylRS 9_6. Predicted mass: 27986.2 Da, found mass: 27987.2 Da.

[0040] Fig. 17. Selection of PylRS variants for (S)-2amino-3-(4-bromothiophen-2-yl)propanoic acid (BrThiA) 10 by tRNA display. a, Chemical structure of BrThiA 10. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+10) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, 25 main sequences, ordered by selectivity, resulting from selection for substrate 10 with selectivities of > 5 and enrichments of > 5. d, Amber suppressor activity data for selected PylRS variants measured by the production of GFP150BrThiA hís6 from GFP150TAG hís6 from cells harboring a pMB1 plasmid encoding wild-type (wt) Mm PylRS or the indicated Mm PylRS variants, and a p15A plasmid encoding GFP150TAG hís6 in the presence and absence of 4 mM of 10. Fluorescence is shown relative to. Petition 870250088848, dated 09 / 30 / 2025, page 38 / 242 24 / 141 cells harboring Mm PylRS wt, Mm tRNAPylcuA expressing GFP from GFP150TAG hís6 in the presence of 2 mM substrate 1. e, Crude mass spectrum for purified GFP150BrThiAHis6 produced with PylRS 10_1. f, Deconvolved mass spectrum resulting from the primary spectrum shown in panel e. Predicted mass 27944.3 Da, found mass 27945.6 Da. The smaller peak labeled -met corresponds to the cleavage of the N-terminal methionine residue. gh, same as eef, but for GFP150BrThiAHis6 produced with PylRS 10_2. Predicted mass 27944.3 Da, found mass 27945.2 Da. ij, same as eef, but for GFP150BrThiAHis6 produced with PylRS 10_3. Predicted mass 27944.3 Da, found mass 27945.6 Da. kl, same as eef, but for GFP150BrThiAHis6 produced with PylRS 10_4. Predicted mass: 27944.3 Da, found mass: 27945.2 Da. mn, same as eef, except for GFP150BrThiAHis6 produced with PylRS 10_5. Predicted mass: 27944.3 Da, found mass: 27944.4 Da.op, equal to aeef, except for GFP150BrThiAHis6 produced with PylRS 10_6. Predicted mass: 27944.3 Da, mass found: 27944.8 Da.

[0041] Fig. 18. Selection of PylRS variants for (2S)-2amino-3-(((2-((1-(6-nitrobenzo[d][1,3]dioxol-5yl)ethyl)thio)ethoxy)carbonyl)amino)propanoic acid (pcDAP) 11 by tRNA visualization. a, Chemical structure of pcDAP 11. b, Spindle diagram resulting from the tRNA display selection, as described in Fig. 8. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+11) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots Petition 870250088848, dated 09 / 30 / 2025, page 39 / 242 25 / 141 are sequences observed in all positive samples and in at least one control sample. The red dots are sequences observed only in the positive samples. c, The top 25 sequences, ordered by selectivity, resulting from selection for substrate 11 with selectivities > 5 and enrichments > 5. d, Amber suppressor activity data for selected PylRS variants measured by producing GFP150pcDAPHis6 from GFP150TAGhis6 from cells harboring a pMB1 plasmid encoding wild-type (wt) Mm PylRS or the indicated Mm PylRS variants, and a p15A plasmid encoding GFP150TAGhis6 in the presence and absence of 4 mM of 11. Fluorescence is shown relative to cells harboring Mm PylRS wt, Mm tRNAPylcuA expressing GFP from GFP150TAGhis6 in the presence of 2 mM of substrate 1. e, Crude mass spectrum for purified GFP150pcDAPHis6 produced with PylRS 11_1.f, Deconvolved mass spectrum resulting from the primary spectrum shown in panel e. Predicted mass of 28,096.4 Da, found mass of 28,093.2 Da. The smaller peak, labeled -met, corresponds to the cleavage of the N-terminal methionine residue. gh, same as eef, except for GFP150pcDAPHis6 produced with PylRS 11_2. Predicted mass of 28,096.4 Da, found mass of 28,096.4 Da. ij, same as eef, except for GFP150pcDAPHis6 produced with PylRS 11_3. Predicted mass of 28,096.4 Da, found mass of 28,097.2 Da. kl, same as eef, except for GFP150pcDAPHis6 produced with PylRS 11_4. Predicted mass of 28,096.4 Da, calculated mass of 28,093.6 Da.

[0042] Fig. 19. Schematic representation of the selection strategy for non-canonical monomers. Library 14 was transformed into BL21 cells and cultured overnight. Cells were cultured to an OD600 of 0.3-0.4. 4 mL of the library culture were added to stock solutions of each ncM. 4 mL of the library culture Petition 870250088848, dated 09 / 30 / 2025, page 40 / 242 26 / 141 cells were also added to a well without ncM. Cells were cultured for 40 min, stmRNAs were induced, and cells were cultured for a further 20 min, and RNA was isolated. Bio-mREX was performed on the isolated RNA from each sample. The experiment was performed in 4 replicates, resulting in 40 cDNA samples. 6 additional cDNA samples were generated for 6 of the RNA entries in bio-mREX. The resulting 46 cDNA samples were sequenced by NGS and analyzed to generate spindle plots and sequence tables.

[0043] Fig. 20. Selection of PylRS variants for (S)-3amino-3-(3-bromophenyl)propanoic acid ((S)e3mBrF)12 by tRNA display. a, Chemical structure of (S)e3mBrF12. b, Spindle diagram resulting from tRNA display selection as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+12) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. The red dots are sequences observed only in the positive samples.c, The 14 sequences, ordered by selectivity, resulting from selection for substrate 12 with selectivities > 5 and enrichments > 5. d, Fluoro-tREX for PylRS variants 12_1 to 12_6. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM of 12. The experiments were performed in triplicate.

[0044] Fig. 21. Selection of PylRS variants for (S)-3 acid Petition 870250088848, dated 09 / 30 / 2025, page 41 / 242 27 / 141 amino-6-(((benzyloxy)carbonyl)amino)hexanoic ((S)—e3CbzK) 13 by tRNA display. a, Chemical structure of (S)—e3CbzK 13. b, Spindle diagram resulting from tRNA display selection as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+13) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, 25 main sequences, ordered by selectivity, resulting from selection for substrate 13 with selectivities > 5 and enrichments > 5. d, FluorotREX for PylRS variants 13_1 to 13_6. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM of 13. The experiments were performed in triplicate.

[0045] Fig. 22. Selection of PylRS variants for (S)-6-acetamido-3-aminohexanoic acid ((S)-e3AcK) 14 by tRNA display. a, Chemical structure of (S)-e3Ac 14. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+14) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichment. Petition 870250088848, dated 09 / 30 / 2025, page 42 / 242 28 / 141 ments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples. c, The 8 sequences, ordered by selectivity, result from selection for substrate 14 with selectivities > 5 and enrichments > 5.

[0046] Fig. 23. Selection of PylRS variants for BocAhx 15 by tRNA visualization. a, Chemical structure of BocAhx 15. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+15) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, 25 main sequences, ordered by selectivity, resulting from selection for substrate 15 with selectivities > 5 and enrichments > 5. d, FluorotREX for variants 15_1 to 15_4 of PylRS, as well as for wild-type PylRS. Experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each MmPylRS and MmtRNAPyl in the presence and absence of 4 mM of 15. Control samples for wild-type PylRS were performed in the presence and absence of 4 mM of 15 and 2 mM of 1. Experiments were performed in triplicate. Petition 870250088848, dated 09 / 30 / 2025, page 43 / 242 29 / 141

[0047] Fig. 24. Selection of PylRS variants for 6(((benzyloxy)carbonyl)amino)hexanoic acid (CbzAhx) 16 by tRNA display. a, Chemical structure of CbzAhx 16. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+16) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, The 5 sequences, ordered by selectivity, resulting from selection for substrate 16 with selectivities > 5 and enrichments > 5. d, Fluoro-tREX for variant 16_1 of PylRS. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each MmPylRS and MmtRNAPyl in the presence and absence of 4 mM of 16. The experiments were performed in triplicate.

[0048] Fig. 25. Selection of PylRS variants for 3amino-2-((1-ethyl-1H-imidazol-5-yl)methyl)propanoic acid (e2NeH) 17 by tRNA display. a, Chemical structure of e2NeH 17. b, Spindle diagram resulting from tRNA display selection as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+17) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the library. Petition 870250088848, dated 09 / 30 / 2025, page 44 / 242 30 / 141 input. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples. c, The 4 sequences, ordered by selectivity, resulting from selection for substrate 17 with selectivities > 5 and enrichments > 5.

[0049] Fig. 26. Selection of PylRS variants for 3-amino4-(4-bromophenyl)butanoic acid (e3pBrhF) 18 by tRNA display. a, Chemical structure of e3pBrhF 18. b, Spindle diagram resulting from tRNA display selection, as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+18) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, The 5 sequences, ordered by selectivity, resulting from selection for substrate 18 with selectivities > 5 and enrichments > 5. d, Fluoro-tREX for PylRS variants 18_1 to 18_2. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and MmtRNAPyl in the presence and absence of 2 mM of the S or R enantiomer of 18. The experiments were performed in duplicate.

[0050] Fig. 27. Selection of PylRS variants for 2-benzyl acid Petition 870250088848, dated 09 / 30 / 2025, page 45 / 242 31 / 141 3-hydroxypropanoic acid (β2OH-F) 19 by means of tRNA display. a, Chemical structure of β2OH-F 19. b, Spindle diagram resulting from the selection of the tRNA display, as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+19) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, The 3 sequences, ordered by selectivity, resulting from selection for substrate 19 with selectivities > 5 and enrichments > 5.

[0051] Fig. 28. Selection of PylRS variants for 3-amino3-phenylpropanoic acid (e3F) 20 by tRNA visualization. a, Chemical structure of e3F 20. b, Spindle diagram resulting from tRNA visualization selection, as described in Fig. 19. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+20) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the samples. Petition 870250088848, dated 09 / 30 / 2025, p. 46 / 242 32 / 141 positive trans. c, The 5 sequences, ordered by selectivity, result from selection for substrate 20 with selectivities > 5 and enrichments > 5.

[0052] Fig. 29. Scheme of tRNA pulldown followed by LC-MS analysis to determine the monomer identity in the target tRNA. tRNAs are extracted from cells expressing the tRNA of interest and the orthogonal cognate aaRS, cultured in the presence of ncM. A biotinylated probe is annealed and the target tRNA is extracted. After washing, ncM is eluted by alkaline deacylation, derivatized with AQC and detected by LC-MS.

[0053] Fig. 30. Schematic representation of the selection strategy for random mutagenesis selections by tRNA display. An error-prone PCR reaction was performed on the active site sequence of PylRS variants 12_1 and 12_2 using the GeneMorph II kit (Agilent). Diversified PCR amplicons were cloned into a fresh ColE1 plasmid structure by Golden Gate assembly, and error-prone stmRNA libraries were transformed into BL21 cells and cultured overnight. Cells were cultured to an OD600 of 0.3–0.4. 2.6 mL of the library culture were added to a 12 stock solution at a concentration of 4 mM. 2.6 mL of the library culture were also added to a ncM-free well. Cells were cultured for 40 min, stmRNAs were induced, and cells were cultured for a further 20 min, and RNA was isolated. Bio-mREX was performed on RNA isolated from each sample.The experiment was performed in four replicates, resulting in eight cDNA samples. Four additional cDNA samples were generated for four of the RNA entries in Bio-mREX. The twelve resulting cDNA samples were sequenced by NGS and analyzed to generate spindle plots and sequence tables.

[0054] Fig. 31. Selection of enhanced PylRS variants for Petition 870250088848, dated 09 / 30 / 2025, page 47 / 242 33 / 141 ((S)e3mBrF) 12 using tRNA display random mutagenesis libraries. a, Parental active site sequences of (S)e3mBrF PylRS 12_1 and 12_2 used as templates for random mutagenesis. b, Spindle plot resulting from tRNA display selection, as described in Fig. 30. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+12) divided by the relative abundance in the control sample (ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 3 and enrichments > 3. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample.Red dots are sequences observed only in positive samples. Spindle diagram resulting from tRNA display selection, as described in Fig. 30. Selectivity is defined as the ratio of the relative abundance of a specific sequence in positive samples (+ 6), divided by the relative abundance in the control sample (ncAA). Enrichment is defined as the ratio of the same sequence in positive samples, divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 3 and enrichments > 3. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples.c, Table with unscheduled mutations of evolved PylRS variants ordered by selectivity calculated from NGS analysis. We characterize the variants as most selective, the most enriched. Petition 870250088848, dated 09 / 30 / 2025, page 48 / 242 34 / 141 as well as the most abundant sequences with selectivities > 3 and enrichments > 3. All sequences were based on the parental variant of PylRS 12_1. d, Fluoro-tREX data of PylRS variants I2_ievo1a and i2_ievo18. tRNAs were isolated from DH10e cells containing a pMB1 plasmid encoding each Mm PylRS and MmtRNAPyl in the presence and absence of 4 mM of 12. Fluoro-tREX was performed on the isolated tRNAs. All experiments were decayed in triplicate with similar results.

[0055] Fig. 32. LC-MS assay detecting (S)e3mBrF 12 derivatized in Mm tRNAPyl in a PylRS variant-dependent manner. A, Full LC-MS spectrum of the data presented in Figs. 3 ee f. An additional control, in which only the derivatizing agent 6-aminoquinolyl-N-hydroxycinnimidyl carbamate (AQC) was characterized, is shown in black. B, LC-MS trace overlay of the authentic derivatized standard, corresponding to 24 nM of 12, along with 12 isolates of the most active PylRS i2_ievo11 acylated tRNA.

[0056] Fig. 33. Characterization of protein production by amber readout with the PylRS(12_1evol1) / tRNAPylcuA pair. A, Attempt to produce GFP3(S)e3mBrFHis6 proteins from GFP3TAGhis6 from cells containing a pMB1 plasmid encoding the PylRS(12_1evol1) / tRNAPylcuA pair and a p15A plasmid encoding GFP3TAGhis6 in the presence and absence of 4 mM of 12. No modified protein could be produced by amber readout at position 3 in GFP. Points represent the mean of two biological replicates, error bars show ± standard deviation. b, Crude mass spectrum for purified GFP150(S)e3mBrFHis6 produced with PylRS 12_1. The deconvolved mass spectrum is shown in Fig. 3 h. c, MS / MS spectra of ncAA-containing peptides obtained after tryptic digestion of GFP150(S)e3mBrFHis6 produced with PylRS i2_ievol1. The precursor ions confirmed the incorporation of 12 at position 150 of GFP. Petition 870250088848, dated 09 / 30 / 2025, page 49 / 242 35 / 141 It is predicted that the fragmentation of each peptide will produce a series of b ions (red) and a series of y ions (blue), as well as ions corresponding to the full-length peptide (green). D, Crude mass spectrum for purified GFP150(S)β3mBrFHis6 produced with PylRS i2_ievol1. e, intact ESI-MS of GFP(150(S)p3mBrF)His6 purified from cells containing PylRS(12_1evol1), Mm tRNAPylCUA and GFP(150TAG)his6 cultured in the presence of 4 mM of 12. Mass found: 27,940.0 Da, predicted mass: 27,938.2 Da. E, MS / MS spectra of ncAA-containing peptides obtained after tryptic digestion of GFP150(S)β3mBrFHis6 produced with PylRS 12_1. Precursor ions confirmed the incorporation of 12 at the 150 position of GFP. Fragmentation of each peptide is predicted to yield a series of b ions (red) and a series of y ions (blue), as well as ions corresponding to the full-size peptide (green).

[0057] Fig. 34. Details of the GFP150(S)β3mBrFHis6 structure (PDB code 8OVY). A, Detail of the protein chain at position 150. The acquired structure for GFP150(S)β3mBrF-His6 (in yellow) is superimposed on the structure of the GFP wt used for refinement (PDB: 2B3P), showing the twist in the main chain induced by the β3-amino acid. B, Detail of the hydrogen bonding network in the beta barrel at position 150. The GFP150(S)β3mBrF-His6 structure (yellow) is superimposed on the structure of the GFP wt (PDB: 2B3P). The twist induced by the incorporation of (S ^mBrF (12)) affects the local hydrogen bonding at this position, but the remaining contacts of the corresponding beta strand remain intact.

[0058] Fig. 35. Shows sequence alignment of several aminoacyl-tRNA synthetases (aaRSs). * indicates residues found in all aligned aaRS sequences, : indicates residues with strong similarity between the aligned sequences, and . indicates residues with some similarity between the aligned sequences. Mm is Petition 870250088848, dated 09 / 30 / 2025, p. 50 / 242 36 / 141 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 the bacterium Gemmatimonadetes; PGA8 is the bacterium Peptostreptococcaceae pGA-8; I2 is Desulfosporosinus sp. I2; Clos is the bacterium Clostridiales; D121 is a Deltaproteobacteria; and D416 is another Deltaproteobacteria. Residues that are not conserved may be regions that tolerate variability. Thus, in acyl-tRNA synthetase modalities described here with a percent identity to the main chain, these regions are more tolerant to main chain variation.

[0059] Fig. 36. tRNA exhibits selection of O-synthetases carrying ncMs. a, ncMs for which selective PylRS mutants were discovered. b, Fluoro-tREX, representative gel for each PylRS variant. Experiments performed in independent triplicates with similar results. c, Selected PylRS variants acylate tRNAPylCUA with 12. LC-MS traces (ion scanning mode in the 6-aminoquinolyl-N-hydroxycinnimidyl carbamate (AQC) adduct of 12) of AQC derivative eluted from tRNA pulldowns. Cells containing the corresponding PylRS variant and tRNAPylCUA (or only tRNAPylCUA (-)) were cultured with 12; pulldowns used a biotinylated probe against tRNAPylCUA, representative traces are shown. d, f, h, j. l, n, p, as in b, but with PylRS and ncMs variants indicated.e, g, i, k, m, o, q, as in c, but with variants of PylRS and ncMs indicated; e, g, i were performed in duplicate, c, k, m, o, q were performed in triplicate, all replicates produced similar results. r, Fluorescence of cells containing GFP(150TAG)His6, indicated variant of PylRS and tRNAPylCUA, and cultured in the presence or absence of ncM (12, A1, A2, A3, A4, A5, A6, A7, 4 mM). The fluorescence is shown as a fraction of that generated by the pair. Petition 870250088848, dated 09 / 30 / 2025, page 51 / 242 37 / 141 PylRS / tRNAPylcuA wt with 4 mM BocK (1) and GFP(150TAG)His6. Bar graphs represent the average of three independent measurements, with individual data points shown as dots, and the error bar being + / - dps ESI-MS of GFP(150(S)α-MePIF)His6 purified from cells containing PylRS(A6_1), tRNAPylCUA and GFP(150TAG)His6 cultured with A6. Mass found: 28,000.5 Da, predicted mass: 28,000.2 Da. Spectra were acquired once.

[0060] Fig. 37. LC-MS assay detecting ncMs 12 and A1-A7 derivatized with 6-aminoquinolyl-N-hydroxyscinimidyl carbamate (AQC) eluted from MmtRNAPylacylated in a PylRS variant-dependent manner (see Fig. 29 for the assay scheme). a, Full LC-MS spectrum of the data presented in Fig. 36c. An authentic and derivatized standard of ncM 12 is shown in gray. Experiments were performed in triplicate with similar results. b, Enlarged LC-MS spectrum shown in a. c, Full LC-MS spectrum of the data presented in Fig. 36e. An authentic and derivatized standard of ncM A1 is shown in gray. Experiments were performed in two replicates with similar results. d, Enlarged LC-MS spectrum shown in c. d, Full LC-MS spectrum of the data presented in Fig. 36g. An authentic, derivatized ncM A2 standard is shown in gray. The experiments were performed in duplicate with similar results. e, Enlarged LC-MS spectrum shown in d.f, full LC-MS spectrum of the data presented in Fig. 36i. An authentic standard derived from ncM A3 is shown in gray. The experiments were performed in two replicates with similar results. g, enlarged LC-MS spectrum shown in f. h, full LC-MS spectrum of the data presented in Fig. 36k. An authentic standard derived from ncM A4 is shown in gray. The experiments were performed in triplicate with re. Petition 870250088848, dated 09 / 30 / 2025, page 52 / 242 38 / 141 similar results. i, Enlarged LC-MS spectrum shown in h. j, Full LC-MS spectrum of the data presented in Fig. 36m. An authentic standard derived from ncM A5 is shown in gray. The experiments were performed in triplicate with similar results. k, Enlarged LC-MS spectrum shown in j. l, Full LC-MS spectrum of the data presented in Fig. 36o. An authentic standard derived from ncM A6 is shown in gray. The experiments were performed in triplicate with similar results. m, Enlarged LC-MS spectrum shown in l. n, Full LC-MS spectrum of the data presented in Fig. 36q. An authentic standard derived from ncM A7 is shown in gray. The experiments were performed in triplicate with similar results.

[0061] Fig. 38. Schematic representation of the two-step selection strategy for non-canonical monomers. Library 14 was transformed into BL21 cells and cultured overnight. Cells were cultured to an ODgqq of 0.3-0.4. 4 mL of the library culture were added to the stock solutions of each ncM. Cells were cultured for 40 min, stmRNAs were induced, and cells were cultured for a further 20 min and RNA was isolated. Bio-mREX was performed on the isolated RNA for each sample. The experiment was performed in four replicates. For each replicate, cDNA was amplified with primers suitable for Golden Gate assembly. Then, all amplicons from the selected libraries for the same ncM were combined in equimolar ratios and cloned into a new ColE1 vector main strand. This created a pre-selected library for each ncM. The pre-selected libraries were transformed into BL21 cells and cultured overnight.The cells were cultured to OD6qq of 0.3-0.4. For each ncM, 4 mL of the respective culture from the pre-selected library were added to the ncM stock solutions. For each pre-selected library, 4 mL of the culture from the tam library. Petition 870250088848, dated 09 / 30 / 2025, page 53 / 242. 39 / 141 cells were also added to a well without ncM. Cells were cultured for 40 min, stmRNAs were induced, and cells were cultured for a further 20 min, and RNA was isolated. Bio-mREX was performed on the isolated RNA for each sample. The experiment was performed in triplicate, resulting in 6 cDNA samples per ncM. Three additional cDNA samples were generated for each ncM using three RNA entries for bio-mREX from the respective pre-selected libraries. For each ncM, the nine resulting cDNA samples were sequenced by NGS and analyzed to generate spindle plots and sequence tables.

[0062] Fig. 39. Selection of PylRS variants for (S)-3amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)e3MDF) (A1) by tRNA display. a, Chemical structure of (S)e3MDF A1. b, Spindle diagram resulting from tRNA display selection, as described in Figure 38. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+ A1) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. The red dots are sequences observed only in the positive samples.c, The 25 sequences, ordered by selectivity, resulting from selection for substrate A1 with selectivities > 5 and enrichments > 5. d, FluorotREX for PylRS variants A1_1 to A1_12. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of tRNA. Petition 870250088848, dated 09 / 30 / 2025, page 54 / 242 40 / 141 cia of 4 mM of A1. The experiments for A1_1 were performed in triplicate.

[0063] Fig. 40. Selection of PylRS variants for (S)-3amino-3-(4-bromophenyl)propanoic acid ((S^3pBrF)(A2)) via tRNA display. a, Chemical structure of (S)e3pBrF A2. b, Spindle diagram resulting from the selection of the tRNA display, as described in Figure 38. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+A2) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, The 25 sequences, ordered by selectivity, resulting from selection for substrate A2 with selectivities > 5 and enrichments > 5. d, FluorotREX for variants A2_1 to A2_12 of PylRS. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A2. The experiments for A2_1 were performed in triplicate.

[0064] Fig. 41. Selection of PylRS variants for (S)-3amino-3-(3,4-difluorophenyl)propanoic acid ((S)e3pmFF) (A3) by tRNA display. a, Chemical structure of (S)e3pmFF A3. b, Spindle diagram resulting from tRNA display selection as described in Figure 38. Selectivity is defined as the ratio between the relative abundance of a specific sequence in positive samples (+ A3), divided Petition 870250088848, dated 09 / 30 / 2025, page 55 / 242 41 / 141 of the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples, divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples. c, The 25 sequences, ordered by selectivity, resulted from selection for the A3 substrate with selectivities > 5 and enrichments > 5. d, FluorotREX for PylRS variants A3_1 to A3_12. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A3.The experiments for A3_1 were performed in triplicate.

[0065] Fig. 42. Selection of PylRS variants for (S)-3amino-3-(2-bromophenyl)propanoic acid ((S)e3oBrF) (A4) by tRNA visualization. a, Chemical structure of (S)e3oBrF A4. b, Spindle diagram resulting from the tRNA display selection, as described in Figure 38. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+ A4) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed Petition 870250088848, dated 09 / 30 / 2025, page 56 / 242 42 / 141 of the positive samples only. c, The 12 sequences, ordered by selectivity, resulting from selection for substrate A4 with selectivities > 5 and enrichments > 5. D, Fluoro-tREX for PylRS variants A4_1 to A4_7. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A4. The experiments for A4_1 were performed in triplicate.

[0066] Fig. 43. Selection of PylRS variants for (S)-3amino-3-(3-(trifluoromethyl)phenyl)propanoic acid (A5) ((S)e3mCF3F) via tRNA display. a, Chemical structure of (S)e3mCF3F A5. b, Spindle diagram resulting from the selection of the tRNA display, as described in Figure 38. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+A5) divided by the relative abundance in the control sample (ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample.The red dots are sequences observed only in positive samples. c, The 25 sequences, ordered by selectivity, resulting from selection for substrate A5 with selectivities > 5 and enrichments > 5. d, Fluoro-tREX for variants A5_1 to A5_8 of PylRS. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A5. The experiments for A5_1 were performed in triplicate.

[0067] Fig. 44. Selection of PylRS variants for (S)-2 acid Petition 870250088848, dated 09 / 30 / 2025, page 57 / 242 43 / 141 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 diagram resulting from tRNA display selection as described in Figure 38. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+A6) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the input library. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in the positive samples.c, The 25 sequences, ordered by selectivity, resulting from selection for substrate A6 with selectivities > 5 and enrichments > 5. d, FluorotREX for PylRS variants A6_1 to A6_9. The experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A6. The experiments for A6_1 were performed in triplicate.

[0068] Fig. 45. Selection of PylRS variants for (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (A7) (OH-(S)e3mClF) by tRNA visualization. a, Chemical structure of OH-(S)e3mClF A7. b, Spindle diagram resulting from the tRNA display selection, as described in Figure 38. Selectivity is defined as the ratio of the relative abundance of a specific sequence in the positive samples (+A7) divided by the relative abundance in the control sample (-ncAA). Enrichment is defined as the ratio of the same sequence in the positive samples divided by the relative abundance in the library of Petition 870250088848, dated 09 / 30 / 2025, page 58 / 242 44 / 141 entry. The boxed region indicates sequences with selectivities > 5 and enrichments > 5. Black dots are sequences observed in all positive samples and in all control samples. Green dots are sequences observed in all positive samples and in at least one control sample. Red dots are sequences observed only in positive samples. c, The 14 sequences, ordered by selectivity, resulting from selection for substrate A7 with selectivities > 5 and enrichments > 5. d, FluorotREX for variants A7_1 to A7_7 of PylRS. Experiments were performed using tRNA extracted from cells containing a pMB1 plasmid encoding each Mm PylRS and Mm tRNAPyl in the presence and absence of 4 mM A7. Experiments for A7_1 were performed in triplicate.

[0069] Fig. 46. Characterization of the in vivo acylation activity of evolved PylRS variants specific for ncM by tREX. We have previously shown that this assay quantitatively reports in vivo acylation. a, tREX for evolved PylRS variants. tREX was performed on Mm tRNAPylex extracted from cells containing a pMB1 plasmid encoding each Mm PylRS variant and Mm tRNAPyl cultured in the presence of 0, 1, 2, or 4 mM of the indicated ncM. Trials were performed in four replicates. b, Quantification of the in vivo acylation activity of evolved PylRS variants. Acylation levels were quantified by dividing the ratio of the upper band (acylated tRNA) by the sum of the signal intensity of the upper and lower bands (acylated and non-acylated tRNA) in the tREX gels shown in a. The data are presented as a fraction of the acylation activity of PylRS wt with 4 mM BocK (55 ± 1%), which was defined as 1.The dots represent the individual data points, the bars represent the mean, and the error bars represent the standard deviation. They observed that this test is performed under conditions in which the complexes... Petition 870250088848, dated 09 / 30 / 2025, p. 59 / 242 45 / 141 acyl-tRNAs are not actively consumed by translation in the cell and can accumulate over time. Under these conditions, enzymes with lower activity in the cell can lead to comparable levels of measured acylation to enzymes with higher activity in the cell. They observed that 12, A2, A5, and A6 are ribosomally incorporated, therefore they must be acylated to a level that supports ribosomal translation; this suggests that the other monomers may well be acylated to a level that supports translation if they are efficient substrates for other parts of the translational machinery. In future work, it may be useful to measure time-dependent acylation; this is challenging to assess in vivo, as the acylation of a tRNA in vivo is likely dominated by the ncM uptake rate, rather than the acylation rate.Currently published in vitro acylation assays for kinetic measurements require access to radioactive monomers or radiolabeled tRNAs; reagents to generate these tRNAs are not currently available; purification of the active soluble synthetase for in vitro measurements is challenging, and tRNAs for in vitro measurements typically do not exhibit post-transcriptional modifications. As a result, in vitro acylation measurements with this system may not provide information about in vivo behavior.

[0070] Fig. 47. Mass spectrometry of ncM containing GFP150Xhis6. a, Crude mass spectrum for purified GFP150(S)α-MePIFhis6 produced from cells containing PylRS A6_1, MmtRNAPylCUA, GFP(150TAG)his6 and 4 mM A6. The deconvolved mass spectrum is shown in Fig. 36s. b, Crude mass spectrum for purified GFP150(S)β3pBrFHis6 produced from cells containing PylRS A2_1, MmtRNAPylCUA, GFP(150TAG)his6 and 4 mM A2. c, Intact ESIMS of GFP(150(S)β3pBrF)His6 purified from cells containing PylRS(A2_1), MmtRNAPylCUA and GFP(150TAG)his6 grown in the presence of 4 mM A2. Mass found: 27,939.0 Da, mass Petition 870250088848, dated 09 / 30 / 2025, page 60 / 242 46 / 141 predicted: 27,938.2 Da d, Crude mass spectrum for purified GFP150((S)β3mCF3F)His6 produced with PylRS A5_1. e, Intact ESI-MS of purified GFP150((S)β3mCF3F)His6 from cells containing PylRS(A5_1), MmtRNAPylCUA and GFP(150TAG)His6 cultured in the presence of 4 mM A5. Mass found: 27,927.0 Da, predicted mass: 27,928.3 Da. f, MS / MS spectra of ncAA-containing peptides obtained after tryptic digestion of GFP^^^mBrFHis6 produced with PylRS A5_1, or GFP150(S)α-Me-pIFHis6 produced with PylRS A6_1, respectively. Precursor ions confirmed the incorporation of A5 or A6 at position 150 of GFP. Fragmentation of each peptide is predicted to yield a series of b ions (red) and a series of y ions (blue), as well as ions corresponding to the full-size peptide.

[0071] Fig. 48. GFP production. a, GFPhís6 production (or GFPhís6 incorporating 1 at position 150) was measured by GFP fluorescence. The cell contained a pMB1 plasmid encoding the PylRS / tRNAPylCUA wt pair and a p15A plasmid encoding GFP3TAGhís6 or GFPhís6 in the presence and absence of 4 mM BocK (1). The yield of GFPhís6 incorporating 1, produced from GFP3TAGhís6, PylRS / tRNAPylCUA wt pair and 1, was 80% of the GFPhís6 wt protein produced from GFPhís6 wt. Fluorescence values ​​shown are normalized by OD. Bars show the average of three biological replicates, individual replicates are indicated by dots, error bars show ± standard deviation. The fluorescence-based yield of GFPhís6 generated by each ncM incorporation is reported relative to the fluorescence of GFPhís6 generated by incorporation 1 in Fig.36r; The percentage yields are: 6.6% (for variant 12_1 and ncM 12 of PylRS), 14.4% (for variant 12_1 and ncM 12 of PylRS), 3.7% (for variant A2_1 and ncM A2 of PylRS), 4.1% (for variant A5_1 and ncM A5 of PylRS) and 37.0% (for variant A6_1 and ncM A6 of PylRS). Petition 870250088848, dated 09 / 30 / 2025, p. 61 / 242 47 / 141 PylRS). For reference, the Ni-NTA purified yield of GFPhís6, expressed from cells containing a pMB1 plasmid encoding the wild-type PylRS / tRNAPylCUA pair and a p15A plasmid encoding GFP3TAGhís6 in the presence of 4 mM BocK (1), was 105 ± 8 mg / l of culture. The purified yields of GFP150Xhís6 with the relevant ncMs were: 8.4 ± 0.2 mg / l of culture for PylRS12_1; 16.2 ± 1.2 mg / l of culture for PylRS12_1evo1; 3.6 ± 0.4 mg / l of culture for PylRSA2_1; 3.1 ± 0.7 mg / l of culture for PylRSA5_1; 35.5 ± 0.9 mg / l of culture for PylRSA6_1. b, Attempt to produce GFP3(12, A1, A2, A3, A4, A5, A6, A7)hís6 protein from GFP3TAGhís6 from cells containing a pMB1 plasmid encoding the indicated PylRS / tRNAPylCUA variant pair and a p15A plasmid encoding GFP3TAGhís6 in the presence and absence of 4 mM of the respective ncM.Fluorescence is shown as a fraction of the fluorescence generated by the PylRS / tRNAPylCUA wt pair with 4 mM BocK (1) and GFP(3TAG)hís6. Bars show the average of three biological replicates, individual replicates are indicated by dots, and error bars show ± standard deviation. DETAILED DESCRIPTION

[0072] The inventors herein have provided methods for measuring the extent of acylation of a tRNA. These methods allow measuring the effect of an agent on the acylation state of a tRNA. For example, the methods can be used to: i) determine whether an agent can charge a tRNA, ii) determine the extent or efficiency with which an agent can charge a tRNA, iii) determine whether an agent has an indirect effect on the charging of a tRNA, or iv) whether an agent can chemically alter a substrate in such a way as to affect charging or deacylation. The agent can be any polypeptide, nucleic acid, or condition that has or is suspected of having an effect on the acylation of a tRNA. Petition 870250088848, dated 09 / 30 / 2025, page 62 / 242 48 / 141

[0073] The methods described here do not require that the charged tRNA be active in a ribosome. Therefore, the methods allow the measurement of tRNA acylation with substrates that are not compatible with ribosomal translation, with substrates for which compatible ribosomes have not yet been identified, or with substrates for which compatible ribosomes have not yet been developed.

[0074] The inventors also provided molecules, tRNAs, nucleic acid constructs and labeled tRNAs for use with such methods. Method comprising split tRNAs

[0075] One aspect of the present description relates to the inventors' demonstration that tRNAs can be split into two or more portions, can be functionally expressed in this format, and can be acylated in this split format. An advantage of splitting tRNAs in this way is that the inventors demonstrate that additional sequences can be covalently linked to at least one of the tRNA portions. This, then, enables methods by which the identity of an individual agent capable of carrying out the loading of a tRNA can be measured, even in large parallel libraries.

[0076] As an example, inventors fused acyl-tRNA synthetase genes to a portion of a tRNA. Under conditions where the tRNA is expressed and the acyl-tRNA synthetase is expressed as a polypeptide, the acylation of the tRNA can then be measured and a direct link between the acylation state and the acyl-tRNA synthetase in question can be established.

[0077] These methods are exemplified in the Examples section of this document. Further details are provided in the priority documents claimed (EP2306393.6, filed April 28, 2023 and EP2400299.0, filed January 9, 2024, each). Petition 870250088848, dated 09 / 30 / 2025, page 63 / 242 49 / 141 of which is incorporated here by reference). These methods enabled the development of acyl-tRNA synthetases that could not have been developed previously. split tRNAs

[0078] The inventors have developed split tRNAs that find utility in methods for determining the acylation state of a tRNA or the acylation efficiency of a tRNA. These products are exemplified in the Examples section of this document. Further details are provided in the priority documents claimed (EP2306393.6, filed April 28, 2023, and EP2400299.0, filed January 9, 2024, each of which is incorporated herein by reference). These products have contributed to the development of acyl-tRNA synthetases that could not have been developed previously. RNA fusion molecules

[0079] The inventors have developed split tRNAs that can tolerate additional fused sequences and still be capable of being loaded. For example, the split tRNAs can comprise additional nucleic acid sequences that encode proteins or nucleic acids. These split RNAs can comprise an RNA molecule that includes a portion of a tRNA and a sequence encoding a polypeptide of interest / nucleic acid of interest. These products are exemplified in the Examples section of this document. Further details are provided in the priority documents claimed (EP2306393.6, filed April 28, 2023, and EP2400299.0, filed January 9, 2024, each of which is incorporated herein by reference). These products have contributed to the development of acyl-tRNA synthetases that could not have been developed previously. Methods for determining the acylation state of a tRNA or the efficiency. Petition 870250088848, dated 09 / 30 / 2025, page 64 / 242 50 / 141 tRNA acylation

[0080] The inventors have further developed methods for labeling acylated tRNAs. These methods allow the identification of charged tRNAs and are particularly sensitive. One embodiment of these methods is referred to here as fluoro-tREX. This method allows a specific acylated tRNA, from a set of tRNAs isolated from cells, to be labeled by primer extension with fluorescent dNTPs. Another embodiment of these methods is referred to here as bio-tREX. Bio-tREX allows the selective isolation of a specific acylated tRNA using primer extension with biotinylated dNTPs and streptavidin pulldown. These methods are exemplified in the Examples section of this document. Further details are provided in the priority documents claimed (EP2306393.6, filed April 28, 2023, and EP2400299.0, filed January 9, 2024, each of which is incorporated herein by reference). Nucleic acid constructs

[0081] The inventors demonstrate here that split tRNAs can be produced from a single gene, in which the two tRNA halves are circularly interchanged and linked by an intervening sequence. The primary transcript of this gene is processed in cells to produce a functional split tRNA, which is acylated by an acyl-tRNA synthetase. These constructs are exemplified in the Examples section of this document. Further details are provided in the priority documents claimed (EP2306393.6, filed April 28, 2023, and EP2400299.0, filed January 9, 2024, each of which is incorporated herein by reference). These constructs have contributed to the development of acyl-tRNA synthetases that could not have been developed previously. Beta-amino acid-acyl-tRNA synthetases Petition 870250088848, dated 09 / 30 / 2025, page 65 / 242 51 / 141

[0082] The inventors used the methods and tools described herein and identified acyl-tRNA synthetases capable of acylating tRNAs with beta amino acids.

[0083] Thus, in a first aspect, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300A, M300C, M300D, M300M or M300S in relation to SEQ ID NO: 1; A302A, A302C, A302D, A302G, A302H, A302L, A302N or A302Y in relation to SEQ ID NO: 1; and N346A, N346C, N346G, N346S, N346T or N346V in relation to SEQ ID NO: 1.

[0084] In one embodiment, the acyl-tRNA synthetase comprises mutations corresponding to: M300A, M300C, M300D, or M300S mutation relative to SEQ ID NO: 1; A302C, A302D, A302G, A302H, A302L, A302N, or A302Y mutation relative to SEQ ID NO: 1; and N346A, N346C, N346G, N346S, N346T, or N346V mutation relative to SEQ ID NO: 1.

[0085] An acyl-tRNA synthetase is capable of specifically acylating a tRNA with a specific substrate when the synthetase preferentially acylates the tRNA with said substrate in the presence of canonical amino acids. In specific embodiments, the acyl-tRNA synthetase can be expressed in a cell in the presence of the substrate and the target tRNA, and, of the target tRNAs acylated within the cell, at least 60%, 70%, 80%, 90%, 95%, or 99% are acylated with the specific substrate. As an illustrative example, the cell could be a bacterial cell, such as an E. coli cell.

[0086] The beta amino acid may be an analogue of S beta 3 phenylalanine. In particular, the beta amino acid may be (S)-3-amino acid. Petition 870250088848, dated 09 / 30 / 2025, page 66 / 242 52 / 141 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)e3MDF). The beta amino acid may be (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S^pBrF). The beta amino acid may be (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S^pmFF). The beta amino acid may be (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^oBrF). The beta amino acid may be (S)-3-amino-3-(3(trifluoromethyl)phenyl)propanoic acid ((S)β3mCF3F).

[0087] In a specific embodiment, acyl-tRNA synthetase comprises mutations corresponding to: M300D and A302H. M300D and A302H were the most common mutations found in screenings of any of the beta-amino acids tested. In a specific embodiment, acyl-tRNA synthetase comprises mutations corresponding to: M300D; A302H; and N346G, N346A, or N346S. In a specific embodiment, acyl-tRNA synthetase comprises mutations corresponding to: M300D; A302H; and N346G / N346A. In a specific embodiment, the M300 mutation is M300D, the A302 mutation is A302H, and the N346 mutation is N346G.

[0088] In this embodiment, particularly when the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: an M300D mutation relative to SEQ ID NO: 1; an A302H, A302Y or A302C mutation relative to SEQ ID NO: 1; and an N346G, N346A or N346S mutation relative to SEQ ID NO: 1.

[0089] In the examples, particularly when the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the M300 mutation is M330D. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S^MDF)), the M300 mutation is M300A, M300C, M300D, or is the wild-type residue. Petition 870250088848, dated 09 / 30 / 2025, p. 67 / 242 53 / 141 (M300M) - M300D was the most common in screening. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S^^BrF), the M300 mutation is M300C, M300D, M300S, or is the wild-type residue (M300M) - M300D was the most common in the screening. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)e3pmFF), the M300 residue is M300A, M300C, M300D, or M300M. Wild-type M300D was the most common in the screening. In the examples, particularly where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)e3oBrF), the M300 residue is M300D, M300S, or M300M - M300D wild type was the most common in screening. In examples, particularly where the beta amino acid is (S)-3amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)e3mCF3F), the mutation is M300D.

[0090] In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the A302 mutation is A302C, A302H, or A302Y. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)e3MDF), the A302 mutation is wild-type (A302A), A302C, or A302H - A302H was the most common in the screening. In the examples, particularly where the beta amino acid is (S)-3-amino-3(4-bromophenyl)propanoic acid ((S)e3pBrF), the A302 mutation is A302G or A302H - A302H was the most common in the screening. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)e3pmFF), the A302 mutation is A302D, A302H, A302L, or A302N - A302H was the most common in the screening. In the examples, particularly where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)e3oBrF), the A302 mutation is A302D or A302H - A302H was the most common in the screening.In the examples, particularly where the beta amino acid is (S)-3-amino-3. Petition 870250088848, dated 09 / 30 / 2025, p. 68 / 242 54 / 141 (3-(trifluoromethyl)phenyl)propanoic ((S)e3mCF3F), the A302 mutation is A302H, A302N or A302Y; or, in particular, A302H or A302Y.

[0091] The amino acid sequence may comprise a mutation at a position corresponding to L305 of SEQ ID NO: 1. In the examples, the mutation is L305C, L305F, L305G, L305H, L305I, L305N, or L305V, or a conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is L305H or L305C, or a conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S^MDF), the mutation is L305C, L305G, L305H, L305I, or L305N, or the wild-type residue; in particular, the mutation is L305C, L305I, or L305L. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S^3pBrF), the mutation is L305C, L305F, L305I, L305N, L305V, or wild-type (L305L); or, in particular, L305F, L305I, L305L, or L305N.In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)p3pmFF), the mutation is L305C, L305F, L305H, L305I, L305V or wild type (L305L); or, in particular, L305C, L305I, L305L or L305V. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β3mCF3F), the mutation is L305F, L305I or L305V or wild type (L305L). The inventors have functionally identified beta-amino acid tRNA synthetases that do not comprise an L305 mutation, which can be referred to as comprising L305L, and therefore this mutation is not present in all embodiments. L305L is particularly relevant for instances where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S ^oBrF). Petition 870250088848, dated 09 / 30 / 2025, p. 69 / 242 55 / 141

[0092] The amino acid sequence may comprise a mutation at a position corresponding to Y306 of SEQ ID NO: 1. In the examples, the mutation is Y306C, Y306F, Y306H, Y306I, Y306L, Y306N, Y306R, or Y306V, or a conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is Y306L, Y306F, Y306R, or Y306C, or a conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S^3MDF), the mutation is Y306H, Y306I, or Y306L, or the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S^^BrF), the mutation is Y306C or Y306I, or is wild-type (Y306Y); or, in particular, Y306I or Y306Y.In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S^pmFF), Y306F, Y306I, Y306L, Y306R, or wild type (Y306Y); or, in particular, Y306I, Y306L, or Y306Y. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^oBrF), the mutation is Y306H, Y306I, Y306L, Y306R, or wild type (Y306Y); or, in particular, Y306H, Y306L, Y306R, or Y306Y. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S )β3mCF3F), the mutation is Y306F, Y306I, Y306L, Y306N, Y306R or Y306V or wild type (Y306Y); it may be referred to as comprising Y306Y, and therefore this mutation is not present in all forms.

[0093] The amino acid sequence may comprise a mutation at a position corresponding to L309 of SEQ ID NO: 1. Nos Petition 870250088848, dated 09 / 30 / 2025, p. 70 / 242 In 56 / 141 examples, the mutation is L309C, L309F, L309G, L309H, L309I, L309N, L309S, L309V, or L309Y, or a conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is L309V or L309C, or a conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S^MDF), the mutation is L309C, L309F, L309H, L309N or L309S, or the wild-type residue; or, in particular, L309C, L309F, L309H, L309N or the wild-type residue. L309F, L309L or L309N.In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S^pmFF), the mutation is L309C, L309G, L309H, L309N, L309S, L309V or wild type (L309L); or, in particular, L309G, L309H, L309L, L309N, L309S or L309V. In the examples, particularly where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^^BrF), the mutation is L309C, L309G, L309H or wild type (L309L); or, in particular, L309G, L309H or L309L. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^^BrF), the mutation is L309C, L309G, L309H or wild type (L309L); or, in particular, L309G, L309H or L309L. (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic ((S)β3mCF3F), the mutation is L309C, L309G, L309H, L309N, L309V or wild type (L309L); or, in particular, L309C, L309L, L309N or L309V. functional beta-amino acid-tRNA synthetases have been identified that do not comprise an L309 mutation, which may be referred to as comprising L309L, and therefore this mutation is not present in all modalities.

[0094] The amino acid sequence may comprise a mutation. Petition 870250088848, dated 09 / 30 / 2025, page 71 / 242 57 / 141 tion at a position corresponding to M344 of SEQ ID NO: 1. In the examples, the mutation is M344E, M344G, or M344Q, or a conservative substitution of said residues or wild-type residue. In the examples, particularly where the beta amino acid is (S)3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)e3MDF), the mutation is M344E or M344Q, or the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S^^BrF), the mutation is M344Q or is wild type. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)e3pmFF), the mutation is M344Q or (M344M) - M344Q wild type was the most common in the screening. In the examples, particularly where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)e3oBrF), the mutation is M344G, M344Q or (M344M) - M344Q wild type was the most common in the screening.In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)e3mCF3F), the mutation is M344Q or (M344M) - wild-type M344M was the most common in screening. The inventors identified functional beta-amino acid tRNA synthetases comprising the wild-type residue at a position corresponding to M344 of SEQ ID NO: 1. This can be termed as comprising M344M. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the sequence comprises M344M.

[0095] In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the N346 mutation is N346G. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)e3MDF), the N346 mutation is N346A, N346C, N346G, N346S, N346T, or N346V; or, in particular, N346G or N346A. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(4. Petition 870250088848, dated 09 / 30 / 2025, page 72 / 242 58 / 141 bromophenyl)propanoic acid ((S)e3pBrF), the N346 mutation is N346A, N346C, N346G, N346S, N346T, N346V; or, in particular, N346A or N346G. In the examples, particularly where the beta amino acid is (S)3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)e3pmFF), the N346 mutation is N346A, N346C, N346G, N346S or N346T - N346G was the most common in the screening. In the examples, particularly where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S)e3oBrF)), the N346 mutation is N346A, N346G, N346S, or N346T; or, in particular, N346A, N346G, or N346S; or, more particularly, N346G or N346S. In the examples, particularly where the beta amino acid is ((S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)e3mCF3F), the N346 mutation is N346G.

[0096] The amino acid sequence may comprise a mutation at a position corresponding to C348 of SEQ ID NO: 1. In the examples, the mutation is C348A, C348F, C348G, C348H, C348I, C348L, C348M, C348N, C348R, C348S, C348T, C348V, or C348Y, or a conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is C348I, C348L, C348V, or C348F, or a conservative substitution of said residues or of the wild-type residue. In specific examples, particularly where the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, the mutation is C348F, C348I, or C348L. In specific examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S)e3MDF), the mutation is C348G, C348I, C348L, C348M, C348N, C348S, C348V, or C348Y; or, in particular, C348G, C348N, C348S, or C348V.In specific examples, particularly where the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)e3pBrF), the mutation is C348F, C348G, C348H, C348I, C348N, or C348S, or it is jungle type. Petition 870250088848, dated 09 / 30 / 2025, p. 73 / 242 59 / 141 gem (C348C); or, in particular, C348C, C348G, C348I or C348S. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S^pmFF)), the mutation is C348A, C348F, C348H, C348I, C348L, C348R, C348S, C348T, C348V or wild type (C348C); or, in particular, C348F, C348L, C348S or C348V. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^BrF)), the mutation is C348G, C348I, C348S, C348V, C348Y or wild type (C348C); or, in particular, the mutation is C348C, C348I, C348S or C348Y. In the examples, particularly where the beta amino acid is (S)-3amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β3mCF3F), the mutation is C348F, C348H, C348I, C348L, C348S, C348V or C348Y or wild type (C348C); or, in particular, C348F, C348H or C348V.The inventors have functionally identified beta-amino acid tRNA synthetases that do not comprise a C348 mutation, which can be referred to as comprising C348C, and therefore this mutation is not present in all embodiments.

[0097] The amino acid sequence may comprise the wild-type residue at a position corresponding to Y384 of SEQ ID NO: 1. This may be referred to as comprising Y384Y.

[0098] The amino acid sequence may comprise the wild-type residue at a position corresponding to S399 of SEQ ID NO: 1. This may be referred to as comprising S399S.

[0099] The amino acid sequence may comprise a mutation at a position corresponding to V401 of SEQ ID NO: 1. In the examples, this residue is any of the 20 canonical amino acids (i.e., V401X). In the examples, the mutation is V401A, V401C, V401F, V401K, V401L, V401S, or V401T, or a conservative substitution of said residues or of the wild-type residue. In the examples, the mutation is V401A, V401C, V401K, V401L, V401S, or V401T, or a Petition 870250088848, dated 09 / 30 / 2025, p. 74 / 242 60 / 141 conservative substitution of said residues or of the wild-type residue. In the specific examples, particularly 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 conservative substitution of said residues or of the wild-type residue. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((S^MDF), the mutation is V401A, V401C, V401K, or V401L, or the wild-type residue (V401V); or, in particular, V401C, V401L, or V401V. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S^^BrF), the mutation is V401A, V401C, V401K, or V401L, or is wild-type (V401V); or, in particular, V401C, V401L, or V401V.In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)p3pmFF), the mutation is V401C, V401F, V401K, V401L, V401T, or wild type (V401V); or, in particular, V401C, V401L, or V401V. In the examples, particularly where the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^^BrF)), the mutation is V401A, V401C, V401K, V401L, or wild type (V401V); or, in particular, V401C or V401V. In the examples, particularly where the beta amino acid is (S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β3mCF3F), the mutation is V401C, V401K, V401L, V401S, or V401T, or the wild type residue; or, in particular, V401S, V401T, or V401V. These comprise a V401 mutation, which may be referred to as comprising V401V, and therefore this mutation is not present in all embodiments.

[00100] In a particular embodiment, the acyl-tRNA synthetase comprises residues corresponding to: M300D, A302H, M344M / M344Q and N346G / N346A, and optionally a C348 mutation. C348 can, Petition 870250088848, dated 09 / 30 / 2025, page 75 / 242 61 / 141 in some embodiments, being any one of C348A, C348F, C348G, C348H, C348I, C348L, C348M, C348N, C348R, C348S, C348T, C348V or C348Y, or more particularly, any one of C348F, C348G, C348H, C348I, C348L, C348N, C348S, C348V or C348Y.

[00101] The inventors provide specific embodiments in Table 1. These embodiments are defined in relation to SEQ ID NO: 1 and, for example, 12_1 comprises M300D, A302H, L305H, etc. The inventors tested each of the mutation sets below when applied to the MmPyIRS backbone (SEQ ID NO: 1) and found that each is functional with respect to loading a tRNA with a beta amino acid. In particular, these embodiments are capable of loading a tRNA with (S)-3-amino-3-(3-bromophenyl)propanoic acid. Table 1 - Mutations of exemplary functional beta-amino acid tRNA synthetases ((S)-3-amino-3-(3-bromophenyl)propanoic acid) ncrnedo Wt M3D0 A 302 L305 Y306 L309 M344 N346 C34E Y3B4 5399 V401 12_1 □ HHYLMGFY 5 C 12_2 □ HLLLMGLY 5 5 12_3 □ HLLLMGLY 5 L 12_4 □ CCFVMGLY 5 C 12_A □ YLRLMGIY 5 V 12_E □ HLRLMGLY 5 V 12_C □ YLCCMGIY 5 V 12_D □ HCFLMGCY 5 K 12_E □ HCFLMGLY 5 A

[00102] Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations in Table 1. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 1. These mutations are particularly suitable for beta-amino acid-tRNA synthetases capable of incorporating the beta-amino acid listed in the table title.

[00103] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, Petition 870250088848, dated 09 / 30 / 2025, p. 76 / 242 62 / 141 wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: M300D, A302H, L305H, N346G, C348F, and V401C. In one embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D, A302H, L305H, Y306Y, L309L, M344M, N346G, C348F, Y384Y, S399S, and V401C.

[00104] In one embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: M300D, A302H, Y306L, N346G, C348L, and V401S. In another embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D, A302H, L305L, Y306L, L309L, M344M, N346G, C348L, Y384Y, S399S, and V401S.

[00105] In one embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: M300D, A302H, Y306L, N346G, C348L, and V401L. In another embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D, A302H, L305L, Y306L, L309L, M344M, N346G, C348L, Y384Y, S399S, and V401L.

[00106] In one embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta amino acid is provided, Petition 870250088848, dated 09 / 30 / 2025, p. 77 / 242 63 / 141 wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: M300D, A302C, L305C, Y306F, L309V, N346G, C348L, and V401C. In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D, A302C, L305C, Y306F, L309V, M344M, N346G, C348L, Y384Y, S399S, and V401C.

[00107] In addition to the sets of mutations and, optionally, wild-type residues discussed above, acyl-tRNA synthetase may also comprise a mutation or any combination of mutations at positions corresponding to the following positions in SEQ ID NO: 1: F295, N307, T364, F366, K375, T387, D414, and G421. In a specific embodiment, the mutation may be any one or any combination of: F295L / F295I, N307K, T364A, F366L, K375R, T387I / T387S, D414V, and G421A, or a conservative substitution of said residues or the wild-type residue. Petition 870250088848, dated 09 / 30 / 2025, p. 78 / 242 64 / 141 Table 2 - Mutations of exemplary functional beta-amino acid tRNA synthetases ((S)-3-amino-3-(benzo[d][1,3]dioxol-5-yl)propanoic acid ((3)β3MϋP)) M3 00 A302 L305 Y30fi L3OS M344 N346 C34B Y3B4I5399 V4O1 C Η O Y Γ» 5 [ ¥ Y 'c- A1_2 □ H L Y H Q G 3. Y y A1_3 D Η I Y £ Q G V Y S 'V A1_4 - D H L Y L E A G Y j V A1_5 □ H L H N M A 3 Y 3 V A1_6 - D H L L N r / G S Y S V A1_7 - D H € V F G G Y S V Μ Η N ! M T I Y S K A1_8 - D H L Y H A S ¥ 3 € D Η I L L ** V V i S A A A L ¥ L IV A M Y 3 V A1_9 - D Η I H &·. A S Y S V D H L r S Q G L - s □ Η N Ϊ M k: G 3 Y s A1_10 — □ H I r L Q G V Y S L | D H L I L M G 3 Y S V D H L f L ΕΠ C S Y 5 L | A1_11 - □ H H Q A 5 Y 5 V D C I L L b A V Y S ” D Η I Y F S G V S V D Η H L fl- V S r S V A1 1 - D H 1 Y Q G N Y i f A1 12- D H C r L Q A S V

[00108] The acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations in Table 2. The acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 2. The acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and, optionally, wild-type residues associated with an identifier (e.g., A1_2) in Table 2. These mutations are particularly suitable for beta-amino acid-tRNA synthetases capable of incorporating the beta-amino acid listed in the table title. Petition 870250088848, dated 09 / 30 / 2025, p. 79 / 242 65 / 141 Table 3 - Mutations of exemplary functional beta-amino acid tRNA synthetases ((S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)ppBrF)) M3D0 A302 L305 Υ3Ο·6 1309 M344 Ί346 C34B Y3B4 5399 V4C1 | A2_2 — D H F í F Q A G / S A2_1 — D H I F M G G f $ V A2_3 — D H I V N M G C V £ L A2_4 D H N Y N Q A s 5 A2_5 — D H I Y N Q A t Y 5 V A2_6 — D H L Y F Q G G Y S A2_7 □ H I Y M Q (= s V 5 vl A2_a — 0 H F Y F Q A G Y 5 c D H L ¥ 1 M G - r 5 L A2_9 — D H F Y F A G íf S L A2_1O — 0 H N ¥ N Q G S T ς V D H I Y 5 k' A c ' / S L 5 6 L. Y V k A G Y 5 7 ¥ A2_11 ™· D H L ¥ L Q G G Y s V D H N Y M V. A 3 Ϊ s V A2_12 — D H I Y C Q G 1 ¥ 5 D H =. c G M A F Y S -.1 D H L ¥ N Q G 5 ¥ 5 V D H I G Q G F V 5 V D H V Y - U G C L S L D H L Y L Q G Γ Y S L D H V ¥ 1 Q A L Y 5 X D H F ¥ F Γ / 6 G ¥ 3 A D H I ¥ 5 M G H 5 L D H I Y 1 M S Γ 5 L C H r Y F M T 1 S A V H L C L Q C h_ Y 5 K s H C ¥ L k* V N Y 3 V D H L Y V k' A N Y s rJ

[00109] Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations in Table 3. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 3. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and, optionally, wild-type residues that are associated with an identifier (e.g., Petition 870250088848, dated 09 / 30 / 2025, p. 80 / 242 66 / 141 A2_2) in Table 3. These mutations are particularly suitable for beta-amino acid tRNA synthetases capable of incorporating the beta-amino acid listed in the table title. Table 4 - Mutations of exemplary functional beta-amino acid tRNA synthetases ((S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((SW3pmFF)) M3C0 A3O2 L3C5 Y30fi L309 M344 N346 £348 ¥334 5399 1 V4O1 A3_2 — D H L G O G F y A3_3 — D H 1 L L Q G S A A3_1 — D H V ¥ L U G V V J» L. A3_4 - D H j_ ¥ N Q G 5 ¥ J V A3_5 — D H L L H Q G 5 ç V A3_6 — D N L ¥ L Q G A ¥ 5 V A3_7 — D H c ¥ V Q G L ¥ 5 V A3_8 — D H L I - Q G L T j y A3 9 — D H 1 ¥ V fr- G L y £ D H L 1 R H k G ò 1: D H ! L H í- G 5 ¥ J J A3_10 — □ H L 7 5 Q G F 5 A H L ¥ L P / T L X i K A3_11 — D H L y L Q 1 <5 S 3 L D L L ¥ L -.1 G T 5 V A3_12 — D H L 7' G G F ¥ S F D H 1 ¥ - F A F ς T D D L L £ r । G R 5 y D H C ¥ G I1.' G L y1 c V C H H Y £ P. * 5 1 Y s c Π H 1 1 C 1 G H T s V 1 í H F L w A V Ϊ - c D H 1 F S A s ¥ V ·* __H 1 1 J. € V ¥ s K

[00110] A acil-tRNA sintetase pode compreender uma sequência de amino acids comprising any of the sets of mutations in Table 4. The acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 4. The acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and, optionally, wild-type residues that are associated with an identifier (e.g., Petition 870250088848, dated 09 / 30 / 2025, p. 81 / 242 67 / 141 A3_2) in Table 4. The acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and, optionally, wild-type residues that are associated with any of the A3_1, A3_2, A3_3, A3_4, A3_5, A3_7, A3_8, A3_9, A3_10, or A3_11 in Table 4. These mutations are particularly suitable for beta-amino acid-tRNA synthetases that are capable of incorporating the beta-amino acid listed in the table title. Table 5 - Mutações de beta-aminoácido-tRNA syntheses funcionais exemplares (ácido (S)-3-amino-3-(2-bromophenil)propanoico ((S^3oBrF\) name do hit M3Ü0 A3O2 L3Ü5 Y3Ü6 L3C9 M344 N346 C34B Y3S4 S399 V401 □ H II. HQGSVL । MJ2 - DH ll 1 1 α G 4 VKDH l C l·1- A w - A A4_3 - DH il LH q G 5 —r C A4_1 - □ DR 1 QG r lí ill A4_4 - DHLH 6 1 G si·1 K A4_5 - 0 H ll 1 QT - 'r A A4_6 - 0 lí HIQG r lr A4_7 - H |i GQS 1 Ll s H L. LGA - s Ll Π 1! IQGGV € DH II LQGV lr L

[00111] Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations in Table 5. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 5. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and, optionally, wild-type residues associated with an identifier (e.g., A4_3) in Table 5. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and, Petition 870250088848, dated 09 / 30 / 2025, p. 82 / 242 68 / 141 optionally, wild-type residues associated with any of the following: A4_1, A4_3, A4_6, or A4_7 from Table 5. These mutations are particularly suitable for beta-amino acid tRNA synthetases capable of incorporating the beta-amino acid listed in the table title. Table 6 - Mutations of beta-aminoacid-tRNA syntheses exemplary functions ((S)-3-amino-3-(3-(trifluorometH)phenyl)propanoic acid ((Sy33mCF3i=Y) name do hit «soo «02 lbqs race Laos N34E C34S Y3B4 S399 V4O1 D Η VC PI GF rt A5_2 - 0 h ii fc GF A5_1 - DHFN [1 IGH r A5_3 - DYVL l 1 GV ϊ A5-4 - DY r VF c GV 5 D Η VVGGF - A5_5 D Η VFLQGV * A5_6 — D Η V Cl! GVT A5_7 0 Η 11 LH 1 GS A5_8 - d Hi FV ri 1 GFDHFL 1 G l' Γ DU . NQG - D Η H 6 - D Η 1 RGVDY | 1 GGL. 1 [> HFN y GYKD Η VF 1 G 1 - DN 1 G IL 2 I 1 DH 1 RH 1 1 G l - DY 1 1 F . í 1 G te 11 “ CL> Η VL il íi G s < DH II VI r1 1 GVD Η VLGD Hi il LGVD Η VFC 1 GL

[00112] Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations in Table 6. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 6. Acyl-tRNA synthetase may Petition 870250088848, dated 09 / 30 / 2025, p. 83 / 242 69 / 141 comprise an amino acid sequence comprising any of the sets of mutations and, optionally, wild-type residues associated with an identifier (e.g., A5_2) from Table 6. The acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and, optionally, wild-type residues associated with any of the following: A5_1, A5_2, A5_3, A5_4, A5_5, A5_6, or A5_8 from Table 6. These mutations are particularly suitable for beta-amino acid-tRNA synthetases capable of incorporating the beta-amino acid listed in the table title.

[00113] In some embodiments, acyl-tRNA synthetase may also comprise any of the following sets of mutations: a) N307K and F366L b) F295I and T387I c) G421A d) T364A and T387I e) T387Se D414V f) F295L and K375R.

[00114] In the experimental data, mutations designated evol1 are defined as a), mutations designated evol2 are defined as b), mutations designated evol3 are defined as c), mutations designated evol5 are defined as d), mutations designated evol7 are defined as e), and mutations designated evol8 are defined as f). For example, 12_1evol1 has the mutations of 12_1 in Table 1 and the mutations from set a) above.

[00115] In a specific embodiment, any of the sets a), b), c), d), e), or f) may be present in combination with any set of mutations and, optionally, wild-type residues in any of Tables 1, 2, 3, 4, 5, or 6. In an embodiment Petition 870250088848, dated 09 / 30 / 2025, page 84 / 242 70 / 141 preferred, any of the sets a), b), c), d), e) or f) is present in combination with the mutations and, optionally, wild-type residues of any set associated with an identifier (e.g., 12_1) in any of Tables 1, 2, 3, 4, 5 or 6.

[00116] The mutations of the first aspect are provided with reference to SEQ ID NO: 1, which is the sequence of the wild-type Methanosarcina mazei pyrrolisyl-tRNA synthetase (MmPylRS) and is provided below. MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVV NNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVK VKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVS TQESVSVPASVSTSISSISTGATASALVKGNTN PITSM SAPVQASAPA LTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAE ERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQI FRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGK EHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVY GDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVK HDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 1)

[00117] To aid alignment, a version of the Mm PylRS sequence where an X is marked at positions 300, 302, 305, 306, 309, 346, 348, 384 and 401 is provided below. MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVV NNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVK VKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVS TQESVSVPASVSTS ISSI STGATASALVKGNTN PITSM SAPVQASAPA LTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAE ERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQI FRVDKNFCLRPXLXPNXXNYXRKLDRALPDPIKIFEIGPCYRKESDGK EHLEEFTMLXFXQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVX GDTLDVMHGDLELSSAXVGPIPLDREWGIDKPWIGAGFGLERLLKVK Petition 870250088848, dated 09 / 30 / 2025, page 85 / 242 71 / 141 HDFKNIKRAARSESYYNGISTNL (SEQ ID NO: 2)

[00118] Thus, in one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, wherein said synthetase comprises an amino acid sequence with 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 said amino acid sequence comprises any of the mutations or sets of mutations described in relation to the first aspect. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity to 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, except for the mutations mentioned.

[00119] The acyl-tRNA synthetase may comprise an amino acid sequence with 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 comprising an M300D mutation, an A302H, A302Y or A302C mutation and an N346G, N346A or N346S mutation. The amino acid sequence may include mutations corresponding to mutations of any of the 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 include residues corresponding to mutations and wild-type residues of any of the 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 mutations corresponding to the mutations listed as sets a), b), c), d), e), or f) herein.

[00120] Acyl-tRNA synthetase may comprise an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence Petition 870250088848, dated 09 / 30 / 2025, p. 86 / 242 72 / 141 of those from SEQ ID NO: 1 or SEQ ID NO: 2 and comprising any of the sets of mutations in any of Tables 1 to 6. The acyl-tRNA synthetase may comprise an amino acid sequence with 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 comprising any of the sets of mutations and wild-type residues in any of Tables 1 to 6.The acyl-tRNA synthetase may comprise an amino acid sequence with 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 comprising any of the sets of mutations, or sets of mutations and wild-type residues, labeled as 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, 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 in Tables 2 to 6. The amino acid sequence may also include mutations corresponding to the mutations listed as sets a), b), c), d), e), or f) here.

[00121] In a preferred embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: M300D, A302H, L305H, N307K, N346G, C348F, F366L, and V401C relative to SEQ ID NO: 1. The acyl-tRNA synthetase may comprise an amino acid sequence comprising 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% Petition 870250088848, dated 09 / 30 / 2025, p. 87 / 242 73 / 141 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, except for the mutations mentioned.

[00122] A skilled individual would also be able to apply the identified mutations to other PylRS backbones, including backbones not described in this document. This is despite the fact that other PylRSs may have low sequence identity with Mm PylRS. To apply the mutations cited with reference to Mm PylRS to other synthases, the sequences must be aligned to identify the corresponding residues. In particular, the sequences representing the catalytic sites of the synthases can be aligned. Thus, the SEQ ID NO: 1 region extending from residue 296 to residue 428 can be aligned to the sequence of another PylRS, and the corresponding mutations can be performed. More information on transferring mutations from one PylRS backbone to another is provided in document WO2013 / 171485 A1 (incorporated herein by reference). An exemplary alignment is provided in Figure 35.

[00123] Thus, in one embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising any mutations and, optionally, wild-type residues described in relation to the first aspect, or any set of mutations and, optionally, wild-type residues described in relation to the first aspect, and wherein the backbone of the acyl-tRNA synthetase is a PylRS.

[00124] Mutations can be applied to the PylRS of Nitrososphaeria archaeon (Nitra). This sequence is provided below. MSKIRFTRGQIHRLIELGAEPTELERDFETEAERDKEFNKIAENLARKN Petition 870250088848, dated 09 / 30 / 2025, p. 88 / 242 74 / 141 LKNIKDFLEQRRKPLVRVIEEKLRTTALRLGFSEVVTPIIIPRLFIKRMGI DEGDPLWKQVMLIDDKRALRPMLAPNLYVLMAKLSNIVRPVKIFEIGP CFRRETGGRYHLEEFTMFNMVELAPEGDPKERLLDYIDTIMRDIGLNY TISVEPSNVYGETLDVVVNGIEVASAAIGPKPIDANWGVREPWIGVGF GVERLAMLVGGYNSIARIAKSLSYLDGSTLSVIKLRW (SEQ ID NO: 3)

[00125] To aid in alignment, a version of the Nitra PylRS sequence is provided below, in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, 401 in the Mm PylRS sequence. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way. MSKIRFTRGQIHRLIELGAEPTELERDFETEAERDKEFNKIAENLARKN LKNIKDFLEQRRKPLVRVIEEKLRTTALRLGFSEVVTPIIIPRLFIKRMGI DEGDPLWKQVMLIDDKRALRPXLXPNXXVLXAKLSNIVRPVKIFEIGP CFRRETGGRYHLEEFTMFXMXELAPEGDPKERLLDYIDTIMRDIGLNY TISVEPSNVXGETLDVVVNGIEVASAXIGPKPIDANWGVREPWIGVGF GVERLAMLVGGYNSIARIAKSLSYLDGSTLSVIKLRW (SEQ ID NO: 4)

[00126] Mutations can be applied to the PylRS of the bacterium Clostridiales (Clos). This sequence is provided below. MENFTITQTERLKQLNCENDVLELEFEDSEARNSKFREIEIGRVKKGK ENIKNLLKEKHITISDEVGNKLSDWLMSKDYTKVLTPTIISKDQLKAMTI DEENHLFSQVFWIDNNKCLRPMLAPNLYIVMRELKRITNEPVKIFEIGS CFRKESQGARHMNEFTMLNMVELASVEDGKQLDTLKALAHEAMESL GVESYELVIEESAVYGSTLDIEIDGIEVASGSYGPHELDANWDIFDTW VGIGFGIERLAMAINGGSTIKKYGRSINFIDGETMKL (SEQ ID NO: 5)

[00127] To aid alignment, a version of the ClosPylRS sequence is provided below, in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384 and 401 in the MmPylRS sequence. Therefore, these are positions to which the respective mutations of the present invention can be applied. Other Petition 870250088848, dated 09 / 30 / 2025, page 89 / 242 75 / 141 corresponding positions can be identified in the same way. MENFTITQTERLKQLNCENDVLELEFEDSEARNSKFREIEIGRVKKGK ENIKNLLKEKHITISDEVGNKLSDWLMSKDYTKVLTPTIISKDQLKAMTI DEENHLFSQVFWIDNNKCLRPXLXPNXXIVXRELKRITNEPVKIFEIGS CFRKESQGARHMNEFTMLXMXELASVEDGKQLDTLKALAHEAMESL GVESYELVIEESAVXGSTLDIEIDGIEVASGXYGPHELDANWDIFDTW VGIGFGIERLAMAINGGSTIKKYGRSINFIDGETMKL (SEQ ID NO: 6)

[00128] Mutations can be applied to the PylRS of Methanomethylophilus sp.1R26 (1R26). This sequence is provided below. MAEHFTDAQIQRLREYGNGTYKDMEFADVSAREKAFTKLMSDASRD NESALKGMIAHPARQGLSRLMNDIADALVADGFIEVRTPIIISKDALAK MTITPDKPLFKQVFWIDDKRALRPMLAPSLYTVMRSLRDHTDGPVKIF EMGSCFRKESHSGMHLEEFTMLNLVDMGPAGDATESLKKYIGIVMK AAGLPDYQLVHEESDVYKETIDVEINGQEVCSAAVGPHYLDAAHDVH EPWAGAGFGLERLLTIRQGYSTVMKGGASTTYLNGAKMD (SEQ ID NO: 7)

[00129] To aid in alignment, a version of the 1R26 PylRS sequence is provided below, where an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, 401 in the Mm PylRS sequence. Therefore, these are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way. MAEHFTDAQIQRLREYGNGTYKDMEFADVSAREKAFTKLMSDASRD NESALKGMIAHPARQGLSRLMNDIADALVADGFIEVRTPIIISKDALAK MTITPDKPLFKQVFWIDDKRALRPXMXAPXXYTXMRSLRDHTDGPVK IFEMGSCFRKESHSGMHLEEFTMLXLXDMGPAGDATESLKKYIGIVM KAAGLPDYQLVHEESDVXKETIDVEINGQEVCSAXVGPHYLDAAHDV HEPWAGAGFGLERLLTIRQGYSTVMKGGASTTYLNGAKMD (SEQ ID NO: 8) Petition 870250088848, dated 09 / 30 / 2025, page 90 / 242 76 / 141

[00130] Mutations can be applied to the PylRS of Methanomassiliicoccus luminyensis 1 (Lum1). This sequence is provided below. MDTRLTPAQAQRIREMGGTVDPSLAFSSEAERESAFQRISADLQGAN LAKIRRCAEAPERHPIGSLENTLACALAAKGFIEVKTPMMIPADGLVK MGIDESHPLWNQVFWVGPKKALRPMLAPNLYFLMRHLRRSVPAPLL LFEIGPCFRKERGSNHLEEFTMLNLVELAPQADATERLKEHIATVMNA VGLPYELVVEGSEVYGTTIDVEVDGCANDLESGAVGPLPMDPHGITE PWAGVGFGLERIALMRTKEQNIKKVGRSLVYVNGARIDI (SEQ ID NO: 9)

[00131] To aid alignment, a version of the Lum1 PylRS sequence is provided below, in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, 401 in the Mm PylRS sequences. Therefore, these are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way. MDTRLTPAQAQRIREMGGTVDPSLAFSSEAERESAFQRISADLQGAN LAKIRRCAEAPERHPIGSLENTLACALAAKGFIEVKTPMMIPADGLVK MGIDESHPLWNQVFWVGPKKALRPXLXPNXXFLXRHLRRSVPAPLLL FEIGPCFRKERGSNHLEEFTMLXLXELAPQADATERLKEHIATVMNAV GLPYELVVEGSEVXGTTIDVEVDGVELASGXVGPLPMDPHGITEPWA GVGFGLERIALMRTKEQNIKKVGRSLVYVNGARIDI (SEQ ID NO: 10)

[00132] Other main chains to which mutations of the present invention can be applied include Mb PylRS (SEQ ID NO: 11), Lum1 PylRS (SEQ ID NO: 12), TronPylRS (SEQ ID NO: 13), Gemm PylRS (SEQ ID NO: 14), PGA8 PylRS (SEQ ID NO: 15), I2 PylRS (SEQ ID NO: 16), D121 PylRS (SEQ ID NO: 17) and D416 PylRS (SEQ ID NO: 18).

[00133] Thus, in one embodiment, a syn-acyl-tRNA is provided. Petition 870250088848, dated 09 / 30 / 2025, page 91 / 242 77 / 141 synthase capable of specifically acylating a tRNA with a beta amino acid, wherein said synthase comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% similarity or identity to the amino acid sequence of any of the SEQ ID NOs: 1 to 18, and said amino acid sequence comprising any of the mutations and, optionally, wild-type residues, or sets of mutations and, optionally, wild-type residues, described in relation to the first aspect. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95% or 99% identity to the amino acid sequence of any of the SEQ ID NOs: 1 to 18. In one embodiment, the amino acid sequence may be identical to any of the SEQ ID NOs: 1 to 18, except for the mutations mentioned.

[00134] In a specific embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, said synthetase comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% similarity or identity to the amino acid sequence of any of the SEQ ID NOs: 1 to 10 and said amino acid sequence comprising any of the mutations and, optionally, wild-type residues, or sets of mutations and, optionally, wild-type residues, described in relation to the first aspect. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of any of the SEQ ID NOs: 1 to 10. In one embodiment, the amino acid sequence may be identical to any of the SEQ ID NOs: 1 to 10, except for the mutations mentioned.

[00135] Thus, acyl-tRNA synthetase may comprise an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the sequence of Petition 870250088848, dated 09 / 30 / 2025, p. 92 / 242 78 / 141 amino acids from any of the SEQ ID NOs: 1 to 18 and comprising mutations corresponding to an M300D mutation, an A302H, A302Y or A302C mutation and an N346G, N346A or N346S mutation in SEQ ID NO: 1 (as indicated by the relevant X in SEQ ID NOs: 2, 4, 6, 8 or 10). The amino acid sequence may include mutations corresponding to mutations in any of the 12_1, 12_2, 12_3, 12_4, 12_A, 12_B, 12_C, or 12_D sets in Table 1. The amino acid sequence may include residues corresponding to mutations and wild-type residues in any of the 12_1, 12_2, 12_3, 12_4, 12_A, 12_B, 12_C, or 12_D sets in Table 1. The amino acid sequence may include residues corresponding to the sets of mutations in any of Tables 1 through 6. The amino acid sequence may include residues corresponding to the sets of mutations and wild-type residues in any of Tables 1 through 6.labeled as A1_1, A1_2, A1_3, A1_4, A1_5, A1_6, the amino acid sequence may also include mutations corresponding to the mutations listed as sets a), b), c), d), e) or f) here.

[00136] In a preferred embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: M300D, A302H, L305H, N307K, N346G, C348F, F366L, and V401C relative to SEQ ID NO: 1. The acyl-tRNA synthetase may comprise an amino acid sequence comprising 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 any of the SEQ ID Nos: 1 to 18. The amino acid sequence may have Petition 870250088848, dated 09 / 30 / 2025, p. 93 / 242 79 / 141 at least 80%, 85%, 90%, 95% or 99% identity with the amino acid sequence of any of the SEQ ID NOS: 1 to 10.

[00137] First-aspect synthetases may comprise sequence changes relative to the wild-type sequence, in addition to the mutations described in more detail herein. Specifically, synthetases may comprise sequence changes at locations that do not significantly compromise the function or operation of the synthetase, as described herein. The function of the synthetase can be tested by operating on it as described, as in the examples section, in order to verify that the function has not been overridden or significantly altered. Thus, provided that the synthetase maintains its function, which can be tested as set out herein, sequence variations may be made to the synthetase relative to the wild-type reference sequence.

[00138] Fig. 35 provides a sequence alignment 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. The synthase can be at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any of the SEQ ID NOs: 1 to 18, where variation is found only in residues not labeled with: i) *; ii) * or :, or iii) or *, : or ., in addition to the specifically cited mutations discussed here.

[00139] Conservative substitutions can be made, for example, according to the table below. Amino acids in the same block in the second column and preferably in the same row in the third column can be substituted for each other: Petition 870250088848, dated 09 / 30 / 2025, pp. 94 / 242 80 / 141 Table 6.1 Aliphatic Non-polar Gapilv Polar - uncharged CSTMNQ Polar - charged Dekr Aromatic HFWY

[00140] The first aspect acyl-tRNA synthetase can be isolated or purified. The first aspect acyl-tRNA synthetase may be a non-natural acyl-tRNA synthetase.

[00141] In a second aspect, the use of an acyltRNA synthetase in a method for generating a polymer comprising a beta amino acid is provided.

[00142] In one embodiment, a method is provided for producing a polymer comprising a beta amino acid, wherein the method comprises: i) the use of an acyl-tRNA synthetase to acylate a tRNA with the beta amino acid, wherein the acyl-tRNA synthetase comprises mutations to allow the acylation of a tRNA with a beta amino acid, and ii) the incorporation of the beta amino acid into a polymer.

[00143] In one embodiment, the use of an acyl-tRNA synthetase of the first aspect is provided in a method for generating a polymer comprising a beta amino acid.

[00144] In one embodiment, a method is provided for producing a polymer comprising a beta amino acid, wherein the method comprises: i) the use of a first-aspect acyl-tRNA synthetase to acylate a tRNA with the beta amino acid, and ii) the incorporation of the beta amino acid into a polymer. Petition 870250088848, dated 09 / 30 / 2025, page 95 / 242 81 / 141

[00145] The beta amino acid may be an analogue 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^MDF), (S)-3-amino-3(4-bromophenyl)propanoic acid ((S^^BrF), (S))-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S^pmFF), (S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^oBrF) or (S)-3-amino-3-(3(trifluoromethyl)phenyl)propanoic acid ((S)β3mCF3F).

[00146] The polymer may comprise one or more canonical amino acids or one or more naturally occurring amino acids. The polymer may comprise one or more non-natural amino acids and / or hydroxy acids. The non-natural amino acid may be an α-amino acid and / or the hydroxy acid may be an α-hydroxy acid.

[00147] The polymer can be formed by the genetic incorporation of monomers using a ribosome. The polymer can be formed by the translation of a nucleic acid sequence by a ribosome. The translation may comprise the binding of a tRNA charged with the beta amino acid to a ribosome and the formation of a linkage between the beta amino acid and a preceding and / or subsequent monomer. Thus, the method may comprise providing a nucleic acid sequence encoding a polymer and using charged tRNAs and a ribosome to translate said sequence to form the polymer.

[00148] The use or method can be performed on a cell. For example, a prokaryotic cell, a bacterial cell, or an E. coli cell.

[00149] In a third aspect, a nucleic acid encoding an acyl-tRNA synthetase from the first aspect is provided. The nucleic acid may be DNA. A vector may comprise the nucleic acid.

[00150] In a fourth aspect, a cell comprehends Petition 870250088848, dated 09 / 30 / 2025, p. 96 / 242 82 / 141 dendo uma acil-tRNA synthetase de primeiro aspecto, umácido nucleic do terceiro aspecto ou um vetor planejadondo umácido nucleic do third aspecto.

[00151] In one particular embodiment, the cell comprises or expresses a first-aspect acyl-tRNA synthetase, and the acyl-tRNA synthetase is orthogonal to endogenous tRNAs. Thus, the acyl-tRNA synthetase does not acylate endogenous tRNAs to an extent that would render the cell unviable. In one embodiment, cell fitness, for example, as measured by proliferation, is reduced by less than 50%, less than 25%, less than 10%, less than 5%, or not reduced at all when the acyl-tRNA synthetase is expressed. disubstituted α,α-amino acid-acyl-tRNA synthetases

[00152] The inventors used the methods and tools described herein and identified acyl-tRNA synthetases capable of acylating tRNAs with disubstituted α,α-amino acids.

[00153] Thus, in a fifth aspect, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a disubstituted α,α-amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: A302C, A302G, A302H or A302S with respect to SEQ ID NO: 1; and N346A, N346C, N346E, N346G, N346T or N346V with respect to SEQ ID NO: 1.

[00154] The definition of specific acylation is given in the section relating to the first aspect.

[00155] In one specific embodiment, the amino acid sequence comprises A302G or A302H and / or N346A, N346C or N346G.

[00156] The amino acid sequence may comprise a mutation at a position corresponding to M300 relative to SEQ ID NO: 1. The mutation may be M300A, M300C, M300D, M300E, M300S Petition 870250088848, dated 09 / 30 / 2025, p. 97 / 242 83 / 141 or M300T. In some strains, the residue is wild type (M300M). The most common mutation in screening was M300D. In specific strains, the mutation is M300D.

[00157] The amino acid sequence may comprise a mutation at a position corresponding to L305 relative to 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 the screening was L305C.

[00158] The amino acid sequence may comprise a mutation at a position corresponding to Y306 relative to SEQ ID NO: 1. The mutation may be Y306D, Y306F, Y306L, or Y306N. In some embodiments, the residue is wild-type (Y306Y). In specific embodiments, the amino acid sequence comprises Y306F or Y306Y.

[00159] The amino acid sequence may comprise a mutation at a position corresponding to L309 relative to 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 specific embodiments, the amino acid sequence comprises L309F or L309L.

[00160] The amino acid sequence may comprise a mutation at a position corresponding to M344 relative to 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.

[00161] The amino acid sequence may comprise a mutation at a position corresponding to C348 relative to SEQ ID NO: 1. The mutation may be C348F, C348G, C348H, C348I, C348L, C348S, or C348V. In some embodiments, the residue is of the jungle type. Petition 870250088848, dated 09 / 30 / 2025, page 98 / 242 84 / 141 gem (C348C). In specific modalities, the amino acid sequence comprises C348G or C348C. The most common mutation in the screening was C348G.

[00162] The amino acid sequence may comprise the wild-type residue at a position corresponding to Y384 of SEQ ID NO: 1. This may be referred to as comprising Y384Y.

[00163] The amino acid sequence may comprise the wild-type residue at a position corresponding to S399 of SEQ ID NO: 1. This may be referred to as comprising S399S.

[00164] The amino acid sequence may comprise a mutation at a position corresponding to V401 of SEQ ID NO: 1. In the examples, the mutation is V401A, V401C, V401K, V401L, or a conservative substitution of said residues. More particularly, the mutation may be V401C or V401L. In some embodiments, the residue is wild-type (V401V). In specific embodiments, the amino acid sequence comprises V401C, V401L, or V401V.

[00165] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a disubstituted α,α-amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D; A302C, A302G, A302H or A302S; or more particularly A302G or A302H; and N346A, N346C, N346E, N346G, N346T or N346V; or more particularly N346A, N346C, N346G.

[00166] In a specific embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a disubstituted α,α-amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: Petition 870250088848, dated 09 / 30 / 2025, page 99 / 242 85 / 141 M300D; A302G or A302H; and N346A, N346C or N346G.

[00167] An acyl-tRNA synthetase capable of specific acylation of a tRNA with a disubstituted α,α-amino acid is provided here, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D; A302G or A302H; an L305 mutation and an N346 mutation (for any residue).

[00168] In a specific embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a disubstituted α,α-amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D; A302G or A302H; an L305 mutation, a Y306 mutation, an L309 mutation, an N346 mutation, and a C348 mutation; and, optionally, a V401 mutation.

[00169] In a specific embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a disubstituted α,α-amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D; A302H; N346A or N346G; and C348G.

[00170] In a specific embodiment, an acyl-tRNA is provided. Petition 870250088848, dated 09 / 30 / 2025, pages 100 / 242 86 / 141 synthetase capable of specifically acylating a tRNA with a disubstituted α,α-amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D; A302G or A302H; L305C, L305F or L305S; N346A, N346C or N346G; and C348C or C348G.

[00171] In a specific embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a disubstituted α,α-amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: 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.

[00172] In a specific embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a disubstituted α,α-amino acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300D; A302G or A302H; L305C; Petition 870250088848, dated 09 / 30 / 2025, pp. 101 / 242 87 / 141 Y306F; L309F N346A or N346G; and C348G.

[00173] In a specific embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a disubstituted α,α-amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: 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.

[00174] The disubstituted α,α-amino acid can be (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S ^-Me-pIF).

[00175] The inventors provide specific embodiments in Table 7. These embodiments are defined in relation to SEQ ID NO: 1. For example, A6_2 comprises M300D, A302H, L305S, etc. Petition 870250088848, dated 09 / 30 / 2025, pp. 102 / 242 88 / 141 Table 7 - Mutations of tRNA synthetases of a,a-disubstituted amino acids exemplary hit name M300 A302 L3O5iT3Qà Liter M344 N346 C34S HS4 S3S9 V401 A6_2 - DHS Ί FGG r ί d AFL 1 - gu i.1' V It K A6_4 - ogcff AGL A6 _5 - OGCFFAG Α6_θ - 0 hc · ' AGC A6J ~ 0 HF r 1 C i[ L A6_7 - D 14 CFFGG 1 r A6_8 - o Η Γ FGG - A6_9 — DGC FFGDL 13 'VIGDL : AND -·. ,r ; A OS NGGFL CHI Ή TL [ ? Η 1 Ί HV $ c OHSFAG 1 SHF h 11 A ii LAHLVQGF — Õ H 1 NV ,1 6 V li OC _ FVA 1 ir '> S Η HL r. 1 V IL HHA 1

[00176] Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations in Table 7. For example, acyl-tRNA synthetase may comprise an amino acid sequence comprising residues corresponding to M300D, A302H, L305S, L309F, N346G, and C348G. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 7. For example, acyl-tRNA synthetase may comprise an amino acid sequence comprising residues corresponding to M300D, A302H, L305S, Y306Y, L309F, M344M, N346G, C348G, Y384Y, S399S, and V401V.

[00177] Acyl-tRNA synthetase can comprise an amino acid sequence comprising any of the sets of mutations Petition 870250088848, dated 09 / 30 / 2025, pp. 103 / 242 89 / 141 labeled as A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8 or A6_9 in Table 7 (for example, M300D, A302H, L305S, L309F, N346G and C348G for A6_2). Acyl-tRNA synthetase can comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues labeled A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7 (e.g., M300D, A302H, L305S, Y306Y, L309F, M344M, N346G, C348G, Y384Y, S399S, and V401V for A6_2).

[00178] The fifth aspect mutations are provided with reference to SEQ ID NO: 1, which is the wild-type Methanosarcina mazei pyrrolisyl-tRNA synthetase (MmPylRS) sequence and is provided here. To aid alignment, a version of the MmPylRS sequence where an X is marked at positions 300, 302, 305, 306, 309, 346, 348, 384, 401 is provided as SEQ ID NO: 2.

[00179] Thus, in one embodiment, an acyl-tRNA synthetase is provided that is capable of specific acylation of a tRNA with a disubstituted α,α-amino acid, wherein said synthetase comprises an amino acid sequence with 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 said amino acid sequence comprising any of the mutations or sets of mutations described in relation to the fifth aspect. 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, except for the mutations mentioned.

[00180] Acyl-tRNA synthetase may comprise an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% similarity or identity to the amino acid sequence Petition 870250088848, dated 09 / 30 / 2025, pp. 104 / 242 90 / 141 of those from SEQ ID NO: 1 or SEQ ID NO: 2 and comprising M300D; A302G or A302H; and N346A, N346C or N346G. The acyl-tRNA synthetase may comprise an amino acid sequence with 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 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 include mutations corresponding to mutations from any of the sets of mutations in Table 7. The amino acid sequence may include mutations corresponding to mutations from any of the sets of mutations 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 comprise residues corresponding to mutations and wild-type residues from any of the following groups: A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8, or A6_9 in Table 7.

[00181] A skilled individual would also be able to apply the identified mutations to other PylRS backbones, including backbones not described in this document. This is despite the fact that other PylRSs may have low sequence identity with MmPylRS. To apply the cited mutations with reference to MmPylRS to other synthases, the sequences must be aligned to identify the corresponding residues. In particular, the sequences representing the catalytic sites of the synthases can be aligned. Thus, the SEQ ID NO: 1 region, which extends from residue 296 to residue 428, can be aligned to the sequence of another PylRS, and the corresponding mutations can be performed. More information on transferring mutations from one PylRS backbone to another is provided in the document. Petition 870250088848, dated 09 / 30 / 2025, p. 105 / 242 91 / 141 WO2013 / 171485 A1 (incorporated herein by reference). An exemplary alignment is provided in Figure 35.

[00182] Thus, in one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a disubstituted α,α-amino acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising any mutations and, optionally, wild-type residues described in relation to the fifth aspect, or any set of mutations and, optionally, wild-type residues described in relation to the fifth aspect, and wherein the acyl-tRNA synthetase backbone is a PylRS.

[00183] Mutations can be applied to the PylRS of Nitrososphaeria archaeon (Nitra). This sequence is provided here as SEQ ID NO: 3. To aid in alignment, a version of the Nitra PylRS sequence is provided here (SEQ ID NO: 4), in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384 and 401 in the Mm PylRS sequence. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00184] Mutations can be applied to the PylRS of the bacterium Clostridiales (Clos). This sequence is provided as SEQ ID NO: 5. To aid in alignment, a version of the Clos PylRS sequence is provided here (SEQ ID NO: 6), in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384 and 401 in the Mm PylRS sequence. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00185] Mutations can be applied to the PylRS of Methanomethylophilus sp.1R26 (1R26). This sequence is provided here (SEQ ID NO: 7). To aid alignment, a version of the sequence of Petition 870250088848, dated 09 / 30 / 2025, pp. 106 / 242 92 / 141 1R26 PylRS is provided herein (SEQ ID NO: 8), in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384 and 401 in the Mm PylRS sequence. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00186] Mutations can be applied to the PylRS of Methanomassiliicoccus luminyensis 1 (Lum1). This sequence is provided in this document (SEQ ID NO: 9). To aid in alignment, a version of the Lum1 PylRS sequence is provided in this document (SEQ ID NO: 10), in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the MmPylRS sequences. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00187] Other main chains to which mutations of the present invention can be applied include Mb PylRS (SEQ ID NO: 11), Lum1 PylRS (SEQ ID NO: 12), TronPylRS (SEQ ID NO: 13), Gemm PylRS (SEQ ID NO: 14), PGA8 PylRS (SEQ ID NO: 15), I2 PylRS (SEQ ID NO: 16), D121 PylRS (SEQ ID NO: 17) and D416 PylRS (SEQ ID NO: 18).

[00188] Thus, in one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with an α,α-disubstituted amino acid, said synthetase 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 any of the SEQ ID NOs: 1 to 18 and said amino acid sequence comprising any of the mutations and, optionally, wild-type residues, or sets of mutations and, optionally, wild-type residues, described in relation to Petition 870250088848, dated 09 / 30 / 2025, p. 107 / 242 93 / 141 fifth aspect. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of any of the SEQ ID NOs: 1 to 18. In one embodiment, the amino acid sequence may be identical to any of the SEQ ID NOs: 1 to 18, except for the mutations mentioned.

[00189] In a particular embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a disubstituted α,α-amino group, said synthetase 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 any of the SEQ ID NOs: 1 to 10 and said amino acid sequence comprising any of the mutations and, optionally, wild-type residues, or sets of mutations and, optionally, wild-type residues, described in relation to the fifth aspect. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of any of the SEQ ID NOs: 1 to 10. In one embodiment, the amino acid sequence may be identical to any of the SEQ ID NOs: 1 to 10, except for the mutations mentioned.

[00190] Thus, in examples, acyl-tRNA synthetase may comprise 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 any of the SEQ ID NOs: 1 to 18 and comprising any of the sets of mutations in Table 7 or any of the sets of mutations 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 comprise residues corresponding to the mutations and wild-type residues from any of the sets in Table 7 or any of the sets of mutations labeled Petition 870250088848, dated 09 / 30 / 2025, pp. 108 / 242 94 / 141 as A6_1, A6_2, A6_4, A6_5, A6_6, A6_7, A6_8 or A6_9 in Table 7.

[00191] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with an α,α-disubstituted amino acid, said synthetase 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 any of the SEQ ID NOs: 1 to 18 and said amino acid sequence comprising: M300D with respect to SEQ ID NO: 1; A302C, A302G, A302H or A302S with respect to SEQ ID NO: 1; or more particularly A302G or A302H; and N346A, N346C, N346E, N346G, N346T or N346V in relation to SEQ ID NO: 1; or more particularly N346A, N346C, N346G. In one embodiment, the amino acid sequence comprises 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.

[00192] Fifth-aspect synthases may comprise sequence changes relative to the wild-type sequence, in addition to the mutations described in more detail herein. Specifically, synthases may comprise sequence changes at sites that do not significantly compromise the function or operation of the synthase, as described in this document. The function of the synthase can be tested by operating on it as described, as in the examples section, in order to verify that the function has not been abolished or significantly altered. Thus, provided that the synthase maintains its function, which can be tested as set forth in this document, sequence variations may be made to the synthase relative to the wild-type reference sequence.

[00193] As discussed in relation to the first aspect, the Figure Petition 870250088848, dated 09 / 30 / 2025, p. 109 / 242 95 / 141 provides a sequence alignment. The synthase can be at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any of the SEQ ID Nos: 1 to 18, where variation is found only in residues not labeled with: i) *; ii) * or :, or iii) or *,: or ., in addition to the mutations specifically cited and discussed in this document.

[00194] Conservative substitutions can be made, for example, according to the table described in relation to the first aspect.

[00195] The fifth aspect acyl-tRNA synthetase can be isolated or purified. The fifth aspect acyl-tRNA synthetase may be a non-natural acyl-tRNA synthetase.

[00196] In a sixth aspect, the use of an acyl-tRNA synthetase is provided in a method for generating a polymer comprising an α,α-disubstituted amino acid.

[00197] In one embodiment, a method is provided for producing a polymer comprising an α,α-disubstituted amino acid, wherein the method comprises: i) the use of an acyl-tRNA synthetase to acylate a tRNA with the α,α-disubstituted amino acid, wherein the acyl-tRNA synthetase comprises mutations to allow the acylation of a tRNA with the α,α-disubstituted amino acid, and ii) the incorporation of the α,α-disubstituted amino acid into a polymer.

[00198] In one embodiment, the use of a fifth-aspect acyl-tRNA synthetase is provided in a method for generating a polymer comprising an α,α-disubstituted amino acid.

[00199] In one embodiment, a method is provided for producing a polymer comprising an α,α-disubstituted amino acid, wherein the method comprises: i) the use of a fifth-aspect acyl-tRNA synthetase for Petition 870250088848, dated 09 / 30 / 2025, page 110 / 242 96 / 141 acylate a tRNA with the α,α-disubstituted amino acid, and ii) the incorporation of the α,α-disubstituted amino acid into a polymer.

[00200] The α,α-disubstituted amino acid can be (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S ^-Me-pIF).

[00201] The polymer may comprise one or more canonical amino acids or one or more naturally occurring amino acids. The polymer may comprise one or more non-natural amino acids and / or hydroxy acids. The non-natural amino acid may be an α-amino acid and / or the hydroxy acid may be an α-hydroxy acid.

[00202] The polymer can be formed by the genetic incorporation of monomers using a ribosome. The polymer can be formed by the translation of a nucleic acid sequence by a ribosome. The translation may comprise the binding of a tRNA charged with the α,α-disubstituted amino acid to a ribosome and the formation of a linkage between the α,α-disubstituted amino acid and a preceding and / or subsequent monomer. Thus, the method may comprise providing a nucleic acid sequence encoding a polymer and using charged tRNAs and a ribosome to translate said sequence to form the polymer.

[00203] Thus, a method is provided for the production of a polymer comprising the genetic incorporation of at least one α,α-disubstituted amino acid, which may be (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S^-Me-pIF).

[00204] The use or method can be performed on a cell. For example, a prokaryotic cell, a bacterial cell, or an E. coli cell.

[00205] In a seventh aspect, a nucleic acid encoding an acyl-tRNA synthetase from the fifth aspect is provided. The nucleic acid may be DNA. A vector may comprise the nucleic acid. Petition 870250088848, dated 09 / 30 / 2025, p. 111 / 242 97 / 141

[00206] In an eighth aspect, a cell comprising an acyl-tRNA synthetase from the fifth aspect, a nucleic acid from the seventh aspect, or a vector comprising a nucleic acid from the seventh aspect is provided.

[00207] In one particular embodiment, the cell comprises or expresses a fifth-aspect acyl-tRNA synthetase, and the acyl-tRNA synthetase is orthogonal to endogenous tRNAs. Thus, the acyl-tRNA synthetase does not acylate endogenous tRNAs to the point of rendering the cell unviable. In one embodiment, cell fitness, for example, as measured by proliferation, is reduced by less than 50%, less than 25%, less than 10%, less than 5%, or is not reduced at all when the acyl-tRNA synthetase is expressed. Beta-hydroxy-acyl-tRNA synthetases

[00208] The inventors used the methods and tools described herein and identified acyl-tRNA synthetases capable of acylating tRNAs with beta-hydroxy acids.

[00209] Thus, in a ninth aspect, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300A, M300D, M300M, M300N or M300S with respect to SEQ ID NO: 1; and A302D, A302G, A302H or A302N with respect to SEQ ID NO: 1.

[00210] The definition of specific acylation is given in the section relating to the first aspect.

[00211] In particular, acyl-tRNA synthetase may comprise a residue corresponding to M300N or M300S. M300N was the most common in the screening. Acyl-tRNA synthetase may comprise a residue corresponding to A302H. A302H was the most common in the screening. Acyl-tRNA synthetase may comprise residues corresponding to both M300N and A302H. Petition 870250088848, dated 09 / 30 / 2025, pp. 112 / 242 98 / 141

[00212] The amino acid sequence may comprise a mutation at a position corresponding to N346 relative to 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 specific embodiments, the mutation is N344A, N346C, or N346G.

[00213] The amino acid sequence may comprise a mutation at a position corresponding to L305 relative to SEQ ID NO: 1. The mutation may be L305C, L305N, L305S, or L305V. In some embodiments, the residue is wild-type (L305L). In specific embodiments, the amino acid sequence may comprise L305L or L305V. The most common residue in the screening was the wild-type.

[00214] The amino acid sequence may comprise a mutation at a position corresponding to Y306 relative to SEQ ID NO: 1. The mutation may be Y306D, Y306F, Y306I, Y306N, Y306R, or Y306S. In some embodiments, the residue is wild type (Y306Y). The most common residue in screening was the wild type, but remarkably high selectivity was achieved by a variant including Y306N.

[00215] The amino acid sequence may comprise a mutation at a position corresponding to L309 relative to 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 remarkably high selectivity was achieved by a variant including L309I.

[00216] The amino acid sequence may comprise a mutation at a position corresponding to M344 relative to 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. Petition 870250088848, dated 09 / 30 / 2025, pp. 113 / 242 99 / 141

[00217] The amino acid sequence may comprise a mutation at a position corresponding to C348 relative to 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 in the screening.

[00218] The amino acid sequence may comprise the wild-type residue at a position corresponding to Y384 of SEQ ID NO: 1. This may be referred to as comprising Y384Y.

[00219] The amino acid sequence may comprise the wild-type residue at a position corresponding to S399 of SEQ ID NO: 1. This may be referred to as comprising S399S.

[00220] The amino acid sequence may comprise a mutation at a position corresponding to V401 of SEQ ID NO: 1. In examples, 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).

[00221] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to M300N and A300H and, optionally, mutation(s) at position(s) N346 and / or C348.

[00222] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300A, M300D, M300M, M300N or M300S; more particularly M300N or M300S; A302D, A302G, A302H or A302N; more particularly A302H; and N346A, N346C, N346E, N346G, N346S or N346V; more particularly Petition 870250088848, dated 30 / 09 / 2025, p. 114 / 242 100 / 141 mind N346A, N346C or N346G.

[00223] In one embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta-hydroxy acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300N or M300S; more particularly, M300N; A302H; and N346A, N346C or N346G; more particularly, N346G.

[00224] In one embodiment, an acyl-tRNA synthetase capable of specific acylation of a tRNA with a beta-hydroxy acid is provided, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to M300N, A302H and N346G; optionally, further comprising a C348 mutation and / or a V401 mutation.

[00225] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300N or M300S; A302H; N346A, N346C or N346G; and a C348 mutation and / or a V401 mutation.

[00226] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300N or M300S; A302H; N346A, N346C or N346G; and a C348 mutation, optionally C348I, C348L, C348T or C348V. Petition 870250088848, dated 09 / 30 / 2025, pp. 115 / 242 101 / 141

[00227] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300N; A302H; and N346G; and a C348 mutation and / or a V401 mutation; optionally wherein the C348 mutation is C348I, C348L, C348T or C348V; and / or wherein the V401 mutation is V401C, V401K, V401L or V401V.

[00228] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300N or M300S; A302H; N346A, N346C or N346G; and C348I, C348L, C348T or C348V; and optionally V401C, V401K, V401L or V401V.

[00229] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300N; A302H; N346A, N346C or N346G; and C348I, C348L or C348V.

[00230] Beta-hydroxy acid can be (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S^mClF).

[00231] The inventors provide particular embodiments in Table 8. These embodiments are defined in relation to SEQ ID NO: 1. For example, A7_2 comprises M300N, A302H, L305L, etc. Petition 870250088848, dated 09 / 30 / 2025, pp. 116 / 242 102 / 141 Table 8 - Exemplary beta-hydroxy-acid-tRNA synthetase mutations M3O0 A3Q2 L3ÜS Y306 1309 M344 N34& 048 Y384 S399 V401 A7_2 NH il NGLL A7_3 - NH 1 / 1 NH G 1 C A7_4 SH li 1 EAT - A7_5 - N Η II 1 Q cr K A7_6 AH iL Q | AL A7_7 N Η V 1' SGIDGSODQ _ s NNNRHQV 5 DN 5 QVV : s DHCF , 5 VADHL 1 R it F 1 TS Η N r η AI íi KC j 1 QE v ​​L

[00232] Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations in Table 8. For example, acyl-tRNA synthetase may comprise an amino acid sequence comprising residues corresponding to M300N, A302H, Y306N, N346G, C348L, and V401L. Acyl-tRNA synthetase may comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues in Table 8. For example, acyl-tRNA synthetase may comprise an amino acid sequence comprising residues corresponding to M300N, A302H, L305L, Y306N, L309L, M344M, N346G, C348L, Y384Y, S399S, and V401L.

[00233] Acyl-tRNA synthetase can comprise an amino acid sequence comprising any of the sets of mutations labeled A7_1, A7_2, K7_A, A7_5, or K7_7 in Table 8. Acyl-tRNA synthetase can comprise an amino acid sequence comprising any of the sets of mutations and wild-type residues labeled A7_1, A7_2, A7_3, A7_4, A7_5, or K7_7 in Table 8. Petition 870250088848, dated 09 / 30 / 2025, pp. 117 / 242 103 / 141

[00234] The ninth aspect mutations are provided with reference to SEQ ID NO: 1, which is the sequence of the wild-type Methanosarcina mazei pyrrolisyl-tRNA synthetase (MmPylRS) and provided in this document. To aid alignment, a version of the MmPylRS sequence where an X is marked at positions 300, 302, 305, 306, 309, 346, 348, 384, 401 is provided as SEQ ID NO: 2.

[00235] Thus, in one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein said synthetase comprises 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, and said amino acid sequence comprising any of the mutations or sets of mutations described in relation to the ninth aspect. 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, except for the mutations mentioned.

[00236] The acyl-tRNA synthetase may comprise 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 and comprising M300A, M300D, M300M, M300N or M300S; more particularly M300N or M300S; A302D, A302G, A302H or A302N; more particularly, A302H; and N346A, N346C, N346E, N346G, N346S or N346V; more particularly, N346A, N346C or N346G. The acyl-tRNA synthetase may comprise 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: Petition 870250088848, dated 09 / 30 / 2025, pp. 118 / 242 104 / 141 and comprising M300N or M300S; more particularly, M300N; A302H; and N346A, N346C or N346G; more particularly, N346G. The amino acid sequence may comprise residues corresponding to mutations and wild-type residues from any of the following groups: A7_1, A7_2, A7_3, A7_4, A7_5 and A7_7 in Table 8.

[00237] A skilled individual would also be able to apply the identified mutations to other PylRS structures, including structures not described in this document. This is despite the fact that other PylRSs may have low sequence identity with MmPylRS. To apply the cited mutations with reference to MmPylRS to other synthases, the sequences must be aligned to identify the corresponding residues. In particular, the sequences representing the catalytic sites of the synthases can be aligned. Thus, the SEQ ID NO: 1 region extending from residue 296 to residue 428 can be aligned to the sequence of another PylRS, and the corresponding mutations can be performed. More information on transferring mutations from one PylRS core structure to another is provided in document WO2013 / 171485 A1 (incorporated here by reference). An exemplary alignment is provided in Figure 35.

[00238] Thus, in one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising any mutations and, optionally, wild-type residues described in relation to the ninth aspect, or any set of mutations and, optionally, wild-type residues described in relation to the ninth aspect, and wherein the main structure of the acyl-tRNA synthetase is a PylRS.

[00239] Mutations can be applied to PylRS of Nitrososphaeria archaeon (Nitra). This sequence is provided in this document. Petition 870250088848, dated 09 / 30 / 2025, pp. 119 / 242 105 / 141 as SEQ ID NO: 3. To aid in alignment, a version of the Nitra PylRS sequence is provided in this document (SEQ ID NO: 4), in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384 and 401 in the Mm PylRS sequence. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00240] Mutations can be applied to the PylRS of the bacterium Clostridiales (Clos). This sequence is provided as SEQ ID NO: 5. To aid in alignment, a version of the Clos PylRS sequence is provided in this document (SEQ ID NO: 6), in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384 and 401 in the Mm PylRS sequence. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00241] Mutations can be applied to the PylRS of Methanomethylophilus sp.1R26 (1R26). This sequence is provided in this document (SEQ ID NO: 7). To aid in alignment, a version of the 1R26 PylRS sequence is provided in this document (SEQ ID NO: 8), in which an X is marked at the positions corresponding to 300, 302, 305, 306, 309, 346, 348, 384, and 401 in the MmPylRS sequence. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00242] Mutations can be applied to the PylRS of Methanomassiliicoccus luminyensis 1 (Lum1). This sequence is provided in this document (SEQ ID NO: 9). To aid in alignment, a version of the Lum1 PylRS sequence is provided in this document (SEQ ID NO: 10), in which an X is marked at the positions corresponding to Petition 870250088848, dated 09 / 30 / 2025, pp. 120 / 242 106 / 141 300, 302, 305, 306, 309, 346, 348, 384 and 401 in the Mm PylRS sequences. These are positions to which the respective mutations of the present invention can be applied. Other corresponding positions can be identified in the same way.

[00243] Other structures to which mutations of the present invention can be applied include MbPylRS (SEQ ID NO: 11), Lum1 PylRS (SEQ ID NO: 12), TronPylRS (SEQ ID NO: 13), Gemm PylRS (SEQ ID NO: 14), PGA8 PylRS (SEQ ID NO: 15), I2 PylRS (SEQ ID NO: 16), D121 PylRS (SEQ ID NO: 17) and D416 PylRS (SEQ ID NO: 18).

[00244] Thus, in one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, said synthetase 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 any of the SEQ ID NOs: 1 to 18 and said amino acid sequence comprising any of the mutations and, optionally, wild-type residues, or sets of mutations and, optionally, wild-type residues, described in relation to the ninth aspect. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of any of the SEQ ID NOs: 1 to 18. In one embodiment, the amino acid sequence may be identical to any of the SEQ ID NOs: 1 to 18, except for the mutations mentioned.

[00245] In a particular embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, said synthetase 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 any of the SEQ ID NOS: 1 to 10 and said sequence Petition 870250088848, dated 09 / 30 / 2025, pp. 121 / 242 107 / 141 amino acid sequences comprising any of the mutations and, optionally, wild-type residues, or sets of mutations and, optionally, wild-type residues, described in relation to the ninth aspect. In particular, the amino acid sequence may have at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of any of the SEQ ID NOs: 1 to 10. In one embodiment, the amino acid sequence may be identical to any of the SEQ ID NOs: 1 to 10, except for the mutations mentioned.

[00246] Thus, in examples, acyl-tRNA synthetase may comprise 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 any of the SEQ ID NOs: 1 to 18 and comprising any of the sets of mutations in Table 8 or any 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 comprise residues corresponding to the mutations and wild-type residues from any of the sets in Table 8 or any of the sets of mutations labeled A7_1, A7_2, A7_3, A7_4, A7_5, and A7_7 in Table 8.

[00247] In one embodiment, an acyl-tRNA synthetase is provided that is capable of specifically acylating a tRNA with a beta-hydroxy acid, said synthetase 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 any of the SEQ ID NOs: 1 to 18 and said amino acid sequence comprising mutations corresponding to: M300A, M300D, M300M, M300N or M300S; more particularly M300N or M300S; A302D, A302G, A302H or A302N; more particularly A302H; and N346A, N346C, N346E, N346G, N346S or N346V; more specifically N346A, N346C or N346G (all relating to SEQ ID NO: 1). A Petition 870250088848, dated 09 / 30 / 2025, pp. 122 / 242 The 108 / 141 amino acid sequence may include mutations corresponding to: M300N or M300S; more particularly M300N; A302H; and N346A, N346C or N346G; more particularly N346G. The amino acid sequence may include M300N, A302H and, optionally, N346 and / or C348 mutations.

[00248] Ninth-aspect synthetases may include sequence changes relative to the wild-type sequence, in addition to the mutations described in more detail herein. Specifically, synthetases may include sequence changes at sites that do not significantly compromise the function or operation of the synthetase, as described in this document. The function of the synthetase can be tested by operating on it as described, as in the examples section, in order to verify that the function has not been abolished or significantly altered. Thus, provided that the synthetase maintains its function, which can be tested as set forth in this document, sequence variations may be made to the synthetase relative to the wild-type reference sequence.

[00249] As discussed in relation to the first aspect, Figure 35 provides a sequence alignment. The synthase can be at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any of the SEQ ID NOs: 1 to 18, where variation is found only in residues not labeled with: i) *; ii) * or :, or iii) or *, : or ., in addition to the specifically cited mutations discussed here.

[00250] Conservative substitutions can be made, for example, according to the table described in relation to the first aspect.

[00251] The ninth aspect acyl-tRNA synthetase can be isolated or purified. The ninth aspect acyl-tRNA synthetase may be a non-natural acyl-tRNA synthetase.

[00252] In a tenth aspect, the use of an acyl Petition 870250088848, dated 09 / 30 / 2025, pp. 123 / 242 109 / 141 tRNA synthetase in a method for generating a polymer comprising a beta-hydroxy acid.

[00253] In one embodiment, a method is provided for producing a polymer comprising a beta-hydroxy acid, wherein the method comprises: i) the use of an acyl-tRNA synthetase to acylate a tRNA with the beta-hydroxy acid, wherein the acyl-tRNA synthetase comprises mutations to allow the acylation of a tRNA with the beta-hydroxy acid, and ii) the incorporation of the beta-hydroxy acid into a polymer.

[00254] In one embodiment, the use of a ninth-aspect acyl-tRNA synthetase is provided in a method to generate a polymer comprising a beta-hydroxy acid.

[00255] In one embodiment, a method is provided for producing a polymer comprising a beta-hydroxy acid, wherein the method comprises: i) the use of a ninth-aspect acyl-tRNA synthetase to acylate a tRNA with beta-hydroxy acid, and ii) the incorporation of beta-hydroxy acid into a polymer.

[00256] Beta-hydroxy acid can be (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S^mClF).

[00257] The polymer may comprise one or more canonical amino acids or one or more naturally occurring amino acids. The polymer may comprise one or more non-natural amino acids and / or additional hydroxy acids. The non-natural amino acid may be an α-amino acid and / or the additional hydroxy acid may be an α-hydroxy acid.

[00258] The polymer can be formed by genetic incorporation of monomers using a ribosome. The polymer can be formed by translation of a nucleic acid sequence by a ribosome. Translation may involve the binding of a charged tRNA with Petition 870250088848, dated 09 / 30 / 2025, pages 124 / 242 110 / 141 the beta-hydroxy acid to a ribosome and the formation of a linkage between the beta-hydroxy acid and a preceding and / or subsequent monomer. Thus, the method may comprise providing a nucleic acid sequence encoding a polymer and using charged tRNAs and a ribosome to translate said sequence and form the polymer.

[00259] Thus, a method is provided for producing a polymer comprising the genetic incorporation of at least one beta-hydroxy acid, which may be (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S^mClF).

[00260] The use or method can be performed on a cell. For example, a prokaryotic cell, a bacterial cell, or an E. coli cell.

[00261] In an eleventh aspect, a nucleic acid encoding an acyl-tRNA synthetase from the ninth aspect is provided.

[00262] The nucleic acid can be DNA. A vector can comprise the nucleic acid.

[00263] In a twelfth aspect, a cell comprising an acyl-tRNA synthetase of the ninth aspect, a nucleic acid of the eleventh aspect, or a vector comprising a nucleic acid of the eleventh aspect is provided.

[00264] In a particular embodiment, the cell comprises or expresses a ninth-aspect acyl-tRNA synthetase, and the acyl-tRNA synthetase is orthogonal to endogenous tRNAs. Thus, the acyl-tRNA synthetase does not acylate endogenous tRNAs to the point of rendering the cell unviable. In one embodiment, cell fitness, for example, as measured by proliferation, is reduced by less than 50%, less than 25%, less than 10%, less than 5%, or is not reduced at all when the acyl-tRNA synthetase is expressed.

[00265] Sequence comparisons can be conducted with the Petition 870250088848, dated 09 / 30 / 2025, pp. 125 / 242 111 / 141 with the aid of readily available sequence comparison programs. These computer programs, publicly and commercially available, can calculate the sequence identity between two or more sequences.

[00266] A skilled technician 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 first necessary to prepare an alignment of the two sequences, followed by the calculation of the sequence identity value. The percentage identity for two sequences can take different values ​​depending on: (i) the method used to align the sequences, for example, the Needleman-Wunsch algorithm (e.g., as applied by Needle(EMBOS S) or Stretcher(EMBOSS), the Smith-Waterman algorithm (e.g., as applied by Water(EMBOS S)), or the LALIGN application (e.g., as applied by Matcher(EMBOS S)); and (ii) the parameters used by the alignment method, for example, local versus global alignment, the matrix used, and the parameters applied to the gaps.In a particular embodiment, the sequence identities described herein can be calculated based on a global alignment of the relevant feature, for example, the comparison of full lengths of sequences encoding a PylRS catalytic site, the full lengths of sequences encoding PylRS C-terminal domains, or the full lengths of sequences encoding PylRSs. In particular, proteins defined herein by reference to a degree of identity with a SEQ ID NO can have that degree of identity compared to the full length of the SEQ ID NO.

[00267] Once the alignment is done, there are several ways to calculate the percentage identity between the two sequences. For example, you can Petition 870250088848, dated 09 / 30 / 2025, pages 126 / 242 112 / 141 if you divide the number of identities by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average sequence length; (iv) the number of positions without gaps; or (v) the number of equivalent positions, excluding overhangs. Furthermore, it should be noted that the percent identity also strongly depends on the length. Therefore, the shorter a pair of sequences, the greater the sequence identity that can be expected to occur by chance.

[00268] A percentile identity calculation between two nucleic acid sequences can then be calculated from an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue and T is the total number of positions compared, including gaps but excluding overhangs.

[00269] Sequence alignment can be pairwise sequence alignment. Suitable services include Needle (EMBOS S), Stretcher (EMBOS S), Water (EMBOS S), Matcher (EMBOS S), LALIGN, or GeneWise. In one example, the identity between two amino acid sequences can be calculated using the Needle (EMBOS S) service defined with the default parameters, e.g., array (BLOSUM62), gap opening (10), gap extension (0.5), end gap penalty (false), end gap opening (10), and end gap extension (0.5). In another example, the identity between two amino acid sequences can be calculated using the Matcher (EMBOSS) service defined with the default parameters, e.g., array (BLOSUM62), gap opening (14), gap extension (4), alternative matches (1).In one example, the identity between two nucleic acid sequences can be calculated using the Needle(EMBOSS) service defined with the default parameters, for example, array (DNAfull), gap opening (10), gap extension (0.5), penalty for end gap (false), opening. Petition 870250088848, dated 09 / 30 / 2025, pages 127 / 242 113 / 141 for end gap (10) and extension to end gap (0.5). In another example, the identity between two nucleic acid sequences can be calculated using the Matcher service (EMBOS S) configured with the default parameters, for example, array (DNAfull), gap opening (16), extension to gap (4), alternative matches (1).

[00270] All features described in this document (including any attached claims, abstracts and drawings) and / or all steps of any method or process so described may be combined with any of the above aspects in any combination, except combinations in which at least some of these features and / or steps are mutually exclusive.

[00271] For a better understanding of the invention and to show how its embodiments can be implemented, we will now refer to the Examples, which are not intended to limit the invention in any way. EXAMPLES Summary of Examples

[00272] The genetic code of living cells has been reprogrammed to allow the specific incorporation of hundreds of non-canonical amino acids (ncAAs) into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides. Current methods for modifying orthogonal (O)-aminoacyl-tRNA synthetases to acylate new monomers rely on translational reads and therefore require the monomers to be ribosomal substrates. O-synthetases cannot be evolved to acylate O-tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates (and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated into O-tRNAs); this codependency creates an evolutionary impasse that has restricted the scope of translation in Petition 870250088848, dated 09 / 30 / 2025, pages 128 / 242 114 / 141 live cells for closely related alpha-L-amino acids and hydroxy acids. Here, we broke this impasse by developing a direct selection for O-synthetases that acylate cognate O-tRNAs with ncMs, regardless of whether the ncMs are ribosomal substrates. We developed split tmRNAs, composed of a non-covalent assembly between the 5' half of an O-tRNA and a fusion between the 3' half of the O-tRNA and the mRNA encoding the cognate synthetase; this couples the synthetase genotype to the acylation of the split tmRNA in cells. We also developed an approach to specifically isolate and enrich acylated split tmRNAs. Combined, these advances in tRNA display allow for direct, rapid, and scalable selection for O-synthetases that acylate their cognate O-tRNA; This approach uses 50 times fewer ncM than translation-based selections, and we parallelized the tRNA display to select efficient synthases for 8 ncAAs.Using tRNA display, we directly selected O-synthetases that acylate their cognate O-tRNA with a β-amino acid. We leveraged this advancement to demonstrate the genetically encoded, site-specific cellular incorporation of a β-amino acid into a protein, thus expanding the chemical scope of the E. coli genetic code. Introduction

[00273] Here, we develop tREX derivatives in which specific acylated tRNAs, isolated from cells, are labeled with dNTP analogs by primer extension; this allows for fluorescent imaging (fluoro-tREX) or capture (bio-tREX) of acylated tRNAs. We create Methanosarcina mazei pyrrolysine tRNAoUA genes (Mm tRNAPylCUA, hereinafter referred to as tRNAPylCUA) that are circularly permuted (which we refer to as cis-split(s)tRNAPyl genes) by joining the 5' and 3' ends via an intermediate sequence and creating new 5' and 3' ends in the anticodon. The se Petition 870250088848, dated 09 / 30 / 2025, pp. 129 / 242 115 / 141 intermediate sequence is processed out of transcription to generate a split stRNA, composed of a 5' and a 3' half, which is acylated by Mm pyrrolysyl-tRNA synthetase (hereinafter referred to as PylRS), the aminoacyl-tRNA synthetase cognate of the original tRNA.

[00274] They connect the genotype responsible for acylation to the acylation itself, fusing the gene for PylRS to the cis stRNAPyl gene, creating cis stmRNAPyl. They demonstrated that they can selectively enrich stmRNAs encoding active PylRS variants by more than 300 times, compared to variants with attenuated activity, using biomREX (a variation of bio-tREX applied to stmRNA). They increased the dynamic range of PylRS enrichment by maximizing the transcription of stmRNAs while minimizing the translation of the PylRS mRNA encoded within the stmRNA.

[00275] They generate stmRNA libraries with combinations of mutations in the PylRS gene and use parallel bio-mREX-based enrichments, in the presence and absence of ncAAs, followed by reverse transcription and NGS to rapidly and scalably define active and selective PylRS variants, through a process we call tRNA display. They use tRNA display to select orthogonal aminoacyl-tRNA synthetases (aaRSs) that specifically acylate their cognate and orthogonal tRNAs with a carboxylic acid, which does not function in translation, and with a β-amino acid, a class of ncMs generally considered poor ribosomal substrates10,11,14 to 18. Furthermore, using the β-amino acid pair, they demonstrated the site-specific cotranslational incorporation of a β-amino acid into a recombinant protein. Example 1 - Sensitive detection and efficient isolation of acylated tRNAs

[00276] They demonstrated that they can determine the aminoacylation status of a specific tRNA isolated from cells by oxidation with Petition 870250088848, dated 09 / 30 / 2025, pages 130 / 242 116 / 141 periodate, followed by primer-mediated selective extension of unoxidized tRNAs with nucleotide derivatives containing a fluorophore (Cy5) or biotin. These experiments demonstrate that the approach, which they call fluorescent tREX (fluoro-tREX), allows the acylation of a tRNA to be accompanied by the generation of a fluorescent signal.

[00277] Next, they replaced Cy5-dCTP with biotinylated dCTP (bio-dCTP) in the fluoro-tREX extension step, thus creating biotin-tREX (bio-tREX). They selectively captured the biotinylated extension product (resulting from tRNA molecules that were protected from periodate oxidation by their aminoacylation) on streptavidin magnetic beads, washed the beads, and eluted the bound tRNA extension products. The presence of the tRNA extension product Pylcua in the eluate was dependent on the presence of PylRS, BocK (1) in the cells and the addition of bio-dCTP to the extension reaction. They concluded that biotin-tREX allowed the selective capture of tRNA extension products from tRNAs that were aminoacylated.

[00278] Further information is provided in the priority documents (EP2306393.6, filed on April 28, 2023 and EP2400299.0, filed on January 9, 2024; incorporated herein by reference). Example 2 - Split tRNAs: assembly, maturation and acylation in vivo.

[00279] Next, they question whether they could split the tRNAPylcUA gene at the anticodon to create a split tRNAPyl (stRNAPyl). They envision generating a system in which the 5' and 3' halves of a split tRNA gene would be transcribed, assembled in vivo through non-covalent interactions (including base pairing), matured by the cellular tRNA processing machinery, and recognized and efficiently acylated by PylRS. Petition 870250088848, dated 09 / 30 / 2025, pages 131 / 242 117 / 141

[00280] First, they designed a series of constructs in which they split the tRNA gene in two at the anticodon. Some constructs resulted in robust aminoacylation, dependent on the presence of both tRNA halves, PylRS and BocK. To their knowledge, these data constitute the first example of a split tRNA being assembled, matured, and acylated in cells. More information is provided in priority papers.

[00281] Next, they turn their attention to the development of an ideal expression system for stRNAPyls, which: (1) would guarantee equimolar stoichiometry of both tRNA halves and (2) would facilitate spatial proximity and therefore assembly of both tRNA halves into a tRNA body that can be acylated. To this end, they have demonstrated that tRNAPylCUA can be cleaved, expressed from a single cis transcript, and that the cleaved tRNA functions as an efficient substrate for acylation by PylRS. More information is provided in priority papers. Example 3 - Covalent linkage of acylation phenotype and genotype

[00282] Next, the coding sequence of PylRS and a 5' end-linking sequence of the 3' half of the cis stRNAPyl expression cassette are fused, creating the stmRNAPyl cassette. Experiments were performed and concluded that the stmRNA construct is functional. The construct is transcribed and processed to generate a split tRNA, in which the 3' half is fused to mRNA synthetase. The synthetase gene is translated 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 adding fluorescent nucleotides to the RNA strand containing the synthetase mRNA. This creates a physical link between the synthetase mRNA sequence, its genotype, and the fluorescent phenotype generated as a result of synthetase activity. Further information is provided in the priority documents Petition 870250088848, dated 09 / 30 / 2025, pages 132 / 242 118 / 141 rivers. Example 4 - Specific efficient enrichment for acylation of PylRS genotypes.

[00283] Next, they aim to selectively isolate acylated stmRNAs from non-acylated stmRNAs and reverse transcribe the PylRS mRNA isolated within the stmRNAs to directly produce the PylRS gene cDNA responsible for acylation. They envision that the resulting cDNA could then be used as a direct readout in a quantitative PCR (q), converted to DNA for further targeted selection and evolution, or sequenced. To selectively isolate acylated stmRNAs from non-acylated stmRNAs, they created biomREX, an adaptation of bio-tREX for stmRNAs. This method was experimentally tested and they concluded that bio-mREX allows for the efficient and selective recovery of stmRNAs and the genes for synthases that acylate the split tRNAs within them. More information is provided in the priority papers.

[00284] In conclusion, by combining the stmRNAvol2 construct with bio-mREX, we developed a pulldown with which we were able to selectively isolate cDNA from active PylRS variants relative to inactive variants across a wide dynamic range. Example 5 - Direct selection for cellular acylation by tRNA display.

[00285] Next, they set up the first method to directly select synthetase enzymes based on their tRNA acylation activity. They predicted that they could efficiently detect active and selective PylRS variants by performing parallel bio-mREX-based selections coupled to deep sequencing by next-generation sequencing (NGS) (Fig. 2a). In this experiment, PylRS active site libraries are cloned into stmRNAvol2 constructs and transformed into E. coli cells. Overnight cultures Petition 870250088848, dated 09 / 30 / 2025, pages 133 / 242 119 / 141 are then diluted in medium in the presence (positive samples) and absence (negative samples) of the chosen monomer, and bio-mREX is performed. The experiments are performed in multiple replications, and the cDNA from the positive and negative samples, as well as the reverse-transcribed cDNA from the input library, are subsequently slash-encoded and sequenced by NGS (Fig. 2a). They concluded this selection approach with tRNA display.

[00286] NGS data from sample selections allow the calculation of two key parameters: (1) enrichment – ​​defined as the average abundance of a specific sequence in the positive samples relative to the abundance of the same sequence in the input RNA – and (2) selectivity – defined as the ratio between the abundance of a sequence in the positive sample and the abundance of the same sequence in the negative sample. The desired PylRS variants correspond to highly enriched and highly selective sequences. Plotting the natural logarithm of enrichment against the natural logarithm of selectivity results in a spindle-shaped distribution. We expect any desired sequences to be in the upper right quadrant of the spindle plots (Fig. 2a) (highly enriched and highly selective).

[00287] To test tRNA display, we generated a small PylRS library, in which we expected many sequences to be active, such as an stmRNAvol2 construct. The library targeted positions Y306, L309, and N346 in PylRS, where mutations that allow efficient incorporation of CbzK (2)26,27 (Fig. 2b and Fig. 6) had already been identified. They transformed the library into E. coli cells and performed a single tRNA display cycle. They observed a large population of highly enriched and selective PylRS variant sequences in the spindle plot derived from the sequencing of this experiment (Fig. 2c). Petition 870250088848, dated 09 / 30 / 2025, pp. 134 / 242 120 / 141

[00288] To assess the predictive power of tRNA display, we measured in vivo production of GFP(150CbzK)His6 from GFP(150TAG)His6 in the presence of tRNAPylCUA and 65 PylRS variants, which appeared to be potential hits based on their position in the spindle graph (Fig. 7). A positive correlation was observed between enrichment derived from NGS data and translational activity derived from GFP production for these hits (Fig. 2d), and the vast majority of these hits were selective (Fig. 7).

[00289] They concluded that tRNA display allows direct, translation-independent identification of active and selective PylRS enzymes from a PylRS sequence library. Example 6 - High-throughput selection for ncAAs by tRNA display.

[00290] To further validate the utility of tRNA display, they performed parallel selections (Fig. 8) using six independent and highly diverse libraries of PylRS active sites (Fig. 6) and ten ncAAs, 2-11 (Fig. 2b). After two selection cycles, we analyzed spindle plots derived from NGS data and identified individual PylRS mutants for ncAAs 2, 3, 4, 7, 8, 9, 10, and 11 that were enriched and selective; the enriched and selective mutants for each of these ncAAs showed convergent sequence motifs (Figs. 8 to 18).

[00291] They demonstrated the incorporation of ncAAs 3, 4, 7, 8, 9, 10, and 11 in response to the amber codon in GFP(150TAG)hís6, in cells containing tRNAPylCUA and the corresponding PylRS mutants identified by tRNA display. GFP production was ncAA-dependent, and ESI-MS confirmed the incorporation of each ncAA into GFP (Fig. 2e-i, Figs. 9 to 18). They observed that the results include an aminoacyl-tRNA synthetase for 7, which allows the first incorporation of this thiophene-containing ncAA into a protein. Of the 30 variants Petition 870250088848, dated 09 / 30 / 2025, pp. 135 / 242 Of the 121 / 141 variants characterized with a selectivity score greater than or equal to ten and an enrichment score greater than or equal to five, 27 were active with their cognate ncAA in protein expression (20 variants showed activities of at least 50% of the PylRS activity with BocK (1)). All 27 variants selectively incorporated their ncAA substrate, as assessed by mass spectrometry (Fig. 2e-i, Figs. 9 to 18).

[00292] In summary, they demonstrated the parallel, scalable, and fast selection of PylRS variants using various libraries and ncAAs through tRNA display. They observed that the amount of ncAA used in each tRNA display selection is 50 to 100 times smaller than that used in current synthase selection methods. Example 7 - Discovery of a β-amino acid specific PylRS variant by tRNA display.

[00293] Next, they challenged tRNA display to discover synthetases for classes of ncMs that cannot be translated or that are known – from in vitro studies – to be weak ribosomal substrates (Fig. 3)10,11,14 to 18. They focused on nine ncMs (12 to 20, Fig. 3a). These monomers include the amino acids β2 and β3 with variable stereochemistry, β-hydroxy acids, and carboxylic acids. 6-((tert-butoxycarbonyl)amino)hexanoic acid (BocAhx, 15) is a known substrate for PylRS21 wt and provided a control for the ncM selections.

[00294] To select PylRS variants for these monomers, parallel tRNA selections were performed (Fig. 19) using a highly diverse library, which mutated residues 300, 302, 305, 306, 309, 344, 346, 348, and 401 in the PylRS active site (Fig. 6) and in each ncM. We analyzed spindle plots derived from NGS data for each ncM to identify PylRS variants that were enriched and selective (Figs. 20 to 28). For 12 and 15, we identified if Petition 870250088848, dated 09 / 30 / 2025, pages 136 / 242 122 / 141 enriched and selective sequences, and the most selective results converged to a distinct sequence pattern for each of these ncM.

[00295] They identified sequences similar to the wild-type PylRS sequence for ncM 15, 6-((tert-butoxycarbonyl)amino)hexanoic acid (BocAhx), indicating that selection converged towards synthetases active for this monomer. The four most selective and active hits of selection, PylRS(15_1) to PylRS(15_4), differ from the wild-type sequence by V401 point mutations; they demonstrated, by fluoro-tREX, that all these selected sequences were active and selective for 15 (Fig. 3b, Fig. 23). As 15 is expected to be more difficult to cleave from tRNA than other monomers, including β-amino acids (Fig. 4), this result provided confidence that tRNA display would allow selection of synthetases for a wide range of other monomers, including β-amino acids.

[00296] Notably, two variants of PylRS, PylRS(12_1) and PylRS(12_2), identified by tRNA display selection, directed the acylation of tRNAPylCUA dependent on (S)-3-amino-3-(3-bromophenyl)propanoic acid ^-mBrF, 12), assessed by fluoro-tREX (Fig. 3c, Fig. 20). To verify the identity of the tRNA-bound monomer by PylRS(12_1) and PylRS(12_2), we captured the acylated tRNAPylCUA on streptavidin beads using a biotinylated probe for tRNAPylCUA, washed the beads, and eluted the ncM by heating under alkaline conditions; then, we derivatized the free ncM and analyzed the sample by LC-MS (Fig. 29). Using this approach, they confirmed that both PylRS variants carried tRNAPylCUA with ncM 12 (Fig. 3e, f). To our knowledge, PylRS(12_1) / tRNAPylCUA and PylRS(12_2) / tRNAPylCUA are the first orthogonal aminoacyl-tRNA synthetase / β-amino acid-specific tRNA pairs described so far. Petition 870250088848, dated 09 / 30 / 2025, pp. 137 / 242 123 / 141

[00297] Next, they increased the activity of PylRS(12_1) and PylRS(12_2) by randomized mutagenesis of the active site region of the PylRS gene within the stmRNA construct, followed by tRNA display-based selection (Figs. 30, 31). From the resulting spindle plot, they identified sequences carrying one to three additional mutations relative to the parental clones, which were enriched and selectively selected. The three most enriched and selective hits, PylRS(12_1evol1-3), were derived from PylRS(12_1). They confirmed the specificity of PylRS(12_1evol1-3) for the acylation of tRNAPylCUA with 12, both by fluoro-tREX and by the LC-MS-based assay (Fig. 3d, f, g, Fig. 29 and 32). PylRS(12_1evol1-3) were notably more active in the acylation of tRNAPylCUA with 12 than PylRS(12_1) (Fig. 3c, d, e, f).

[00298] Next, they combined PylRS(12_1) or PylRS(12_1evQl1) with tRNAPylCUA and GFP150TAG hís6 in cells. Although β-amino acids are expected to be poor ribosomal substrates10,11,14 to18, they found that GFP production depended on the addition of 12 to the cells. GFP fluorescence was 3 times higher with PylRS(12_1evQl1) than with PylRS(12_1), consistent with the higher acylation activity of PylRS(12_1evQl1). They confirmed the incorporation of 12 at position 150 in GFPhís6 purified from cells with PylRS(12_1), or PylRS(12_1evQl1), by ESI-MS and MS / MS. In the absence of 12, they observed some GFP production resulting from the incorporation of However, in the presence of 12 (Fig. 3g,h, Fig. 33), they produced more GFP and detected only the incorporation of 12 by intact MS and MS / MS. They concluded that, in the presence of 12, the prior incorporation of Phe observed in the absence of 12 is effectively overcome by competition.Similar observations have been made previously for efficient and selective ncAA embedding systems28 and the fidelity of the natural code is also known to depend on competition. Petition 870250088848, dated 09 / 30 / 2025, pages 138 / 242 124 / 141 ção29. They noted that they did not observe incorporation of 12 at position 3 of GFP in experiments using GFP3TAGhís6 (Fig. 33), consistent with β-amino acids being poor ribosomal substrates10'11'14 to 18 and the fact that β-amino acids may not be tolerated at all positions in a protein.

[00299] Next, they performed two selection cycles with lib14 and ncMs A1, A2, A3, A4, A5 (β-amino acids with varied side chains), A6 (an α,α-disubstituted amino acid), and A7 (a β-hydroxy acid) (Fig. 36a, Fig. 38). From the resulting spindle plots (Figs. 39 to 45), they identified enriched and selective sequences, and most selective hits converged to a distinct sequence pattern for each ncM. They observed that the synthases selected for all six β-amino acids differ in sequence but contain common M300D and A302H mutations (Figs. 20, 39 to 43). The sequence pattern observed for the β-hydroxy acid A7 is similar to that observed for the β-amino acids. However, the residue at position 300 - which may be in direct proximity to the amine / hydroxy group - is changed from aspartic acid to asparagine (Fig. 45).The PylRS variants, identified by tRNA display selection, directed specific acylation of tRNAPylcuA by its cognate monomer, as assessed by fluoro-tREX and the LC-MS-based assay (Fig. 36d-q). We quantified the fractional acylation as a function of ncM concentration (Fig. 46). To our knowledge, we have discovered the first aminoacyl-tRNA synthetase / tRNA pairs specific for three distinct classes of ncM: β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids. Example 8 - ncMs mercury binding in proteins

[00300] To investigate the incorporation of β-amino acids, disubstituted α,α-amino acids, and β-hydroxy acids into proteins, they combined the orthogonal synthetase / orthogonal tRNA pairs they discovered for eight ncMs, 12, A1, A2, A3, A4, A5, A6, A7, with GFP150TAGhís6 in Petition 870250088848, dated 09 / 30 / 2025, pages 139 / 242 125 / 141 cells, and added the cognate ncM. They observed ncM-dependent GFP production for 12, A2, A5, and A6, with isolated yields ranging from 3 to 35 mg per liter of culture, and mass spectrometry confirmed the incorporation of these β-amino acids and disubstituted α,α-amino acids into GFP (Figs. 3h, 33, 36r, 36s, 47, and 48). In the absence of 12, they observed some GFP production resulting from the incorporation of natural amino acids (Fig. 36r). However, in the presence of 12, they produced more GFP, and detected only the incorporation of 12 by intact MS and MS / MS (Figs. 3h, 33, 47). They concluded that, in the presence of 12, the preceding incorporation, observed in the absence of 12, was effectively overcome; We made similar observations for A2, A5, and A6 (Fig. 3h, 36s, 33, 47). Similar observations have been made previously for efficient and selective ncAA embedding systems, and natural code fidelity is also known to depend on competition.They concluded that 12, A2, A5, and A6 are incorporated site-specifically with high fidelity. They did not observe incorporation of ncMs 12, A2, A5, or A6 at position 3 of GFP (from GFP3TAGhís6) (Fig. 48), indicating that these ncMs are not tolerated at all positions of a protein; similar site-dependent incorporation efficiency has been previously observed for ncAAs.

[00301] They noted that they did not observe a ncM-dependent increase in GFP production from GFP150TAG hís6 or GFP3TAG hís6 with A1, A3, A4, or A7 when the cells received these ncMs and their cognate orthogonal synthetase / orthogonal tRNA pairs (Figs. 36r, 48). These observations are consistent with the fact that these ncMs are poor substrates for ribosomal polymerization. For A4 and A7, they observed a decrease in GFP production after ncM addition (Fig. 36r); this is consistent with the fact that these ncMs, once acylated onto orthogonal tRNA, inhibit amber codon reading. The discovery Petition 870250088848, dated 09 / 30 / 2025, pages 140 / 242 The 126 / 141 orthogonal synthetases specific for these ncMs provide a starting point for the selection of ribosomes that polymerize them. Example 9 - Structure of the β-amino acid protein

[00302] To better characterize the incorporation of 12 at position 150 of GFP, we solved the structure of GFP(150(S^mBrF^se) at 1.5 Å by X-ray crystallography (the protein was purified from cells harboring PylRS(12_1) and tRNAPylcuA (Fig. 3i)). The electron density shows two consecutive carbon atoms (C2 and C3) in the protein backbone at position 150; the meta-bromophenyl substituent is attached to C3 of the β-amino acid, and the stereochemistry at C3 corresponds to the expected (S) stereoisomer. The structure confirms the site-specific incorporation of the expected amino acid (S)e3mBrF into the protein. The introduction of (S)e3mBrF leads to a notable twist in the beta barrel of GFP, compared to the selvage-type protein (Fig. 34). Interestingly, the linkage networks of The hydrogen atoms of the residues immediately before and after the β-amino acid in the polypeptide chain remain essentially unchanged; this indicates that this beta-barrel can accommodate the ε3-amino acid in this position (Fig.34). To our knowledge, this represents the first structure for an in vivo produced protein containing a β-amino acid. Taken together, the data demonstrate the site-specific incorporation of a β-amino acid into a cell-produced protein. Example 10 - Discussion

[00303] The in vivo incorporation of modified monomers into the main structure is an old and unresolved challenge in expanding the scope of cellular polymer synthesis beyond α-L amino acids and their close analogues4'30. The codependency of tRNA acylation and ribosomal polymerization31 in the translational readouts commonly used to select tRNA acylation12'13 or ribosomal polymerization creates an evolutionary impasse; this impasse Petition 870250088848, dated 09 / 30 / 2025, pages 141 / 242 127 / 141 has significantly limited the range of monomers that can be used to acylate specific orthogonal tRNAs in vivo.

[00304] tRNA display breaks this impasse based on codependency. It achieves this by creating a selectable acylation phenotype and coupling this phenotype to the gene sequence of the corresponding synthase; this allows for the direct selection of orthogonal aminoacyl-tRNA synthetases that acylate their cognate orthogonal tRNAs with ncMs, without any requirement that the ncM be a ribosomal substrate or act in translation. Using tRNA display, we rapidly and scalably selected efficient aaRS systems for eight ncAAs; these selections consume orders of magnitude fewer compounds than previous approaches. Furthermore, we used tRNA display to select and improve tRNA acylation systems for ncMs; these systems could not have been discovered using previous approaches.

[00305] Having established that tRNAPylCUA was acylated with ncM 12 in cells, we were able to identify a site in GFP where the incorporation of this ncM was tolerated to perform the genetically encoded incorporation of a β-amino acid into a protein. In future work, we will leverage cellular acylation of tRNAs with ncMs to enable translation-based selections for orthogonal ribosomes28,31,33,34 that can polymerize ncMs at a wider range of sites. Translation-based selections31 can also generate orthogonal ribosomes that facilitate the cellular synthesis of polymers composed of more diverse ncMs. We anticipate that the repertoire of ncMs that can be used to construct modified proteins and non-canonical polymers can be further expanded by combining approaches for direct coding of ncMs with elegant approaches to increase the chemical scope of peptides and proteins through post-translational modification and ligation35-39de Petition 870250088848, dated 09 / 30 / 2025, pages 142 / 242 128 / 141 proteins.

[00306] Genetic encoding of β-amino acids may allow the creation of protease-resistant proteins40-42 in cells. Future developments may combine strategies for encoding non-canonical polymers in cells5 with an expansion in the range of monomers that can be encoded to allow the encoded biosynthesis of foldmers43, composed of β-amino acids and other ncMs, which can form secondary, tertiary, and quaternary structures44'45. We predict that it will be possible to complement and augment the canonical functions of cells through the design and targeted evolution of genetically encoded foldmers.

[00307] In addition to ncM 12, using tRNA display, they discovered orthogonal synthetase / orthogonal tRNA pairs that are selective for eight new ncMs, including β-amino acids, disubstituted α,α-amino acids, and β-hydroxy acids, thus directly facilitating the genetic coding and specific incorporation of β-amino acids and disubstituted α,α-amino acids into proteins in a living organism. Example 9 - Methods Tampons Resuspension buffer (RB): 100 mM NaOAc, 50 mM NaCl, 0.1 mM EDTA, pH 5.0 Deacylation buffer (DB): 50 mM Bicine pH 9.6, 1 mM EDTA. D-buffer (D): 50 mM NaOAc pH 5, 150 mM NaCl, 10 mM Mg2Cl, 0.1 mM EDTA. Hybridization buffer (HB): 10 mM Tris-HCl, 25 mM NaCl, pH 7.4 Klenow (exo-) main mixture with Cy5-11-dCTP (KMM-Cy5): 17 μL of water, 5 μL of 10x NEBuffer 2.0, 1 μL of Klenow exo(-), 1 dNTPS-dCTP (10 mM), 1 μL of Cy5-11-dCTP (20 pM). Orange filler dye (OLD): 8 M urea, Orange G Petition 870250088848, dated 09 / 30 / 2025, pages 143 / 242 129 / 141 Klenow (exo-) main mixture with Cy5-11-dCTP mini (KMM-Cy5mini): 1.1 μL water, 1.2 μL 10x NEBuffer 2.0, 0.2 μL Klenow exo(-), 0.5 dNTPS-dCTP (5 mM), 1 μL Cy5-11-dCTP (5 μM). Klenow (exo-) main mixture with Bio-11-dCTP (KMM-Bio): 17 μL of water, 5 μL of 10x NEBuffer 2.0, 1 μL of Klenow exo(-), 1 dNTPSdCTP (10 mM), 1 μL of Bio-11-dCTP (20 μM). Wash buffer (WB): 10 mM Tris-HCl, 150 mM LiCl, 1 mM EDTA, 0.05% v / v Tween20, pH 7.5 Binding buffer (BB): 20 mM Tris-HCl, 1 M LiCl, 2 mM EDTA, 0.05% v / v Tween20, pH 7.5 Formamide loading buffer (FMB): 90% formamide Lysis buffer (SLB): 100 mM NaOAc, 50 mM NaCl, 0.1 mM EDTA, pH 5.0, 1% (w / w) SDS Alkaline washing buffer (AWB): 25 mM NaOH, 4 mM EDTA, 0.05% Tween20 Reverse transcription hybridization (RHM) mixture: 1 μL of DNA primer (2 μM), 1 μL of 10 mM dNTPs, 1 μL of 10x HB, 10 μL of water. RT master mix (RMM): 4 μL SSIV buffer, 1 μL RNAse Out, 1 μL SSIV RT, 10.1 M dithiothreitol (DTT) Acid washing buffer 1 (aWB1): 100 mM NaOAc pH 5 Acid Wash Buffer 1 plus Tween20 (aWB1-T): 100 mM NaOAc pH 5, Tween-20 0.01% (v / v) Acid wash buffer 2 (aWB2): 20 mM NaOAc pH 5 Means SOC: Super Optimal Broth plus glucose 2xYT-s: Tryptone yeast extract supplemented with 75 μg / mL of spectinomycin. 2xYT-st: Tryptone yeast extract supplemented with 75 μg / mL spectinomycin and 10 μg / mL tetracycline. Petition 870250088848, dated 09 / 30 / 2025, pages 144 / 242 130 / 141 2xYT-s-ap: Tryptone yeast extract supplemented with 75 μg / mL spectinomycin and 50 μg / mL apramycin. 2xYT-am: Tryptone yeast extract supplemented with 75 μg / mL of ampicillin. Chemical Products

[00308] NcM 1 and 2 were acquired from Bachem. NcM 4 was acquired from Fluorochem. NcM 5 was acquired from Ambeed. NcMs 6, 8, 13, 17, and 18 were acquired from Enamine. NcM 7 was acquired from aaBlocks. NcMs 9, 10, 15, and 16 were acquired from Merck. NcMs 12 and 20 were acquired from BLD. NcM 14 was acquired from Advanced ChemBlock. NcM 19 was acquired from AstaTech. NcMs 3 was synthesized as previously described (Spinck, M. et al. s. Nature Chemistry 15, 61-69 (2023)) and ncM 11 was custom synthesized as previously described (Tang, S. et al. Nature 602, 701-707 (2022)). NcMs 13 and 18 were deprotected by Boc in dioxane-concentrated HCl. Cloning of DNA constructs

[00309] Standard cloning was performed by Gibson assembly using NEBuilder® HiFi DNA Assembly Master Mix (NEB), according to the manufacturer's guidelines. Libraries were generated by enzymatic reverse PCR, as previously described. Briefly, a standard plasmid was amplified by PCR using two primers (see primer list) containing degenerate codons at the desired mutagenesis sites and a BsaI cleavage site. In the case of custom mixtures, primers containing different codons were manually mixed and used for PCR reactions. PCR products were purified on gel and digested using BsaI and DpnI. Subsequently, the samples were purified, ligated using T4 Ligase, and transformed into electrocompetent E. coli DH10β cells, ensuring a minimum transformation efficiency of 10⁹. Individual colonies (>10) were evaluated using the sequence Petition 870250088848, dated 09 / 30 / 2025, pages 145 / 242 131 / 141 Sanger sequencing for quality control of library assembly. Total plasmid DNA was prepared from the resulting culture, sequenced as a volume using Sanger sequencing, and used for subsequent experiments. General Protocols NGS data analysis

[00310] NGS was performed on a MiSeq system (in the case of the evolution test with library 1 and substrate 1) or on a NextSeq2000 system (in all other cases). The resulting cDNA from the tRNA display was amplified using NGS A(1-8) and NGS_B(1-8) oligos containing different combinations of Nextera sequencing barcodes via PCR. Samples were purified, quantified, and combined in equimolar amounts. Paired-end reads were first paired using PEAR7e aligned to a Mm PylRS reference sequence using Bowtie28. Relevant library positions were extracted and translated into amino acids, and the resulting variants were counted using an R script. Subsequent operations were performed using the frequency of each variant in each library, which was calculated as the count value divided by the total number of counts for that library.Using the R script, enrichment and selectivity scores were calculated for all variants as follows. First, variants present only in all positive replications were considered (tables were merged using the AND operator). Assuming that highly enriched sequences might potentially not be covered by the negative and input samples, but could still be of interest, negative and naive replications were merged into the positive table using an OR operator. A placeholder value of 0.95 count was adopted in cases where a replication did not cover a specific variant. Petition 870250088848, dated 09 / 30 / 2025, pages 146 / 242 The resulting 132 / 141 dataset was used to calculate the average enrichments in the presence and absence of the respective substrate, calculated as the quotient of the average frequencies in one condition and the input condition. The resulting positive and negative enrichments were used to calculate the selectivity value for each variant (equivalent to the quotient of the positive and negative frequencies). For further analysis, the variants were filtered using an empirically determined threshold value for the normalized standard deviation of the positive frequency (dispersion error in the positive substrate condition). tRNA pulldown and ncM identification by LC-MS

[00311] tRNAs were isolated from 8 mL of cells following general protocol B, omitting oxidation with NaIO4. The RNA pellet was resuspended in 90 μL of buffer D and RNA concentrations were adjusted to match the lowest concentration in the compared samples. 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 min. 40 μL of Streptavidin Dynabeads MyOne C1 (Invitrogen) were washed twice with DT buffer and added to 10 μL of buffer D. The beads were added to the hybridization reaction and the probe was attached to the beads for at least 30 min at 4 °C with head-over-tail rotation. 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 support. 24 μL of DB were added and the beads were incubated at 42 °C for one hour.12 μL of the deacylation mixture were added to 3 μL of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC - 3 mg / mL in acetonitrile) and the reaction was incubated at 55 °C for 15 min. Samples were analyzed on an Agilent Technologies 6130 quad-pole LC / MS using single-ion monitoring. Petition 870250088848, dated 09 / 30 / 2025, pages 147 / 242 133 / 141 Activity assay of GFP(150X)hís6 and GFP(3X)hís6, where X represents any ncM.

[00312] Chemically competent DH10β cells containing a p15A plasmid encoding GFP150TAG hís6 or GFP3TAGhís6 (two versions of the p15A plasmid with a tetracycline or apramycin resistance cassette, which led to similar results, were used interchangeably) were transformed with a pMB1 plasmid encoding Mm PylRS, or a mutant thereof, and Mm tRNAPylCUA, rescued in SOC and cultured overnight in 2xYTs-t or 2xYTs-ap. 20 μL of the overnight culture were diluted in 480 μL of 2xYTs-t or 2xYTs-ap containing 0.2% L-arabinose in the presence and absence of 2 to 4 mM of the respective ncM, in 96-well plates. The cells were cultured for 16 to 20 ha at 37 °C and 700 rpm. The plates were centrifuged for 12 min at 4200 rcf and 4 °C, and the cells resuspended in 150 μL of PBS. 100 μL of the resuspended cells were transferred to a 96-well Costar flat-bottom plate and to the OD600, and GFP fluorescence was measured using a PHERAstar FS plate reader. Isolation of GFP(150X)hís6 for MS analysis

[00313] Three replications of the protein produced as described above were combined in a 1.5 mL Eppendorf tube, centrifuged at 4200 rcf for 3 min, frozen at -20 °C, thawed, and resuspended in 150 μL of BugBuster (Millipore). The cells were lysed for 1 h with head-over-tail rotation. The lysed cells were centrifuged for 20 min at 20,000 rcf at 4 °C, and the lysate was added to 20 μL of NiNTA beads. GFP(150)h6 was bound to the beads for 20 min at room temperature with head-over-tail rotation. 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. Petition 870250088848, dated 09 / 30 / 2025, pages 148 / 242 134 / 141

[00314] For low ncM 12 activity mutants, 5 to 15 mL of cell culture were used for protein production. Bug Buster volumes were adjusted proportionally, all other volumes were kept constant. Mass spectrometry

[00315] ESI-MS and MS / MS were obtained as described24,27 previously. Protein expression, purification, and crystallization

[00316] Chemically competent DH10β cells containing a p15A plasmid encoding GFP150TAG hs6 and a pMB1 plasmid encoding Mm PylRS(12_1) and Mm tRNAPyl were transformed, recovered in SOC and cultured overnight in 2xYTs-ap. 10 mL of the overnight culture were diluted in 1 L of 2xYTs-ap containing 0.2% L-arabinose in the presence of 5 mM 12.

[00317] A 1 L bacterial pellet of GFP150(S)e3mBrF-His6 expression culture was lysed by sonication, centrifuged at 142,000 rcf for 30 minutes, and the supernatant bound to NiNTA beads (Qiagen). The beads were washed three times before protein elution and subsequently 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 using Vivaspin 20, 10,000 MWCO (Sartorius) at 6 mg / mL. The sample was digested with Sequential Grade modified trypsin (Promega) at a 50:1 ratio. The sample was incubated for 1 hour at 37 °C and centrifuged at 21,000 rcf for 10 minutes before seeding onto crystal trays. Crystallization trays with multiple commercial kits were performed in 96-well vapor diffusion plates (Molecular Dimensions) at 18 °C and configured with a Mosquito HTS robot (TTP Labtech).Ratio of drops of 0.2 μL of protein solution plus 0.2 μL of solution. Repetition 870250088848, dated 09 / 30 / 2025, page 149 / 242. 135 / 141 conservatories were used for screening. The only useful dataset was collected from a crystal obtained from the melt screen (Molecular Dimensions) with the following composition: 37.5% PEG 3350 / PEG 1K / MPD (1:1:1), 0.1 M Bicine / Trizma pH 8.5, 0.8% (w / v) Morpheus III Alkaloids, and 0.12 M Morpheus Alcohols. The crystals were collected and flash-frozen in liquid nitrogen. Collection of diffraction data, processing, and structural solution.

[00318] Diffraction data were collected at the ESRF on beamline ID23-2 at an energy of 14.2 keV. Data were processed with XDS using the autoProc information source (Global Phasing Ltd.). The structure was solved by molecular substitution with MolRep using the homologous PDB 2B3P model. Interactive construction was performed with Coot, refinement with REFMAC5, and validation with Molprobity. Structure figures were prepared with PyMOL (PyMOL Molecular Graphics System, Schrodinger, LLC). Selection for ncAAs

[00319] Selection was performed as described in Figure 8. RNA was isolated and oxidized as described in general procedure A. BiomREX was performed as specified in the general procedure. After the first selection cycle, new libraries were assembled from the amplified cDNA, as described above. After the second selection cycle, NGS samples were prepared from the isolated cDNA, as described above, NGS was run using a 600-cycle P2 cartridge, and the data were analyzed as specified above. Selection for ncMs

[00320] Selection was performed as described in Figure 19. RNA was isolated and oxidized as described in general procedure A. BiomREX was performed as specified in the general procedure. NGS samples were prepared from the isolated cDNA as Petition 870250088848, dated 09 / 30 / 2025, pages 150 / 242 136 / 141 described above, the NGS was run using a 600-cycle P1 cartridge and the data were analyzed as specified above. Selection for substrate 12 using a randomized mutagenesis library

[00321] The concentrations of pMB1 plasmids encoding PylRS hits 12_1 and 12_2 were measured using a Qubit 2 Fluorometer (Life Technologies) and the Qubit 1x dsDNA HS Assay Kit (Invitrogen), and the plasmids were combined in equimolar amounts. The combined plasmids were used for an error-prone active site PCR of PylRS using Golden Gate primers and the GeneMorph II kit (Agilent) under conditions that led to the maximum number of randomized mutations. Amplicons were cloned into a novel pColE1 structure by two-piece Golden Gate assembly, according to NEB (New England Biolabs) guidelines. Selection was performed as described in Figure 30. RNA was isolated and oxidized as described in general procedure A. Bio-mREX was performed as specified in the general procedure.NGS samples were prepared from isolated cDNA as described above, NGS was run using a 600-cycle P2 cartridge, and the data were analyzed as specified above. Code availability

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Claims

1. Acyl-tRNA synthetase capable of specifically acylating a tRNA with a beta amino acid, characterized in that the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300A, M300C, M300D, M300M or M300S with respect to SEQ ID NO: 1; A302A, A302C, A302D, A302G, A302H, A302L, A302N or A302Y with respect to SEQ ID NO: 1; and N346A, N346C, N346G, N346S, N346T or N346V with respect to SEQ ID NO:

1.

2. Acyl-tRNA synthetase, according to claim 1, characterized in that it comprises M300A, M300C, M300D or M300S; A302C, A302D, A302G, A302H, A302L, A302N or A302Y; and N346A, N346C, N346G, N346S, N346T or N346V.

3. Acyl-tRNA synthetase, according to claim 1, characterized in that it comprises M300D; A302H; and, optionally, N346A, N346G or N346S.

4. Acyl-tRNA synthetase, according to claim 3, characterized in that it comprises N346G.

5. Acyl-tRNA synthetase capable of specifically acylating a tRNA with a beta amino acid, characterized in that optionally the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, wherein the acyl-tRNA synthetase comprises an amino acid sequence comprising mutations corresponding to: an M300D mutation relative to SEQ ID NO: 1, an A302H, A302Y or A302C mutation relative to SEQ ID NO: 1, and an N346G, N346A or N346S mutation relative to SEQ ID NO:

1. Petition 870250088848, dated 09 / 30 / 2025, p. 157 / 242 2 / 18 6. Acyl-tRNA synthetase, according to claim 5, characterized in that the A302 mutation is A302H and the N346 mutation is N346G.

7. Acyl-tRNA synthetase, according to any one of claims 1 to 6, characterized in that the amino acid sequence comprises any one or any combination of mutations at positions corresponding to the following positions in SEQ ID NO: 1: F295, L305, Y306, N307, L309, M344, N346, C348, T364, F366, K375, T387, V401, D414 and G421.

8. Acyl-tRNA synthetase, according to claim 7, characterized in that the amino acid sequence comprises any one, any combination of, or all of the following: 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 M344Q; C348A, C348C, C348F, C348G, C348H, C348I, C348L, C348M, C348N, C348R, C348S, C348T, C348V or C348Y; and V401A, V401C, V401F, V401K, V401L, V401S, V401TOU or V401V.

9. Acyl-tRNA synthetase, according to claim 7, characterized in that optionally the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, wherein the amino acid sequence comprises any one or any combination 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.

10. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that optionally the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, in Petition 870250088848, dated 09 / 30 / 2025, p. 158 / 242 3 / 18, wherein the amino acid sequence comprises any one of the following sets of mutations at positions corresponding to the positions in SEQ ID NO: 1: 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.

11. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that optionally the beta amino acid is ((S)-3-amino-3-(benzo[d][1,3]dioxol-5yl)propanoic acid ((S^MDF)), wherein the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, the residues: M300D; A302H; optionally L305C, L305I or L305L; optionally Y306H, Y306I, Y306L or Y306Y; optionally L309C, L309F, L309H, L309L or L309N; C348, optionally C348G, C348N, C348S or C348V; 12. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that optionally the beta amino acid is ((S)-3-amino-3-(4-bromophenyl)propanoic acid ((S^^BrF)), wherein the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, the residues: M300D; A302H; optionally L305F, L305I, L305L or L305N; optionally Y306I or Y306Y; optionally L309C, L309F, L309L or Petition 870250088848, dated 09 / 30 / 2025, page N346A or N346G; and optionally C348C, C348G, C348I or C348S; optionally Y384Y; 13. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that optionally the beta amino acid is ((S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S^pmFF)), wherein the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, the residues: M300D; A302H; optionally L305C, L305I, L305L or L305V; optionally Y306I, Y306L or Y306Y; optionally L309G, L309H, L309L, L309N, L309S or L309V; C348L, C348S or C348V; optionally Y384Y; 14. Acyl-tRNA synthetase according to any one of claims 1 to 9, characterized in that optionally the beta amino acid is ((S)-3-amino-3-(2-bromophenyl)propanoic acid ((S^^BrF)), wherein the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, the residues: M300D or M300M; A302D or A302H, optionally L305L, optionally Y306H, Y306L, Y306R or Y306Y; optionally L309G, L309H or L309L; optionally M344Q; N346G or N346S; S399S; and optionally V401C or V401V.

15. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that optionally the beta amino acid is ((S)-3-amino-3-(3-(trifluoromethyl)phenyl)propanoic acid ((S)β3mCF3F)), wherein the amino acid sequence comprises, at positions corresponding to the positions in SEQ 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.

16. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any one of the sets of mutations in any one of Tables 1 to 6.

17. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any one of the sets of mutations and wild-type residues in any one of Tables 1 to 6.

18. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any one of the sets of mutations associated with an identifier (e.g., 12_1) in any one of Tables 1 to 6.

19. Acyl-tRNA synthetase, according to any one of claims 1 to 9, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any one of the sets of mutations and wild-type residues associated with an identifier (e.g., 12_1) in any one of Tables 1 to 6.

20. Acyl-tRNA synthetase, according to any one of claims 1 to 19, characterized in that the amino acid sequence comprises any of the following sets of mutations at positions corresponding to the positions in SEQ ID NO: 1: a) N307K and F366L; b) F295I and T387I; c) G421A; d) T364A and T387I; e) T387S and D414V; or f) F295L and K375R.

21. Acyl-tRNA synthetase, according to any one of claims 1 to 20, characterized in that optionally the beta amino acid is (S)-3-amino-3-(3-bromophenyl)propanoic acid, wherein the amino acid sequence comprises mutations corresponding to A302H, L305H, N307K, N346G, C348F, F366L and V401C relative to SEQ ID NO:

1.

22. Acyl-tRNA synthetase, according to claim 5, characterized in that the amino acid sequence comprises mutations corresponding to A302H, L305H, Y306Y, N307K, L309L, M344M, N346G, C348F, F366L, Y384Y, S399S and V401C relative to SEQ ID NO:

1.

23. Acyl-tRNA synthetase, according to any one of claims 1 to 22, characterized in that 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.

24. Acyl-tRNA synthetase, according to any one of claims 1 to 23, characterized in that 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 any one of the SEQ ID NOs: 1 to 18.

25. Acyl-tRNA synthetase, according to any of claims 1 to 24, characterized in that the amino acid sequence is a PylRS amino acid sequence.

26. Use of an acyl-tRNA synthetase, as defined in any one of claims 1 to 25, in a method for generating a polymer, characterized in that it comprises a beta amino acid.

27. Method for producing a polymer comprising a beta amino acid, characterized in that it comprises: i) the use of an acyl-tRNA synthetase to acylate a tRNA with the beta amino acid and ii) the incorporation of the beta amino acid into a polymer.

28. Method for producing a polymer comprising a beta amino acid, characterized in that it comprises: i) the use of an acyl-tRNA synthetase, as defined in any one of claims 1 to 25, to acylate a tRNA with the beta amino acid and ii) the incorporation of the beta amino acid into a polymer chain.

29. Acyl-tRNA synthetase, according to any one of claims 1 to 25, use according to claim 26, or method according to claim 27 or 28, characterized in that the beta amino acid is an analog of S-beta-3-phenylalanine.

30. Acyl-tRNA synthetase, according to any one of claims 1 to 25, use according to claim 26, or method according to claim 27 or 28, characterized in that 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)p3MDF), (S)-3-amino-3-(4-bromophenyl)propanoic acid ((S)p3pBrF), (S)-3-amino-3-(3,4-difluorophenyl)propanoic acid ((S)p3pmFF), (S)-3-amino-3-(2 Petition 870250088848, dated 09 / 30 / 2025, p. 163 / 242 8 / 18 bromophenyl)propanoic acid ((S)p3oBrF) or (S)-3-amino-3-(3(trifluoromethyl)phenyl)propanoic acid ((S)p3mCF3F).

31. Nucleic acid, characterized in that it encodes an acyl-tRNA synthetase as defined in any one of claims 1 to 25.

32. Host cell, characterized in that it comprises an acyl-tRNA synthetase as defined in any one of claims 1 to 25, or a nucleic acid as defined in claim 31.

33. Host cell according to claim 32, characterized in that the acyl-tRNA synthetase is orthogonal to endogenous tRNAs.

34. Host cell, according to claim 32 or 33, characterized in that the host cell is viable.

35. Acyl-tRNA synthetase capable of specifically acylating a tRNA with a disubstituted α,α-amino acid, characterized in that the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: A302C, A302G, A302H or A302S with respect to SEQ ID NO: 1; and N346A, N346C, N346E, N346G, N346T or N346V with respect to SEQ ID NO:

1.

36. Acyl-tRNA synthetase, according to claim 35, characterized in that the amino acid sequence comprises a residue corresponding to M300A, M300C, M300D, M300E, M300M, M300S or M300T with respect to SEQ ID NO:

1.

37. Acyl-tRNA synthetase, according to claim 35, characterized in that the amino acid sequence comprises a residue corresponding to M300D relative to SEQ ID NO:

1. Petition 870250088848, dated 09 / 30 / 2025, p. 164 / 242 9 / 18 38. Acyl-tRNA synthetase, according to claim 35, characterized in that the amino acid sequence comprises residues corresponding to: M300D; A302G or A302H; and N346A, N346C or N346G.

39. Acyl-tRNA synthetase, according to any one of claims 35 to 38, characterized in that the amino acid sequence comprises any one or any combination of mutations at positions corresponding to the following positions in SEQ ID NO: 1: F295, M300, L305, Y306, N307, L309, M344, N346, C348, T364, F366, K375, T387, V401, D414 and G421.

40. Acyl-tRNA synthetase, according to claim 39, characterized in that the amino acid sequence comprises any one, any combination of, or all of the following: 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 M344Q; C348C, C348F, C348G, C348H, C348I, C348L, C348S or C348V; Y384Y; S399S; and V401A, V401C, V401K, V401L or V401V.

41. Acyl-tRNA synthetase, according to any one of claims 35 to 40, characterized in that the amino acid sequence comprises residues corresponding to: M300D; A302G or A302H; L305C, L305F or L305S; N346A, N346C or N346G; and C348C or C348G.

42. Acyl-tRNA synthetase, according to any one of claims 35 to 40, characterized in that the amino acid sequence comprises residues corresponding to: 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.

43. Acyl-tRNA synthetase, according to any one of claims 35 to 40, characterized in that the amino acid sequence comprises residues corresponding to: 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.

44. Acyl-tRNA synthetase, according to claim 35, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any of the sets of mutations in Table 7.

45. Acyl-tRNA synthetase, according to claim 35, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, Petition 870250088848, dated 09 / 30 / 2025, p. 166 / 242 11 / 18, any of the sets of mutations and wild-type residues in Table 7.

46. ​​Acyl-tRNA synthetase, according to claim 35, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any of the sets of mutations associated with an identifier (e.g., A6_2) in Table 7.

47. Acyl-tRNA synthetase, according to claim 35, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any of the sets of mutations and wild-type residues associated with an identifier (e.g., A6_2) in Table 7.

48. Acyl-tRNA synthetase, according to any one of claims 35 to 47, characterized in that the amino acid sequence comprises any one of the following sets of mutations at positions corresponding to the positions in SEQ ID NO: 1: a) N307K and F366L; b) F295I and T387I; c) G421A; d) T364A and T387I; e) T387S and D414V; or f) F295L and K375R.

49. Acyl-tRNA synthetase according to any one of claims 35 to 48, characterized in that 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.

50. Acyl-tRNA synthetase, according to any one of claims 35 to 49, characterized in that the amino acid sequence of Petition 870250088848, dated 09 / 30 / 2025, p. 167 / 242 12 / 18 has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% similarity or identity with the amino acid sequence of any of the SEQ ID NOs: 1 to 18.

51. Acyl-tRNA synthetase, according to any one of claims 35 to 50, characterized in that the amino acid sequence is a PylRS amino acid sequence.

52. Use of an acyl-tRNA synthetase, as defined in any one of claims 35 to 51, characterized in that it is in a method of generating a polymer comprising a disubstituted α,α-amino acid.

53. Method for producing a polymer comprising a disubstituted α,α-amino acid, characterized in that it comprises: i) the use of an acyl-tRNA synthetase to acylate a tRNA containing the disubstituted α,α-amino acid, and ii) the incorporation of the disubstituted α,α-amino acid into a polymer.

54. Method for producing a polymer comprising a disubstituted α,α-amino acid, characterized in that it comprises: i) the use of an acyl-tRNA synthetase as defined in any one of claims 35 to 51, to acylate a tRNA containing the disubstituted α,α-amino acid, and ii) the incorporation of the disubstituted α,α-amino acid into a polymer chain.

55. Acyl-tRNA synthetase, according to any one of claims 35 to 51, use according to claim 52, or method according to claim 53 or 54, characterized in that the disubstituted α,α-amino acid is (S)-2-amino-3-(4-iodophenyl)-2-methylpropanoic acid ((S)α-Me-plF). Petition 870250088848, dated 09 / 30 / 2025, pp. 168 / 242 13 / 18 56. Nucleic acid, characterized in that it encodes an acyl-tRNA synthetase, as defined in any one of claims 35 to 51.

57. Host cell, characterized in that it comprises an acyl-tRNA synthetase, as defined in any one of claims 35 to 51, or a nucleic acid, as defined in claim 56.

58. Host cell, according to claim 57, characterized in that the acyl-tRNA synthetase is orthogonal to endogenous tRNAs.

59. Host cell, according to claim 57 or 58, characterized in that the host cell is viable.

60. Acyl-tRNA synthetase capable of specifically acylating a tRNA with a beta-hydroxy acid, characterized in that the acyl-tRNA synthetase comprises an amino acid sequence comprising residues corresponding to: M300A, M300D, M300M, M300N or M300S with respect to SEQ ID NO: 1; and A302D, A302G, A302H or A302N with respect to SEQ ID NO:

1.

61. Acyl-tRNA synthetase, according to claim 60, characterized in that the amino acid sequence comprises a residue corresponding to M300D, M300N or M300S.

62. Acyl-tRNA synthetase, according to claim 60 or 61, characterized in that the amino acid sequence comprises a residue corresponding to N346A, N346C, N346E, N346G, N346N, N346S or N346V.

63. Acyl-tRNA synthetase, according to claim 62, characterized in that the amino acid sequence comprises a residue corresponding to N346A, N346C or N346G. Petition 870250088848, dated 09 / 30 / 2025, pp. 169 / 242 14 / 18 64. Acyl-tRNA synthetase, according to any one of claims 60 to 63, characterized in that the amino acid sequence comprises residues corresponding to M300N and A302H relative to SEQ ID NO:

1.

65. Acyl-tRNA synthetase, according to claim 64, characterized in that the amino acid sequence comprises mutation(s) at N346 and / or C348 relative to SEQ ID NO:

1.

66. Acyl-tRNA synthetase, according to claim 60, characterized in that the amino acid sequence comprises residues corresponding to: M300N or M300S; A302H; and N346A, N346C or N346G.

67. Acyl-tRNA synthetase, according to claim 60, characterized in that the amino acid sequence comprises residues corresponding to M300N, A302H, and N346G.

68. Acyl-tRNA synthetase, according to any one of claims 60 to 67, characterized in that the amino acid sequence comprises any one or any combination of mutations at positions corresponding to the following positions in SEQ ID NO: 1: F295, L305, Y306, N307, L309, M344, N346, C348, T364, F366, K375, T387, V401, D414 and G421.

69. Acyl-tRNA synthetase, according to claim 68, characterized in that the amino acid sequence comprises any one, any combination of, or all of the following: L305C, L305L, L305N, L305S or L305V; Y306D, Y306F, Y306I, Y306N, Y306R, Y306S or Y306Y; L309D, L309H, L309I, L309L, L309R or L309S; M344E, M344M or M344Q; C348C, C348F, C348I, C348L, C348T or C348V; Y384Y; S399S; and V401A, V401C, V401K, V401L, V401S, V401T and V401V. Petition 870250088848, dated 09 / 30 / 2025, pages 170 / 242 15 / 18 70. Acyl-tRNA synthetase, according to any one of claims 60 to 69, characterized in that the amino acid sequence comprises residues corresponding to: M300N or M300S; A302H; N346A, N346C or N346G; and a C348 mutation, which is optionally C348I, C348L, C348T or C348V.

71. Acyl-tRNA synthetase, according to any one of claims 60 to 69, characterized in that the amino acid sequence comprises residues corresponding to: 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.

72. Acyl-tRNA synthetase, according to any one of claims 60 to 69, characterized in that the amino acid sequence comprises residues corresponding to: M300N or M300S; A302H; L305L or L305V; Y306N or Y306Y; L309I, L309L or L309S; M344E, M344M or M344Q; N346A, N346C or N346G; Petition 870250088848, dated 09 / 30 / 2025, p. 171 / 242 16 / 18 C348I, C348L, C348T or C348V; Y384Y; S399S; and V401C, V401K, V401L or V401V.

73. Acyl-tRNA synthetase, according to claim 60, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any of the sets of mutations in Table 8.

74. Acyl-tRNA synthetase, according to claim 60, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any of the sets of mutations and wild-type residues in Table 8.

75. Acyl-tRNA synthetase, according to claim 60, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any of the sets of mutations associated with an identifier (e.g., A7_2) in Table 8.

76. Acyl-tRNA synthetase, according to claim 60, characterized in that the amino acid sequence comprises, at positions corresponding to the positions in SEQ ID NO: 1, any of the sets of mutations and wild-type residues associated with an identifier (e.g., A7_2) in Table 8.

77. Acyl-tRNA synthetase, according to any one of claims 60 to 76, characterized in that the amino acid sequence comprises any one of the following sets of mutations at positions corresponding to the positions in SEQ ID NO: 1: a) N307K and F366L; b) F295I and T387I; Petition 870250088848, dated 09 / 30 / 2025, p. 172 / 242 17 / 18 c) G421A; d) T364A and T387I; e) T387S and D414V; or f) F295L and K375R.

78. Acyl-tRNA synthetase, according to any one of claims 60 to 77, characterized in that 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.

79. Acyl-tRNA synthetase, according to any one of claims 60 to 78, characterized in that 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 any one of the SEQ ID NOs: 1 to 18.

80. Acyl-tRNA synthetase, according to any one of claims 60 to 79, characterized in that the amino acid sequence is a PylRS amino acid sequence.

81. Use of an acyl-tRNA synthetase as defined in any one of claims 60 to 80, characterized in that it is in a method for generating a polymer comprising a beta-hydroxy acid.

82. Method for producing a polymer comprising a beta-hydroxy acid, characterized in that it comprises: i) using an acyl-tRNA synthetase to acylate a tRNA with the beta-hydroxy acid and ii) incorporating the beta-hydroxy acid into a polymer.

83. Method for producing a polymer comprising a beta-hydroxy acid, characterized in that it comprises: i) use of an acyl-tRNA synthetase as defined in any one of claims 60 to 80, to acylate a tRNA with the beta-hydroxy acid and ii) incorporation of the beta-hydroxy acid into a polymer chain.

84. Acyl-tRNA synthetase, according to any one of claims 60 to 80, use according to claim 81, or method according to claim 82 or 83, characterized in that the beta-hydroxy acid is (S)-3-(3-chlorophenyl)-3-hydroxypropanoic acid (OH-(S)p3mCIF).

85. Nucleic acid, characterized in that it encodes an acyl-tRNA synthetase, as defined in any one of claims 60 to 80.

86. Host cell, characterized in that it comprises an acyl-tRNA synthetase, as defined in any one of claims 60 to 80, or a nucleic acid, as defined in claim 85.

87. Host cell according to claim 86, characterized in that the acyl-tRNA synthetase is orthogonal to endogenous tRNAs.

88. Host cell, according to claim 86 or 87, characterized in that the host cell is viable.

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