Genetic Incorporation of Diverse Noncanonical Amino Acids for Histidine Substitution.

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Learn more: PMC Disclaimer | PMC Copyright Notice J Am Chem Soc . 2026 Mar 26;148(13):13619–13632. doi: 10.1021/jacs.5c19599 Search in PMC Search in PubMed View in NLM Catalog Add to search Genetic Incorporation of Diverse Noncanonical Amino Acids for Histidine Substitution Anton Natter Perdiguero Anton Natter Perdiguero 1 Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Find articles by Anton Natter Perdiguero 1 , Sandro Fischer Sandro Fischer 1 Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Find articles by Sandro Fischer 1 , Alrika R Lischke Alrika R Lischke 1 Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Find articles by Alrika R Lischke 1 , Benjamin P Manser Benjamin P Manser 1 Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Find articles by Benjamin P Manser 1 , Alexandria Deliz Liang Alexandria Deliz Liang 1 Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Find articles by Alexandria Deliz Liang 1, * Author information Article notes Copyright and License information 1 Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland * Email: [email protected] . Received 2025 Nov 5; Accepted 2026 Mar 2; Revised 2026 Feb 27; Collection date 2026 Apr 8. © 2026 The Authors. Published by American Chemical Society This article is licensed under CC-BY 4.0 PMC Copyright notice PMCID: PMC13067346  PMID: 41885601 Abstract Using genetic code expansion, canonical amino acid residues can be site-specifically substituted by noncanonical amino acids (ncAAs) with modified chemical properties. This technique has enabled detailed enzymatic studies, the design of enzymes that catalyze novel reactions, and the engineering of enzymes with improved function. In proteins, histidine can play versatile roles in catalysis, including as an acid, a base, a nucleophile, and a coordinating ligand to a catalytic metal. However, the current scope of histidine-like ncAAs that can be incorporated is limited. Herein, we develop a toolkit consisting of nine new aminoacyl-tRNA synthetase/tRNA pairs for the site-specific genetic encoding of an expanded set of 12 new histidine-like ncAAs. The 12 ncAAs feature broadly tuned nitrogen p K a H, alternative heterocycles, and varying substitution patterns. We profile the substrate specificity of the developed aaRS/tRNA pairs and uncover many mutually orthogonal substrate specificities, which we validate for six combinations of dual encoded histidine-like ncAAs. We expect that the tools presented herein will be broadly applicable to study histidine residues in catalysis and to tune the properties of histidine residues for enzyme engineering and design. Introduction Among canonical amino acids, histidine has the highest catalytic propensity and is often found in the active site of enzymes. Histidine can play versatile roles in catalysis including as an acid, a base, a nucleophile, and a coordinating ligand to a catalytic metal. The imidazole side chain of histidine contains two nitrogen atoms that can participate in such chemistry N τ and N π (see Note for further clarification of the nomenclature). Because histidine is structurally and chemically distinct from other canonical amino acids, its substitution with traditional mutagenesis methods is typically not a viable strategy to study or tune its role in catalysis. Alternatively, histidine residues can be substituted by histidine-like noncanonical amino acids (ncAAs). Several methods to incorporate histidine-like ncAAs into proteins exist. Global replacement of histidine has been leveraged to incorporate a selection of histidine-like ncAAs, but the ncAAs are incorporated indiscriminately at all histidine residues. − In contrast, genetic code expansion enables the site-specific incorporation of ncAAs into proteins, allowing for more atomistic control. The most widely employed method for genetic code expansion uses engineered aminoacyl-tRNA synthetase (aaRS) and tRNA pairs to selectively incorporate ncAAs in response to a reassigned codon, typically the amber stop codon (TAG). This method enables expansion of the accessible chemical diversity in proteins and has been applied to carry out detailed enzymatic studies, − to design enzymes that catalyze novel reactions, − and to engineer enzymes with improved function. − Despite the importance of histidine in catalysis and extensive efforts to engineer systems for incorporation of diverse histidine-like ncAAs, the current accessible scope of such ncAAs remains limited, ,,− particularly compared to tyrosine derivatives, lysine derivatives, and larger aromatic ncAAs. The most widely used histidine-like ncAA is N π -methyl- l -histidine ( πMH , also referred to as NMH). ,,,,, The extensive use of this system in several different domains highlights the value of such histidine-like ncAAs. Expanding the scope available for histidine substitution to include different coordination chemistry, nucleophilicity, and acid/base reactivity would enable new modalities for studying and engineering of histidine-containing proteins. Additionally, a systematic assessment of the resulting engineered aaRS/tRNA pairs could illuminate features contributing to substrate recognition and enable incorporation of previously intractable ncAAs. Thus, to expand the available chemistry, we explored the genetic encoding of a large panel of diverse histidine-like ncAAs in Escherichia coli . Through extensive engineering of various aaRS/tRNA pairs, we identify nine aaRS/tRNA systems for the site-specific genetic incorporation of a panel of 12 histidine-like ncAAs ( Figure a). The 12 ncAAs feature broadly tuned nitrogen p K a H, alternative heterocycles, and varying substitution patterns. We note that Figure a provides predicted aqueous p K a H as a general reference. However, protein microenvironments can dramatically alter the p K a H values of amino acid side chains. − Within the set of engineered aaRS/tRNA pairs is the highly sought-after and elusive system for incorporation of N τ -methyl- l -histidine ( τMH ). 1. Open in a new tab Targeting histidine-like ncAAs for site-selective incorporation by genetic code expansion. Classical aaRS engineering strategies were applied to expand the genetic code with a diverse set of histidine-like ncAAs with tuned chemical properties. (a) The p K a H scale for the side chains of histidine and histidine-like ncAAs targeted in this study. Previously incorporated ncAAs are illustrated in gray, whereas newly incorporated ncAAs reported herein are shown in black. The p K a H values provided are predicted for an aqueous environment. Protein microenvironments can dramatically alter side chain p K a H values; − thus, aqueous p K a H values are provided as a general reference. (b) Schematic of ncAA incorporation by amber suppression using genetic code expansion. (c) General schematic for classical aaRS evolution, including aaRS library creation, positive selection to identify aaRS variants that incorporate the ncAA or a canonical amino acid, negative selection to remove variants that incorporate a canonical amino acid, a second positive selection for further enrichment, screening to quantify ncAA-dependent protein production, MS analysis to confirm the identity of the incorporated ncAA, and validation in an optimized plasmid designed for high production of the tRNA and the evolved aaRS. (d) Amber suppression benchmarking of the first seven aaRS/tRNA pairs evolved in this study. Suppression efficiencies in sfGFP150 TAG were measured with and without the respective ncAA. The ncAA concentrations are listed in Supporting Table 1 . The data are presented as % of wt sfGFP, defined as normalized fluorescence (excitation at 480 nm and emission at 510 nm, normalized to the optical density at 600 nm) as percentage of a wt sfGFP reference. The mean and standard deviation of three biological replicates are shown. These new incorporation systems were identified through multiple complementary strategies: classical directed evolution for de novo selections, screening of existing variant libraries, focused library design informed by substrate profiling, and in vivo mutagenesis. Herein, we describe each approach and further elucidate features of these new incorporation systems, including profiling the substrate scope of the evolved aaRS/tRNA pairs and describing orthogonal substrate combinations that enable the double-encoding of several histidine-like ncAAs. We expect that the tools presented herein will be broadly applicable to study histidine residues in catalysis and to tune the properties of histidine residues for enzyme engineering applications and enzyme design. Results Toward expanding the scope for histidine mutagenesis, we identified several key properties that were missing from the genetic code expansion toolkit for histidine-like ncAAs: (i) a diversity of heterocycles for fine-tuning reactivity, (ii) histidine-like ncAAs with preserved N π and N τ and increased p K a H, (iii) histidine-like ncAAs with preserved N π and N τ and decreased p K a H, and (iv) N τ -substituted histidine derivatives, of which the only current incorporable ncAA contains a large photocleavable protecting group, making it excellent for photoactivation but preventing more widespread use in enzyme engineering, such as that seen for πMH. To address these limitations, we selected a panel of target ncAAs spanning a broad p K a H range that complement existing ncAAs and encompass a variety of desirable properties and substitutions ( Figure a). This panel included new heterocyclic ncAAs that can enable alternative reactivity compared to imidazoles (1,2,3-triazol-4-yl- l -alanine: 123Trz-4A , oxazol-4-yl- l -alanine: 4OxzA , oxazol-5-yl- l -alanine: 5OxzA , 1,2,4-triazol-3-yl- l -alanine: 124Trz-3A , and 3-pyrazolyl- l -alanine: 3PzA ), two stereoisomers of the useful 3-pyridyl- l -alanine ( 3PyA ) to enable directional control of the reactive nitrogen (2-pyridyl- l -alanine: 2PyA and 4-pyridyl- l -alanine: 4PyA ), ncAAs containing substituted imidazole rings for tuning the p K a H (5-nitro- l -histidine: 5NO 2 H and 2-ethyl-5-methyl- l -histidine: 2E5MH ), two new N τ -substituted histidine-like ncAAs (1-benzyl-1,2,3-triazol-4-yl- l -alanine: 1Bn123Trz-4A and N τ -benzyl- l -histidine: τBnH ), and finally, the long-sought, but elusive τMH. From this target panel, we aimed to enable genetic code expansion ( Figure b) through the development of orthogonal aaRS/tRNA pairs to encode each of these ncAAs site selectively ( Figure c). Classical aaRS Engineering Approaches Enable the Incorporation of New ncAAs For the target ncAAs, we carried out de novo selections on a set of site-saturation mutagenesis (SSM) libraries ( Supporting Table 2 ) generated from the PylRS from Methanosarcina mazei ( Mm PylRS) and a chimeric PylRS variant that we previously evolved in an unrelated project, termed chPylRS E7 , which derives from a fusion of the PylRSs from Methanohalobium evestigatum and M. mazei ( Note ). As illustrated in Figure c, libraries were subjected to three alternating rounds of positive and negative selection against Mm tRNA Pyl CUA in the presence or absence of the given ncAA, followed by screening of ncAA-dependent suppression in sfGFP150 TAG by fluorescence. The concentrations used for selections and screening can be found in Supporting Table 1 . With 123Trz-4A, 5NO 2 H, 3PzA, and 2E5MH, distinct PylRS mutants were identified that incorporated each of the corresponding ncAAs ( Supporting Table 7 ). The derived synthetase for 3PzA exhibited low incorporation efficiency, but further optimization was achieved by replacing its N-terminal domain with that of Mb PylRS carrying the “IPYE” mutations (V31I/T56P/H62Y/A100E). These previously reported mutations can enable increased amber suppression efficiency of PylRS variants in some cases. , Transfer of the engineered pairs onto our optimized plasmid for genetic code expansion application (pOS1T) afforded suppression systems that incorporate the given ncAA into sfGFP150 TAG with incorporation efficiencies ranging between 30% and 85% compared to wt sfGFP ( Figure d, first four aaRSs). Incorporation of the ncAAs was confirmed by LC-MS analysis of intact sfGFP150 TAG and LC-MS/MS of trypsinized sfGFP150 TAG ( Supporting Figures 2a–d and 3a–d , respectively). Using LC-MS/MS analysis, we searched for the desired ncAA modification and also possible, unwanted canonical amino acids incorporated in response to the amber stop codon to provide a more rigorous analysis of incorporation fidelity. For 123Trz-4A, 3PzA, and 2E5MH, no misincorporation was observed. In contrast, for 5NO 2 H, we observed very weak signals corresponding to incorporation of phenylalanine and glutamine, which were not detected by intact mass analysis. Based on comparison of the sfGFP production in the presence and absence of the ncAA, the intact mass spectra, and the low intensity of the misincorporation signals, our results strongly suggest that the misincorporation is very low, with a purity greater than 97% ( Supporting Tables 9 and 10 ). Although LC-MS/MS searches for misincorporation are not commonly reported in genetic code expansion efforts, these results highlight the importance of robust MS/MS analysis in rigorously characterizing incorporation fidelity. Based on this finding, we conducted LC-MS/MS misincorporation analysis for the previously reported ncAA-aaRS/tRNA systems explored herein and our newly evolved ncAA-aaRS/tRNA systems to understand and approximate the extent of low-level misincorporation in these systems ( Supporting Table 10 ). The final aaRS/tRNA pair found through the classical approach was for 4PyA. We envisioned that this ncAA might be incorporated through engineering of a recently described chimeric pair derived from a fusion between a pyrrolysyl and phenylalanine system (chPheRS/3C11-chPheT CUA ). , Through screening a set of SSM libraries ( Supporting Table 5 ), we identified a variant carrying the mutations Q365N and A507S (chPheRS 4PyA‑v0 , Supporting Table 7 ). This mutant was further improved by three rounds of error-prone PCR followed by positive selection and screening. The final variant (chPheRS 4PyA , Supporting Table 7 ) contains 12 additional coding mutations throughout both the N- and C-terminal domains of the chPheRS and has an approximately 21-fold activity improvement compared to chPheRS 4PyA‑v0 ( Supporting Figure 5 ). Transfer of the chPheRS 4PyA /3C11-chPheT CUA pair into a pOS1T plasmid afforded a suppression system that incorporates 4PyA with low background activity and wt-like incorporation efficiency ( Figure d). Incorporation of 4PyA was confirmed by LC-MS analysis of intact sfGFP150 TAG and LC-MS/MS analysis of trypsinized sfGFP150 TAG ( Supporting Figures 2g and 3g ). The improvements with 4PyA highlight the value of random mutagenesis for improving activity from a weakly active starting point. We note that while our work was in progress, a report was published describing incorporation of 4PyA using polyspecific Methanocaldococcus jannaschii tyrosyl tRNA synthetase and Methanogenic archaeon ISO4-G1 PylRS ( G1 PylRS) mutants, albeit at lower efficiencies (<20% of wt sfGFP). Although classical engineering succeeded for several targets, some ncAAs remained elusive. We therefore turned to an alternative strategy: screening our growing panel of evolved aaRS/tRNA pairs. Panel Screening Can be Leveraged to Identify aaRS/tRNA Pairs for New ncAAs For the remaining ncAAs within the target set, we screened an in-house panel of aaRS/tRNA pairs, which consists of previously reported aaRS/tRNA pairs and some unreported pairs that have been derived for various ongoing projects. From this panel, we evaluated sfGFP150 TAG production in the presence and absence of the target ncAAs, and we were able to identify aaRS/tRNA pairs for three additional ncAAs: 2PyA, τBnH, and 1Bn123Trz-4A. In the case of 2PyA, two reported synthetases for the incorporation of ortho-substituted phenylalanine derivatives , displayed promiscuous activity with 2PyA. However, the activity was low, and the background in the absence of 2PyA was high ( Supporting Figure 4 ). Thus, additional classical engineering was required to achieve selective incorporation. Mutations of two residues located near the ncAA side chainN346 and C348often impart important changes in substrate recognition but are insufficient to enable robust incorporation. Thus, to optimize the incorporation of 2PyA further, we constructed libraries of chPylRS E7 in which N346 and C348 were fixed to mimic these mutations in the weakly active aaRS/tRNA pairs and an additional five positions within the ncAA binding site were randomized by SSM ( Supporting Table 4 ). Through screening and selection, we identified a more active and selective mutant, chPylRS 2PyA ( Supporting Table 7 ). Transfer of the chPylRS 2PyA / Mm tRNA Pyl CUA pair into a pOS1T plasmid afforded a suppression system that incorporates 2PyA with low background activity and approximately 50% yield compared to wt sfGFP ( Figure d). Incorporation of 2PyA was confirmed by LC-MS analysis of intact sfGFP150 TAG and LC-MS/MS analysis of trypsinized sfGFP150 TAG ( Supporting Figures 2f and 3f ). In the case of τBnH and 1Bn123Trz-4A, we found that both of these ncAAs were incorporated by an unreported Mm PylRS mutant from the panel (hereafter Mm PylRS τBnH , Supporting Table 7 ). Expression from sfGFP150 TAG yielded incorporation efficiencies of approximately 20% and 10%, respectively. However, there was very low background suppression in the absence of ncAA ( Figure d). Additionally, incorporation of the desired ncAAs was confirmed by LC-MS analysis of intact sfGFP150 TAG . LC-MS/MS of trypsinized sfGFP150 TAG exhibited low misincorporation levels and indicated that the fidelity was higher for τBnH than 1Bn123Trz-4A ( Supporting Figures 2h,i and 3h,i and Supporting Tables 9 and 10 ). Based on the low background misincorporation, further engineering was not conducted. Between the classical approach and panel screening, we successfully incorporated eight new ncAAs with seven new aaRSs. However, several high-value targets remained elusive, prompting us to explore substrate profiling as a strategy to understand substrate specificity and guide further engineering. Substrate Profiling Reveals Surprising Substrate Specificities With a panel of aaRS/tRNA pairs that direct the incorporation of diverse histidine analogs in hand, we profiled the substrate specificity of different ncAA-aaRS combinations. We expected that such a panel might reveal some incorporation trends and identify aaRS/tRNA pairs with beneficial properties for incorporation of our remaining ncAAs. We augmented our newly derived set with several previously reported aaRS/tRNA pairs that have been described for the incorporation of histidine analogs: Mm PylRS IFGFF / Mm tRNA Pyl , which carries mutations grafted from Mb PylRS IFGFF , and G1 PylRS MIFAF / G1 tRNA ΔΝPyl , which carries mutations from Ma PylRS MIFAF , both of which were developed for πMH incorporation. Additionally, we included the Mm PylRS FLF / Mm tRNA Pyl and Mm PylRS 8_2 / Mm tRNA Pyl pairs described for 3PyA incorporation, , the Mb PylRS 4Thz / Mm tRNA Pyl described for 4-thiazolyl- l -alanine (4ThzA) incorporation and the Mm PylRS QF / Mm tRNA Pyl and Mm PylRS 7_1 / Mm tRNA Pyl pair described for 3-thienyl- l -alanine (3ThA) incorporation. , We tested each suppressor system with the ncAAs in Figure a as well as 2-thienyl- l -alanine (2ThA) and 3ThA. As a reporter, we used sfGFP150 TAG , and we quantified the sfGFP production in the presence or absence of the given ncAA using the concentrations reported in Supporting Table 1 and our optimized plasmid system ( Figure a and Supporting Figure 6 ). 2. Open in a new tab aaRS-ncAA substrate profiling and incorporation of additional histidine-like ncAAs by polyspecificity. aaRS substrate preferences are often nonintuitive: both strict specificity and functional polyspecificity are observed, enabling immediate access to new histidine-like ncAAs and informing future enzyme engineering strategies. (a) Substrate specificity profiling for different aaRS-ncAA combinations. sfGFP150 TAG production was measured for different aaRS/tRNA pairs in the presence or absence of the indicated histidine-like ncAA, and the relative sfGFP production in the presence versus absence of ncAA was determined. Each column corresponds to a distinct ncAA and each row to a distinct aaRS. The entry for the “cognate” ncAA of the aaRS/tRNA pair is outlined with a thick black border. The ncAA concentrations are reported in Supporting Table 1 . The data represent the mean of 2–3 biological replicates. (b) Hierarchical clustering of aaRSs based on substrate profiling. In the dendrogram, the distance at which the branches connect reflects similarity of substrate preferences: shorter distances indicate greater similarity. (c) Chemical structure of 124Trz-3A. (d) Incorporation of 124Trz-3A into sfGFP150 TAG using the chPylRS 3PzA system. (e) Chemical structure of 5OxzA. (f) Incorporation of 5OxzA into sfGFP150 TAG using the Mm PylRS IFGFF system. The data in panels d and f are presented as % of wt sfGFP (as defined for Figure ). The data represent the mean and standard deviation of three biological replicates. Within the panel, we observed high substrate specificity for aaRS/tRNA pairs with their cognate ncAAs (meaning the ncAAs for which they were originally evolved) compared to canonical amino acids. Furthermore, for most previously evolved aaRS/tRNA pairs, we find that they can efficiently incorporate other ncAAs in addition to the cognate substrate. This observation, termed polyspecificity, is a common result for aaRSs obtained from directed evolution. , Within genetic code expansion, polyspecificity refers to the ability of an aaRS/tRNA pair to efficiently direct incorporation of several ncAAs, often ncAAs with similar side chains. Although the terms are used loosely, the term polyspecificity is distinct from both promiscuity and nonspecificity. Promiscuity is a larger umbrella term that includes polyspecificity but also includes weak substrate recognition that might not result in efficient catalysis. Nonspecificity implies a completely lack of molecular discrimination. Polyspecificity and weak promiscuity can each be useful features in enzyme chemistry. Within genetic code expansion, polyspecificity can enable direct incorporation of a desired ncAA without further directed evolution, and weak promiscuity can be leveraged to access new chemical space through additional directed evolution. As expected, polyspecificity is often found for ncAAs that are chemically highly similar to the cognate substrate. Notably, there was significantly less polyspecificity within the newly engineered aaRS/tRNA pairs reported herein, compared to previously described aaRS/tRNA pairs ( Figure a). The reason for this is unclear but could reflect the ease of incorporation for previously described histidine-like ncAAs, differences in application of the classical engineering approach, or lower chemical similarity of the new ncAA set, which was designed to fill gaps in the existing histidine-like ncAA set. Despite the generally higher specificity observed for the newly engineered aaRSs, we observe polyspecificity in selected cases. For example, two distinct synthetases were evolved for 123Trz-4A and 3PzA, Mm PylRS 123Trz‑4A and chPylRS 3PzA , respectively. Based on the chemical similarity between these ncAAs, we expected that both synthetases could be polyspecific for 123Trz-4A and 3PzA, which was confirmed experimentally. Notably, Mm PylRS 123Trz‑4A has a broader substrate scope, also efficiently incorporating 4ThzA and πMH, substrates for which chPylRS 3PzA has no activity. Interestingly, the same libraries were used to derive Mm PylRS 123Trz‑4A as chPylRS 3PzA , but Mm PylRS 123Trz‑4A was not found in the 3PzA screening. This result points to the stochastic nature of multistep selection and screening for such large libraries, beyond even the randomness of large library creation itself. Moreover, regarding ease of incorporation, we observed that πMH was incorporated by many different aaRS/tRNA pairs, and both Mm PylRS 123Trz‑4A and chPylRS 5NO2H incorporated πMH more efficiently than their cognate substrate ( Supporting Figure 6 ). Analogously, chPylRS 5NO2H also incorporates 3PyA at wt-like levels, more efficiently than the previously reported G1 PylRS MIFAF and chPylRS FLF ( Supporting Figure 6 ). Collectively, these results also highlight the ability of many different evolutionary solutions to efficiently address a single enzyme engineering goal. Overall, we observe many nonobvious substrate selectivities, with some aaRS variants capable of distinguishing substrates with highly similar structures and some that seem to act more as generalists. Toward mapping these differences more systematically, we performed hierarchical clustering analysis based on substrate profiles ( Figure b). The hierarchical analysis revealed that the aaRSs can be separated into distinct clades based on their substrate profiles. Both chPylRS 2PyA and Mb PylRS 4Thz have highly similar substrate profiles that are also the most different from the other aaRSs. Additionally, both chPheRS 4PyA and chPylRS 2E5MH are outliers with unique profiles within the data set. In the case of chPheRS 4PyA , this observation is perhaps not so surprising given that this system is derived from a PheRS system and not a PylRS system like all the others. In terms of future engineering campaigns, we expect that selecting several parent sequences from disparate substrate profile clades may improve the chances of successful aaRS evolution, such as how sequence similarity networks are used in general enzyme engineering campaigns. Beyond describing these substrate profiles, the ability to predict the substrate tolerance of an aaRS would be of extremely high value; thus, we studied if incorporation parameters were correlated with computationally predicted binding affinities. However, we found that the predicted binding affinitieseven when accounting for logP valuesare a very poor indicator for aaRS substrate preference within this set of ncAAs ( Supporting Figure 7 ). These results highlight the complexity of aaRS activity and selectivity, particularly for relatively small, polar ncAAs with highly similar structures. In addition to exploring substrate preferences, we were able to identify two additional pairs with robust suppression efficiencies for new ncAAs. In the case of 124Trz-3A, we discovered that this ncAA is a substrate for chPylRS 3PzA . Indeed, production of sfGFP in the presence of elevated 124Trz-3A concentrations showed incorporation of 124Trz-3A with an approximately 20% yield of wt sfGFP and a low background in the absence of 124Trz-3A ( Figure d). The incorporation was confirmed by LC-MS analysis of intact sfGFP150 TAG , and LC-MS/MS analysis of trypsinized sfGFP150 TAG indicated low levels of misincorporation ( Supporting Figures 2e and 3e, Supporting Tables 9 and 10 ). We also identified 5OxzA as a substrate for Mm PylRS IFGFF , albeit with low protein yields compared to wt sfGFP (∼12%, Figure f). Incorporation of 5OxzA was confirmed by LC-MS analysis of intact sfGFP150 TAG , and LC-MS/MS analysis of trypsinized sfGFP150 TAG also indicated low misincorporation ( Supporting Figures 2j and 3j, Supporting Tables 9 and 10 ). In all, from the comprehensive substrate profiling, we were able to identify distinct clades of engineered PylRSs that can be used for increasing the diversity of future engineering campaigns, and we identified two additional histidine-like ncAAs that are accepted by existing aaRS/tRNA pairs. Moreover, beyond revealing general substrate preferences, we hypothesized that careful analysis of the profiling data could guide smarter library design for our most challenging targets: 4OxzA and τMH. Substrate Profiling Can be Leveraged to Conduct Smarter Engineering From the aaRS-ncAA substrate profiling, we carefully examined the incorporation profiles for ncAAs for which we had failed to identify an aaRS/tRNA pair from the methods above, namely 4OxzA and τMH. We identified two aaRS/tRNA pairs with minute (<1.2-fold) but reproducible activity above background ( Mb PylRS 4ThzA and chPylRS 2PyA , respectively). We sought to identify mutations that might be shared between these weakly active pairs, reasoning that such mutations, if genuinely important for substrate recognition, could serve as starting points for focused library design. We identified A314Q in chPylRS 2PyA (corresponding to A302Q in Mm PylRS and A267Q in Mb PylRS) and A267Q in Mb PylRS 4ThzA as a shared mutation in these two systems. We thus generated libraries of chPylRS E7 in which we fixed the mutation A314Q ( Figure a,b and Supporting Table 6 ). We carried out selections with these libraries on 4OxzA and τMH as described above. Although no hit was found for τMH, we identified two aaRS/tRNA pairs that incorporate 4OxzA (chPylRS 4OxzA and chPylRS 4OxzA‑2 ). In the pOS1T vector, chPylRS 4OxzA ( Figure c) showed a superior ncAA-dependent sfGFP production in the presence of 4OxzA, compared to chPylRS 4OxzA‑2 ( Supporting Figure 10 ). Incorporation of 4OxzA with chPylRS 4OxzA was confirmed by LC-MS analysis of intact sfGFP150 TAG and LC-MS/MS of trypsinized sfGFP150 TAG ( Supporting Figures 2k and 3k, and Supporting Table 9 ). These results highlight the value of substrate profiling, particularly in light of the extremely weak starting point that could only be identified reproducibly above background based on this profiling. 3. Open in a new tab Evolution of additional aaRS/tRNA pairs for histidine-like ncAAs. Activity trends from substrate profiling were leveraged to guide smarter aaRS engineering strategiesincluding focused library design and parent sequence selection for in vivo mutagenesisto access ncAAs that are otherwise refractory to incorporation. (a) Chemical structure of 4OxzA. (b) Schematic of directed evolution workflow for identification of chPylRS 4OxzA . (c) Incorporation of 4OxzA into sfGFP150 TAG using the chPylRS 4OxzA system. (d) Chemical structure of τMH. (e) Schematic of directed evolution workflow for identification of Mb (IPYE)­PylRS τMH . (f) Incorporation of τMH into sfGFP150 TAG using the Mb (IPYE)­PylRS τMH system. (g) The substrate specificity profiles of chPylRS 4OxzA and Mb (IPYE)­PylRS τMH , showing the relative sfGFP production in the presence versus in the absence of the ncAA. The data were collected and analyzed as indicated in Figure a. (h) Alphafold3 model of the C-terminal domain of the evolved Mb (IPYE)­PylRS τΜH in complex with ATP. The domain is shown in cartoon representation, and ATP is shown in stick representation. The mutated residues are shown as spheres: “QSW” mutations present in Mb PylRS 4ThzA (light cyan), mutations obtained from MutaT7 mutagenesis (dark cyan), and the rational S364T mutation (purple). One of the mutations is in the unstructured linker between the N- and C-terminal domains and is therefore not depicted in the cartoon. The data in panels c and f are depicted as % of wt sfGFP (as defined for Figure ). The data represent the mean and standard deviation of three biological replicates. In Vivo Mutagenesis Succeeds Where Other Strategies Failed From our initial ncAA targets, τMH was the ncAA that was most widely sought after, elusive, and frankly puzzling, not only to our lab but to others as well. ,,,, The lack of successful aaRS engineering suggested that the challenges might be related to steps ancillary to aminoacylation (such as cell uptake or metabolism) or postaminoacylation (such as EF-Tu binding, ribosomal recognition, ribosomal incorporation, etc.). However, intracellular accumulation of τMH was supported by LC-MS analysis, suggesting that τMH can accumulate in E. coli (data not shown). Additionally, although postaminoacylation issues might be consistent with previous results, such a constraint seemed unlikely given the relatively innocuous structure and canonical backbone. Given these puzzling failures, we re-evaluated our previous strategies, which included classical screening using more than ten libraries from SSM and error-prone PCR, substrate walking from τBnH and Mm PylRS τBnH with progressively smaller N τ substituents, and the polyspecificity approach described for 4OxzA from Mb PylRS 4ThzA . Most of these strategies relied on our limited structural knowledge of PylRS constructs. Thus, based on recent success stories with in vivo evolution, we turned to a facile strategy for in vivo random mutagenesis, the MutaT7-transition system. Although MutaT7-transition has some limitations compared to other newer (near-)­continuous evolution systems, it was easier to establish, and the integration with our classical selection method was reliable. Based on our analysis of the substrate profiling, we selected Mb PylRS 4ThzA carrying the “IPYE” mutations as a parent sequence and performed ten rounds of passaging. These passages were coupled to positive selection in the presence of τMH, followed by selection on agar plates and screening in the presence and absence of the ncAA ( Figure d,e). A variant, termed Mb (IPYE)­PylRS p10 , was identified which showed an approximately 2.7-fold increase in sfGFP150 TAG suppression in the presence of τMH ( Supporting Figure 11 ). From this initial variant, we confirmed incorporation of τMH into sfGFP150 TAG by LC-MS and LC-MS/MS, but phenylalanine misincorporation was significant as determined by an LC-MS/MS search ( Supporting Figures 2l and 3l ). To reduce the observed misincorporation of phenylalanine, we introduced a rational mutation, S364T, designed to reduce the size of the substrate binding pocket. This mutation significantly reduced the background incorporation of phenylalanine and increased the ncAA-dependent sfGFP150 TAG production to approximately 5.5-fold ( Figure f). The final engineered aaRS ( Mb (IPYE)­PylRS τMH ) enabled incorporation of τMH into sfGFP150 TAG , as determined by LC-MS of intact sfGFP150 TAG and low misincorporation is suggested by LC-MS/MS of trypsinized sfGFP150 TAG ( Supporting Figures 2m and 3m, and Supporting Tables 9 and 10 ). We additionally evaluated the substrate profile for both chPylRS 4OxzA and Mb (IPYE)­PylRS τMH ( Figure g and Supporting Figure 6 ), which revealed polyspecificity that enables incorporation of 4ThzA and 2PyA for both variants, the cognate ncAAs for the parent aaRSs used for evolution of both chPylRS 4OxzA and Mb (IPYE)­PylRS τMH . Evaluation of the Mb (IPYE)­PylRS τMH sequence and predicted structure yielded surprising results: all three mutations derived from MutaT7-transition were not within what is typically considered the active site based on crystallography of homologous proteins , and structure predictions , ( Figure h). However, current structural models lack full information regarding the linker between the N- and C-termini and the interaction between the two domains. Such mutations could be potentially important for modulating enzyme dynamics, the orientation of other active-site residues, interaction with the tRNA, or direct interactions with the ncAA through conformational changes induced during catalysis. Further mechanistic and structural studies are necessary to uncover their chemical role and enable a deeper understanding of activation. This system for τMH incorporation represents a significant advance, providing the first genetic incorporation of this long elusive ncAA and highlighting the difficulty in pinpointing limiting factors when there are challenges encountered incorporating new ncAAs. Although the current system displays lower efficiency and fidelity than that for πMH, we anticipate that this initial system will enable proof-of-concept studies for proteins with high expression levels, and the mutations identified here may serve as a foundation for future optimization efforts to enable incorporation in more challenging to express target proteins. Mutually Orthogonal Pairs Enable Dual Incorporation of Unique Histidine-Like ncAAs Upon further considering our substrate profiling results, the distinct substrate specificities observed in our profiling suggested a potential opportunity to enable dual incorporation of histidine-like ncAAs. Thus, we sought to leverage this potential orthogonality between given aaRS-ncAA combinations to enable dual histidine-like ncAA encoding ( Figure a). 4. Open in a new tab Dual histidine-like ncAA incorporation. Six combinations of two histidine-like ncAAs were incorporated into sfGFP by exploiting mutually orthogonal aaRS/tRNA pairs. (a) Schematic representation of double suppression at amber and opal codons in sfGFP40 TAG 150 TGA . (b–g) Dual histidine-like ncAA incorporation into sfGFP40 TAG 150 TGA . The side chains of the histidine-like ncAAs incorporated at the amber codon (blue) and opal codon (yellow) are shown. The data are presented as % of wt sfGFP (as defined for Figure ). The data represent the mean and standard deviation of three biological replicates. Several PylRS/tRNA Pyl that are mutually orthogonal in their aminoacylation specificity have previously been used to incorporate two or more ncAAs into a single protein. An established system is the combination of an N PylRS/ Ms tRNA Pyl (using a PylRS with an N-terminal domain) and a ΔN PylRS/tRNA ΔNPyl pair (using a PylRS lacking an N-terminal domain). ,,, All aaRS pairs that we engineered in this study are N PylRS-type pairs, and we expected that we could combine them with a ΔN PylRS pair to incorporate two histidine-like ncAAs simultaneously. We selected the Mm PylRS/ Ms tRNA NPyl CUA and G1 PylRS MIFAF / Ma tRNA ΔNPyl (8) UCA as well as Mm PylRS/ Ms tRNA NPyl CUA and Ma PylRS IFGFF / Ma tRNA ΔNPyl (8) UCA for potential dual encoding of a subset of histidine-like ncAA combinations. We confirmed the opal suppression activity of the ΔN PylRS/tRNA ΔNPyl UCA pairs ( Supporting Figure 14 ) and chose to proceed with G1 PylRS MIFAF / Ma tRNA ΔNPyl UCA based on its superior activity. Additionally, we confirmed the orthogonality relationship between the chPheRS 4PyA /3C11-chPheT system toward other systems tested in our study ( Supporting Figure 15 ). We found that the chPheRS 4PyA /3C11-chPheT and Mm PylRS/ Ms tRNA Pyl are not fully orthogonal, which is expected, as chPheRS 4PyA is a chimera of a PheRS and an N PylRS. Gratifyingly, the chPheRS 4PyA /chPheT system is orthogonal toward the G1 PylRS/ Ma tRNA ΔNPyl (8) pair. Upon validating suitable pairs for dual incorporation, we defined approximately 20 possible dual combinations consisting of one of the N PylRS construct and G1 PylRS MIFAF from the aaRS-ncAA substrate profile (which incorporates both πMH and 3PyA). We explored dual suppression for a subset of six aaRS-ncAA combinations ( Supporting Table 11 ) at TAG and TGA codons in a sfGFP40 TAG 150 TGA reporter. For all of the six ncAA combinations ( Figure b–g), we observed sfGFP production at efficiencies of 15–40% of wt sfGFP only in the presence of both ncAAs. We confirmed the presence of the desired ncAA at the given position using LC-MS/MS of trypsinized sfGFP40 TAG 150 TGA samples ( Supporting Figure 16 ). When the ncAA for opal suppression was not supplied, ncAA-independent readthrough was more pronounced than when the ncAA for amber suppression was not supplied. LC-MS/MS analysis of sfGFP150 TGA and sfGFP40 TAG 150 TGA expressed without ncAA supplementation showed background incorporation of Trp and Cys at the opal codon ( Supporting Figure 17 ),​ consistent with earlier reports.​​ When​ the amber and opal codon positions were exchanged (sfGFP40 TGA 150 TAG ), we observed that the suppression efficiencies are reduced. However, concomitantly the undesired background suppression was also reduced ( Supporting Figure 18 ). Nonetheless, we typically observed near quantitative incorporation of the respective ncAAs, as determined by LC-MS/MS ( Supporting Figures 16 and 19 ). Two recently reported strategies could be envisioned to potentially circumvent or more reliably reduce any background suppression: quadruplet recoding or use of the Ochre recoded cell line. However, because the incorporations were typically quantitative by LC-MS/MS, these strategies were not deemed necessary in this case. Collectively, we encoded six combinations of histidine-like ncAAs using an N PylRS or chPheRS pair together with a ΔN PylRS pair and confirmed the orthogonality of the substrate selectivity of these aaRS-ncAA combinations. We anticipate that such combinations could facilitate the generation of highly tailored metal coordination sites in a manner that is so far only accessible through small molecule catalysts. Discussion In this work, we have significantly expanded the diversity of histidine-like ncAAs accessible through genetic code expansion, developing nine novel aaRS/tRNA pairs for the site-specific incorporation of 12 histidine-like ncAAs with systematically varied properties. These ncAAs span a wide range of nitrogen p K a H values, including five alternative heterocycles beyond imidazole and N τ -substituted variants. This expanded toolkit addresses key gaps in the genetic code expansion landscape and provides researchers with significantly expanded chemical diversity for studying and engineering histidine-dependent reactivity. The development of this toolkit required diverse engineering strategies. Classical directed evolution from de novo selections using SSM succeeded for several targets (123Trz-4A, 5NO 2 H, 3PzA, 2E5MH, and 4PyA), while others could be found through screening of existing variant libraries (1Bn123Trz-4A, τBnH, 124Trz-3A, and 5OxzA), focused libraries with rationally fixed mutations (2PyA and 4OxzA), or in vivo mutagenesis (τMH). The particularly challenging case of τMH illustrates that structural similarity to successfully incorporated ncAAs does not guarantee ease of incorporation. Through extensive engineering, this work provides not only a toolkit but also highlights the versatility of engineering strategies that canand sometimes mustbe leveraged to access high-value incorporation targets. Importantly, the systematic LC-MS/MS analysis revealed low-level misincorporation for some systems that was not detectable by intact mass analysis alone, which has also been previously reported. While the misincorporation levels are low and unlikely to interfere with most applications, these findings underscore the importance of rigorous characterization. We recommend that researchers incorporating ncAAs for genetic code expansion applications conduct similar LC-MS/MS searches with their target protein to estimate potential misincorporation. A striking finding from our substrate profiling is the remarkable orthogonality between many of the newly evolved aaRS variants. Unlike previously reported aaRS systems for phenylalanine, tyrosine, or lysine derivatives, which often show broad substrate promiscuity, the new variants for histidine-like ncAAs frequently discriminate between structurally similar substrates. We propose that this heightened specificity may be consequence of engineering aaRSs to accept small, polar substrates. In the case of aaRSs evolved for large ncAAs, less competition from canonical amino acids may allow for less stringent evolutionary solutions allowing greater polyspecificity. In contrast, these smaller, polar ncAAs may necessitate more customized substrate binding pockets with less tolerance for substrate variation. This hypothesis could explain why incorporation of diverse histidine-like ncAAs has lagged behind other ncAA classes and why we observe significant substrate orthogonality with these new systems. However, the surprising substrate profiles highlight blind spots in our understanding of aaRS activities, reinforce the continued importance of empirical screening, and underscore the unresolved need for medium/high-throughput computational methods that can robustly predict suitable variants for complex enzyme reactions. Although the high substrate specificity made individual ncAA incorporation more challenging, the high degree of orthogonality was advantageous for dual encoding. We anticipate that such combinations could enable the design of highly tailored metal coordination geometries and cooperative catalytic motifs that are currently inaccessible in natural proteins and difficult to achieve even with small molecule catalysts. The ability to install two distinct histidine-like ncAAs at defined positions provides experimental control over both the electronic properties and spatial arrangement of catalytic residues. Moreover, based on the discovery of multiple mutually orthogonal PylRS pairs and the ability to encode multiple distinct ncAAs simultaneously, , we expect that many more combinations of histidine-like ncAAs could be accessible. Given the importance of histidine in enzyme systems, the toolkit presented here significantly expands the accessible chemical space for the study and engineering of proteins. The nine aaRS/tRNA pairs, 12 ncAAs, and validated dual incorporation systems provide researchers with powerful new methods for interrogating the roles of histidine residues in enzyme catalysis and for engineering proteins with novel or enhanced functions. Additionally, the starkly different substrate scopes of these aaRSs provide valuable tools for further mechanistic studies of aaRS activity. We anticipate that these tools will find broad application in mechanistic enzymology, enzyme engineering, genetic code expansion, and the design of artificial enzymes. Methods General Instrumentation and Materials Absorbance and fluorescence measurements were measured on a Tecan Infinite M Nano+. 1 H NMR spectra were recorded in CDCl 3 , D 2 O, DMSO- d 6 or MeOD on a Bruker AV-AV-400 (400 MHz), chemical shift d in ppm relative to solvent signals ( d = 7.26 ppm for CDCl 3 , 4.79 ppm D 2 O, 2.50 for DMSO- d 6 , 3.31 for MeOD), coupling constants J are given in Hz. 13 C NMR spectra were recorded in D 2 O on a Bruker AV-AV-400 (400 MHz). All purchased chemicals were used without further purification. Automated flash column chromatography was performed on a Biotage Isolera One system using either Biotage Sfär Silica or Biotage Sfär C18 D columns. Preparative HPLC chromatography was performed on an Agilent 1260 Infinity II system using either a Gemini 5 μm NC-C18 110 Å or Zorbax NH2 7 μM column. All sequencing was conducted by Microsynth (Balgach, CH). Mass Spectrometry Methods During aaRS/tRNA screening, initial high-throughput liquid chromatography–mass spectrometry (LC-MS) of sfGFP was conducted using an Agilent 1290 Infinity II LC system coupled to a single quadrupole mass spectrometer (ESI). Verification of all proteins discussed in the text was additionally carried out by high-resolution LC-MS carried out at the Functional Genomics Center Zürich (FGCZ). Briefly, samples were resolved on an ACQUITY UPLC@BioResolve-RP-mAb (2.7 μm, 2.1 mm × 150 mm, 450 Å) column at a constant flow rate of 0.2 mL/min, with a column temperature of 60 °C. The LC gradient started at 95% buffer A (0.1% DFA) and 5% buffer B (25% acetonitrile/75% iso-propanol with 0.1% DFA). The proportion of buffer B was increased to 20% within 2 min, then ramped to 70% over 14 min, followed by a wash at 80% for 2 min. The gradient was then returned to 5% buffer B to re-equilibrate the column. The analysis was performed on a calibrated Waters Synapt G2-Si mass spectrometer directly coupled to the Waters H-Class UPLC. MS spectra were acquired in the positive-ion mode by scanning the m / z range from 400 to 5000 Da with a scan duration of 1 s and an interscan delay of 0.1 s. The spray voltage was set to 3 kV, the cone voltage to 50 V, and the source temperature to 100 °C. The data were recorded with the MassLynx 4.2 Software. The recorded m / z data of single peaks were deconvoluted into mass spectra by applying the maximum entropy algorithm MaxEnt1 (MassLynx 4.2) with a resolution of the output mass 0.5 Da/channel and Uniform Gaussian Damage Model at the half height of 0.7 Da. To confirm the incorporation at the peptide level, LC-MS/MS was carried out by the FGCZ. Purified protein (approximately 1 mg/mL) was digested in solution by mixing 5 μL of sample with 40 μL digestion buffer (10 mM Tris, 2 mM CaCl 2 , pH 8.2). Protein was reduced and alkylated by 0.9 μL 100 mM Tris­(2-carboxyethyl)­phosphine +1.4 μL 100 mM chloroacetamide. Two μL trypsin (100 ng/μL in 10 mM HCl) were added and microwave assisted digestion was carried out at 60 °C for 30 min. The samples were dried and dissolved in 20 μL ddH 2 O + 0.1% formic acid. Samples were analyzed on Waters M-class UPLC coupled to a calibrated Q-Exactive mass spectrometer (Thermo). Data-dependent (DDA) method was used in this analysis. Samples were loaded onto a nanoEase M/Z Symmetry C18 trap column (180 μm × 20 mm, 100 Å, 5 μm particle size) and separated on a nanoEase M/Z HSS C18 T3 column (75 μm × 250 mm, 100 Å, 1.8 μm particle size), at a constant flow rate of 300 nL/min, with a column temperature of 50 °C. The LC gradient started at 5% solvent B (100% acetonitrile with 0.1% formic acid) and was increased to 35% over 42 min, then ramped to 60% within 5 min, followed by a wash at 95% for 10 min. The gradient was then returned to 5% buffer B to re-equilibrate the column. For MS setting, one scan cycle comprised of a full scan MS survey spectrum, followed by HCD (higher-energy collision dissociation) fragmentation on the 12 most intense signals for cycle. Full-scan MS spectra (350–1500 m / z ) were acquired at a resolution of 70,000 at 400 m / z , while HCD MS/MS spectra were recorded in the FT-Orbitrap at a resolution of 35,000. HCD MS/MS spectra were performed with a target value of 1 × 10 5 using a normalized collision energy 25%. The samples were acquired using internal lock mass calibration on m / z 371.1010 and 445.1200. The MS raw files were searched against sfGFP sequence sequences by Byonic 5.2 (Proteinmetrics, USA) with the consideration of carbamidomethylation at cysteine residues and oxidation at methionine residues. In addition, the mutated site was set to J (default mass is 100 Da) in sfGFP sequence. The mass increase was set to different ncAA and normal amino acids as variable modifications. For example, +47.0789 at J indicated Phenylalanine. If misincorporation could be identified with the manual inspection of MS spectra, the extracted ion chromatograms (XIC) for each observed amino acid were integrated to provide a rough approximation of the misincorporation. We highlight two caveats that make this analysis an approximation not a direct quantitation: (i) ionization efficiencies between point mutations of peptides can vary and (ii) in some cases mutations can change the efficiency of trypsin digestion. Thus, we provide relative integrations only as an extremely coarse estimate. Small-molecule LC-MS analysis and UPLC analysis were both conducted with an Agilent 1290 Infinity II LC system coupled to a single quadrupole mass spectrometer (ESI). p K a Calculations For the p K a estimations ( Figure a) literature reported p K a s were only available for seven of the 17 structures. Three p K a estimators were evaluatedQupkake, MolGpka, and Chemicalize from ChemAxon ( https://chemicalize.com/ )using the neutral amino acid, the zwitterionic form of the amino acid, and the side chain. Each single method and voting combinations of multiple methods were evaluated to determine the tool most accurate for recapitulating literature values. The most accurate method consisted of an averaging of the values from Qupkake for the zwitterionic form, Qupkake for the side chain, and Chemicalize. aaRS SSM Library Construction Libraries were created using modified Golden Gate cloning. A pSL plasmid encoding the corresponding synthetase was amplified using inverse PCR with primers carrying a BsaI restriction site and an additional 6 bp overhang at the 5′ end ( Supporting Tables 12–14 ) using Q5 Hot Start High-Fidelity DNA polymerase. PCR products were purified using an NEB Monarch PCR cleanup kit. The purified PCR products were digested with DpnI and BsaI in 1X rCutSmart. Digests were carried out at 37 °C overnight. Digested products were purified using an NEB Monarch PCR cleanup kit. DNA ligation reactions contained T4-ligase and 1× T4-ligase-buffer. Ligation was carried out at 16 °C overnight. Ligation products were purified using an NEB Monarch PCR cleanup kit. Electrocompetent NEB10β E. coli (100 μL) were transformed with ligation product by electroporation in a cuvette with 2 mm gap on an Eppendorf Eporator with 250 Ω and 2500 V with pulse times of approximately 5 ms. The cells were recovered in SOC media for 1 h at 37 °C with shaking at 220 rpm. The number of transformants was estimated by dilution series plating of LB-agar with 50 μg/mL kanamycin. The recovered cells were transferred to 10 mL LB media with 50 μg/mL kanamycin and grown for 5 h. The cells were harvested by centrifugation, and the DNA was isolated using an NEB Monarch Plasmid Miniprep Kit. The library quality was confirmed by Sanger sequencing of 2–3 individual clones. aaRS Error-Prone PCR Library Construction Libraries were created using modified Golden Gate cloning. The backbone of a pSL plasmid encoding the corresponding synthetase was amplified using PCR with primers carrying a BsaI restriction site and an additional 6 bp overhang at the 5′ end ( Supporting Table 15 ) using Q5 Hot Start High-Fidelity DNA polymerase. The synthetase region to be mutagenized was amplified using PCR with primers carrying a BsaI restriction site and an additional 6 bp overhang at the 5′ end using the JBS Error-Prone Kit (Jena Biosciences) according to the manufacturers protocol. PCR products were purified using an NEB Monarch PCR cleanup kit. The purified PCR products were pooled and digested with DpnI and BsaI in 1× rCutSmart. Digests were carried out at 37 °C overnight. Digested products were purified using an NEB Monarch PCR cleanup kit. DNA ligation reactions contained T4-ligase and 1× T4-ligase-buffer. Ligation was carried out at 16 °C overnight. Ligation products were purified using an NEB Monarch PCR cleanup kit. Electrocompetent NEB10β E. coli (100 μL) were transformed with ligation product by electroporation in a cuvette with 2 mm gap on an Eppendorf Eporator with 250 Ω and 2500 V with pulse times of approximately 5 ms. The cells were recovered in SOC media for 1 h at 37 °C with shaking at 220 rpm. The number of transformants was estimated by dilution series plating of LB-agar with 50 μg/mL kanamycin. The recovered cells were transferred to 10 mL LB media with 50 μg/mL kanamycin and grown for 5 h. The cells were harvested by centrifugation, and the DNA was isolated using an NEB Monarch Plasmid Miniprep Kit. aaRS Library Selections For positive selections, electrocompetent NEB10β cells (100 μL) carrying the corresponding pDPS2 plasmid were transformed with 250 ng of a given library by electroporation in a cuvette with 2 mm gap on an Eppendorf Eporator at 250 Ω and 2500 V with pulse times of approximately 5 ms. The cells were recovered in SOC media for 1 h at 37 °C with shaking and transferred to 10 mL LB media with 50 μg/mL kanamycin, 10 μg/mL tetracycline, and ncAA at the given concentration. The cells were grown for 1–2 h, harvested by centrifugation, resuspended in 250 μL LB media and plated on LB-agar with 0.4% arabinose, 50 μg/mL kanamycin, 10 μg/mL tetracycline, 100 μg/mL chloramphenicol, 0.4% arabinose, and ncAA at the given concentration ( Supporting Table 1 ). Plates were incubated for 24 h at 37 °C. If additional selection rounds were carried out, cells were collected by washing the plate with 10 mL LB media and harvesting the cells by centrifugation. Plasmid DNA was isolated using an NEB Monarch Plasmid Miniprep Kit. DNA was digested with AgeI in 1x rCutsmart buffer for 18 h at 37 °C to remove the selection plasmid and purified using an NEB Monarch PCR cleanup kit. If additional selection rounds were not carried out, we proceeded as though the step was the final positive selection round described further below. For negative selections, electrocompetent NEB10β cells (100 μL) carrying the corresponding pBARN plasmid were transformed with 50 ng of library DNA by electroporation in a cuvette with 2 mm gap on an Eppendorf Eporator at 250 Ω and 2500 V with pulse times of approximately 5 ms. The cells were recovered in SOC media for 1 h at 37 °C with shaking and transferred to 10 mL LB media with 50 μg/mL kanamycin and 35 μg/mL chloramphenicol. The cells were grown in the presence of the antibiotics for 1–2 h. The cells were harvested by centrifugation, resuspended in 250 μL LB media and plated on LB-agar with 0.4% arabinose, 50 μg/mL kanamycin, and 35 μg/mL chloramphenicol. Plates were incubated for 24 h at 37 °C. Cells were collected by washing the plate with 10 mL LB media; cells were harvested by centrifugation; and plasmid DNA was purified using an NEB Monarch Plasmid Miniprep Kit. DNA was digested with AgeI in 1× rCutsmart buffer. Digests were carried out at 37 °C overnight to the remove selection plasmid and purified using an NEB Monarch PCR cleanup kit. For screening after the final round of positive selection, individual colonies were used to inoculate 96-well plates with 250 μL 2xYT media with 50 μg/mL kanamycin, 10 μg/mL tetracycline. Cultures were grown for 24 h at 37 °C with shaking at 400 rpm. Subsequently, 25 μL of each culture was used to inoculate 96-well plates with 250 μL 2xYT media with 50 μg/mL kanamycin, 10 μg/mL tetracycline, 0.4% arabinose, and ncAA at the given concentration. Cultures were grown for 24 h at 37 °C with shaking at 400 rpm. 100 μL of each culture were transferred to a 96-well clear well plate. Fluorescence (excitation at 480 nm and emission at 510 nm) and absorbance at 600 nm were measured. DNA from cultures with high Fluorescence/OD 600 ratios was isolated using an NEB Monarch Plasmid Miniprep Kit and analyzed by Sanger sequencing to identify mutations in the PylRS gene. Evolution of Mb (IPYE)­PylRS 4ThzA for τMH Incorporation NEB10β E. coli were cotransformed with pSLdT7- Mb (IPYE)­PylRS 4ThzA , pDPS2- Ms tRNA Pyl CUA and pDae079so by heat-shock, recovered in SOC for 1 h at 37 °C and plated on LB-agar with 50 μg/mL kanamycin and 10 μg/mL tetracycline and 100 μg/mL spectinomycin. An individual colony was inoculated into LB-media with 50 μg/mL kanamycin and 10 μg/mL tetracycline and 100 μg/mL spectinomycin and grown overnight. Subsequently, cells were diluted 1:100 into LB-media with 50 μg/mL kanamycin and 10 μg/mL tetracycline and 100 μg/mL spectinomycin, 0.2% arabinose, 5 mM τMH and grown for 8h. Cells were diluted and grown four additional times with addition of 35–100 μg chloramphenicol. Finally, cells were harvested by centrifugation for 10 min at 4 °C and 4200 g . Plasmid DNA was isolated using an NEB Monarch Plasmid Miniprep Kit and digested with AgeI in 1× rCutsmart buffer for 18 h at 37 °C to remove the selection and mutagenesis plasmids and purified using an NEB Monarch PCR cleanup kit. A positive selection round was carried out as described in the section “aaRS library selection”. Lastly, NEB10β E. coli were cotransformed with the pSLdT7- Mb (IPYE)­PylRS p10 and pDPS2- Ms tRNA Pyl CUA by heat shock (42 °C, 30 s), recovered in SOC for 1 h at 37 °C and plated on LB-agar with 50 μg/mL kanamycin and 10 μg/mL tetracycline. Individual colonies were used to inoculate 5 mL autoinduction media with 100 μg/mL kanamycin, 20 μg/mL tetracycline, 0.4% arabinose and 20 mM τMH. The cultures were grown for 24 h at 37 °C with shaking at 240 rpm. The cells were harvested by centrifugation for 10 min at 4 °C and 4200 g ; the supernatant was decanted; and the cell pellets were stored at −80 °C. To isolate the sfGFP, the cells were thawed at room temperature and resuspended in lysis buffer (500 μL, 20 mM Tris, 300 mM NaCl, pH 7.2 at 4 °C, 0.2% n -octyl β - d -thioglucopyranoside, 4 mg/mL Lysozyme). The lysis was conducted at 22 °C for 4 h. The sfGFP was isolated by purification with Ni-NTA resin (HisPur from Thermo Scientific) according to the manufacturer’s instructions. The purified sfGFP was analyzed by LC-MS and LC-MS/MS as indicated in the Mass Spectrometry section. sfGFP Amber Suppression Assay for aaRS/tRNA Orthogonality Screening NEB10β E. coli were cotransformed with pDPS2 and corresponding pSL plasmid by heat shock (42 °C, 30 s), recovered in SOC for 1 h at 37 °C and plated on LB-agar with 50 μg/mL kanamycin and 10 μg/mL tetracycline. Plates were incubated for 24 h at 37 °C. Individual colonies were used to inoculate 250 μL autoinduction media with 100 μg/mL kanamycin, 20 μg/mL tetracycline, and ncAA at the indicated concentrations. Cultures were grown for 24 h at 37 °C with shaking at 400 rpm. wt sfGFP was analogously expressed from pBAD-sfGFP by omission of 20 μg/mL tetracycline addition for the culturing of cells. 100 μL of each culture was transferred to a 96-well clear well plate. Fluorescence (excitation at 480 nm and emission at 510 nm) and absorbance at 600 nm were measured. The data were described as fluorescence corresponding to sfGFP production (excitation at 480 nm and emission at 510 nm) normalized to the optical density at 600 nm. sfGFP Amber or Opal Suppression Assay NEB10β E. coli were cotransformed with pBAD-sfGFP150 TAG , pBAD-sfGFP150 TGA , or pBAD-sfGFP40 TGA (modified from Addgene #85483, a gift from Ryan Mehl’s lab) and the corresponding aaRS/tRNA expression plasmid by heat shock (42 °C, 30 s), recovered in SOC for 1 h at 37 °C and plated on LB-agar with 50 μg/mL kanamycin and 10 μg/mL tetracycline (for amber suppression) or 35 μg/mL chloramphenicol (for opal suppression). Plates were incubated for 24 h at 37 °C. Individual colonies were used to inoculate 250 μL LB media with 50 μg/mL kanamycin, 10 μg/mL tetracycline. Cultures were grown for 24 h at 37 °C with shaking at 400 rpm. Subsequently, cultures were diluted into 250 μL autoinduction media with 100 μg/mL kanamycin, 20 μg/mL tetracycline (for amber suppression) or 70 μg/mL chloramphenicol (for opal suppression), 0.4% arabinose and ncAA at the indicated concentrations. For expressions with pBAD-sfGFP40 TGA and 3PyA, increased 3PyA concentrations were used (10 mM). wt sfGFP was analogously expressed from pBAD-sfGFP by omission of tetracycline addition for the culturing of cells. 100 μL of each culture were transferred to a 96-well clear well plate. Fluorescence (excitation at 480 nm and emission at 510 nm) and absorbance at 600 nm were measured. The data were analyzed as % of wt sfGFP, derived from the normalized fluorescence (excitation at 480 nm and emission at 510 nm normalized to the optical density at 600 nm) as percentage of a wt sfGFP reference. Additionally, the data were analyzed as relative sfGFP production ± ncAA, which was calculated from the normalized fluorescence in the presence of the ncAA divided by the normalized fluorescence in the absence of the ncAA. sfGFP Dual Suppression Assay NEB10β E. coli were cotransformed with pBAD-sfGFP40 TAG 150 TGA or pBAD-sfGFP40 TGA 150 TAG (modified from Addgene #85483) and the corresponding aaRS/tRNA expression plasmids by heat shock (42 °C, 30 s), recovered in SOC for 1 h at 37 °C, and plated on LB-agar with 50 μg/mL kanamycin and 10 μg/mL tetracycline and 35 μg/mL chloramphenicol. Plates were incubated for 24 h at 37 °C. Individual colonies were used to inoculate 250 μL LB media with 50 μg/mL kanamycin, 10 μg/mL tetracycline, 35 μg/mL chloramphenicol. Cultures were grown for 24 h at 37 °C with shaking at 400 rpm. Subsequently, cultures were diluted into 250 μL autoinduction media with 100 μg/mL kanamycin, 20 μg/mL tetracycline, 70 μg/mL chloramphenicol, 0.4% arabinose and ncAA at the indicated concentrations. Cultures were grown for 24 h at 37 °C with shaking at 400 rpm. wt sfGFP was analogously expressed from pBAD-sfGFP by omission of tetracycline and chloramphenicol addition for the culturing of cells. 100 μL of each culture were transferred to a 96-well clear well plate. Fluorescence (excitation at 480 nm and emission at 510 nm) and absorbance at 600 nm were measured. The data were described as % of wt sfGFP, determined by normalized fluorescence (excitation at 480 nm and emission at 510 nm normalized to the optical density at 600 nm) as percentage of a wt sfGFP reference. Analysis of ncAA Incorporation in sfGFP150 TAG NEB10β E. coli were cotransformed with pBAD-sfGFP150 TAG and the corresponding aaRS/tRNA expression plasmid by heat shock (42 °C, 30 s), recovered in SOC for 1 h at 37 °C and plated on LB-agar with 50 μg/mL kanamycin and 10 μg/mL tetracycline. Plates were incubated for 24 h at 37 °C. Individual colonies were used to inoculate 5 mL autoinduction media with 100 μg/mL kanamycin, 20 μg/mL tetracycline, 0.4% arabinose and ncAA at the indicated concentration. The cultures were grown for 24 h at 37 °C with shaking at 240 rpm. The cells were harvested by centrifugation for 10 min at 4 °C and 4200 g ; the supernatant was decanted; and the cell pellets were stored at −80 °C. To isolate the sfGFP, the cells were thawed at room temperature and resuspended in lysis buffer (500 μL, 20 mM Tris, 300 mM NaCl, pH 7.2 at 4 °C, 0.2% n -octyl β- d -thioglucopyranoside, 4 mg/mL Lysozyme). The lysis was conducted at 22 °C for 4 h. The sfGFP was isolated by purification with Ni-NTA resin (HisPur from Thermo Scientific) according to the manufacturer’s instructions. The purified sfGFP was analyzed by LC-MS and LC-MS/MS as indicated in the Mass Spectrometry section. Analysis of Dual ncAA Incorporation in sfGFP NEB10β E. coli were cotransformed with pBAD-sfGFP40 TAG 150 TGA or pBAD-sfGFP40 TGA 150 TAG (modified from Addgene #85483) and the corresponding aaRS/tRNA expression plasmids by heat shock (42 °C, 30 s), recovered in SOC for 1 h at 37 °C, and plated on LB-agar with 50 μg/mL kanamycin and 10 μg/mL tetracycline and 35 μg/mL chloramphenicol. Plates were incubated for 24 h at 37 °C. Individual colonies were used to inoculate 5–8 mL autoinduction media with 100 μg/mL kanamycin, 20 μg/mL tetracycline, 70 μg/mL chloramphenicol, 0.4% arabinose and ncAAs at the indicated concentrations. For expressions with pBAD-sfGFP40 TGA 150 TAG and 3PyA, increased 3PyA concentrations were used (10 mM). The cultures were grown for 24 h at 37 °C with shaking at 240 rpm. The cells were harvested by centrifugation for 10 min at 4 °C and 4200 g ; the supernatant was decanted; and the cell pellets were stored at −80 °C. To isolate the sfGFP, the cells were thawed at room temperature and resuspended in lysis buffer (500 μL, 20 mM Tris, 300 mM NaCl, pH 7.2 at 4 °C, 0.2% n -octyl β- d -thioglucopyranoside, 4 mg/mL Lysozyme). The lysis was conducted at 22 °C for 4 h. The sfGFP was isolated by purification with Ni-NTA resin (HisPur from Thermo Scientific) according to the manufacturer’s instructions. The purified sfGFP was analyzed by LC-MS and LC-MS/MS as indicated in the Mass Spectrometry section. Intracellular τMH Concentration Determination A glycerol stock of NEB10β E. coli was used to inoculate 5 mL 2xYT media. Cultures were grown for 16 h at 37 °C. Cultures were diluted 1:100 into 5 mL 2xYT media with or without 5 mM τMH. Cultures were grown for 16h at 37 °C. The OD 600 of each culture was measured. The cells were harvested by centrifugation for 5 min at 4 °C and 4200 g ; the supernatant was decanted; and the cells were washed four times with phosphate buffered saline (pH 7.4). The cells were harvested by centrifugation for 5 min at 4 °C and 4200 g and resuspended in 400 μL H 2 O/MeOH (2:3). Approximately 300 mg of 0.1 mm Glass Beads (Scientific Industries, Inc.) were added, and cells were vortexed for 5 min. Lysates were clarified by centrifugation for 30 min at 4 °C and 20,000 g . Supernatants were filtered through a Millipore Amicon Ultra Centrifugal Filter with 30 kDa MW cutoff by centrifugation for 15 min at 4 °C and 14,000 g . Lysates were analyzed on an ACQUITY UPLC BEH C18 1.7 μm column using an Agilent 1290 Infinity II LC system coupled to a single quadrupole mass spectrometer (ESI) in single-ion-monitoring mode ( m / z = 170). The LC gradient started at with a flow of 0.4 mL/min and 100% buffer A (H 2 O) for 1 min, followed by a gradient to 95% buffer B (MeCN) for 2 min, followed by a gradient to 100% buffer A for 30 s, followed by 100% buffer A for 1 min. ncAA-aaRS Affinity Prediction with Boltz2 Structure and affinity predictions were initially conducted with Boltz2 were carried out using a Google Colab implementation. It was observed that the stereochemistry of the ncAA (but not ATP) was sometimes inverted during the prediction. Thus, the Boltz2 predicted protein structures with ATP bound were used to perform affinity predictions with Gnina, a fork of autodock vina. We also docked the AA-AMP adducts to the predicted protein structure in the absence of ATP, but we observed a similar lack of correlation between predicted binding affinities and incorporation efficiencies. Log P values were calculated in python using the RDKit ( https://www.rdkit.org ). Supplementary Material ja5c19599_si_001.pdf (15.2MB, pdf) Acknowledgments For funding, the authors thank the University of Zurich, the Swiss National Science Foundation (grant no. 10002490 and 10000399), and the UZH CanDoc Award (ANP: grant no. FK-24-093, SF: grant no. FK-22-086). The authors thank the Functional Genomics Center Zurich (FGCZ) for assistance with high-resolution LC-MS and LC-MS/MS data collection and analysis. Source and raw data files are available on Zenodo (10.5281/zenodo.18773764). Key plasmids for the application of the aaRS/tRNA pairs reported here are deposited on Addgene. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c19599 . Figures and tables, synthetic methods, DNA and protein sequences, and plasmid construct sequences ( PDF ) †. A.N.P. and S.F. contributed equally to this work. The authors declare no competing financial interest. Footnotes 1 Note 1, Nomenclature: Herein, we use the IUPAC nitrogen labels. For completeness, N τ (or τ-N) is also sometimes referred to as ε-N, 1-N, or 3-N, and N π (or π-N) is also sometimes referred to as δ-N, 1-N, or 3-N. 2 Note 2, chPylRS E7 evolution: We reasoned that a more efficient PylRS construct might beneficial for the incorporation of “difficult-to-incorporate” ncAAs. We focused on the optimization of a chimeric PylRS (chPylRS) construct. A small selection of chPylRS constructs were prepared by fusing the N-terminal domain of PylRS from thermophilic archaea and bacteria to the C-terminal domain of PylRS from Methanosarcina mazei (83–454), a mesophilic archaeon ( Supporting Table 3 ). The constructs were expressed under control of a gln S promoter (plasmid: pSL) and tested for the incorporation of the near-native substrate N ε -(tert-butoxycarbonyl)- l -lysine (BocK) at low concentrations (0.2 mM). Among the tested constructs, chPylRS Me‑Mm was the only active construct but gratifyingly displayed slightly increased suppression efficiencies with Mm tRNA Pyl CUA compared to Mm PylRS and chPylRS Mb (IPYE)‑ Mm , a previously reported chPylRS system. chPylRS Me‑Mm consists of the N-terminal domain of the PylRS from Methanohalobium evestigatum (1–94) and the C-terminal domain of PylRS from M. mazei (83–454). To further improve the activity of chPylRS Me‑Mm , we pursued a strategy analogous to a previous PylRS engineering campaign. In short, residues 1–94 of chPylRS Me‑Mm were subjected to high error rate error-prone PCR (3–7 mutations/kb), positive selection and screening of fluorescence intensity at low BocK concentrations (0.2 mM). The best performing clones were pooled and subjected to a second round of error-prone PCR, positive selection, and screening of fluorescence intensity at low BocK concentrations (0.2 mM). After two rounds, a clone carrying the mutations S18C/K45E/P68Q/K70I/V74A/N80S/K93I (chPylRS E7 ) emerged. From our selection plasmid, chPylRS E7 displayed approximately 4.3-fold improved suppression efficiencies at low BocK concentrations (0.2 mM) compared to chPylRS Me‑Mm and approximately 6.9-fold improved suppression efficiencies compared to Mm PylRS ( Supporting Figure 1 ). Notably, upon transference of chPylRS E7 to our optimized plasmid for robust production of aaRS/tRNA pairs (pOS1T), the advantage over Mm PylRS was lost, suggesting that these mutations are beneficial for expression from the selection/screening plasmid (pSL) but not in the final GCE plasmid (pOS1T). Interestingly, in our hands, this outcome also appears to be the case for several other reported engineered aaRS/tRNA pairs, highlighting the role of the plasmidlikely through attenuation of protein or tRNA productionin observed engineering outcomes. Nonetheless, the improvements in the selection plasmid were significant, and thus we hypothesized that this variant might be better suited for selection and screening with new aaRSs. References Bartlett G. J., Porter C. T., Borkakoti N., Thornton J. M.. Analysis of Catalytic Residues in Enzyme Active Sites. J. Mol. Biol. 2002;324(1):105–121. doi: 10.1016/S0022-2836(02)01036-7. [ DOI ] [ PubMed ] [ Google Scholar ] Schlesinger S., Schlesinger M. J.. The Effect of Amino Acid Analogues on Alkaline Phosphatase Formation in Escherichia Coli K-12. I. Substitution of Triazolealanine for Histidine. J. Biol. Chem. 1967;242(14):3369–3372. doi: 10.1016/S0021-9258(18)95919-3. [ DOI ] [ PubMed ] [ Google Scholar ] Soumillion P., Fastrez J.. Incorporation of 1,2,4-Triazole-3-Alanine into a Mutant of Phage Lambda Lysozyme Containing a Single Histidine. Protein Eng., Des. Sel. 1998;11(3):213–217. doi: 10.1093/protein/11.3.213. [ DOI ] [ PubMed ] [ Google Scholar ] Ikeda Y., Kawahara S., Taki M., Kuno A., Hasegawa T., Taira K.. Synthesis of a Novel Histidine Analogue and Its Efficient Incorporation into a Protein in Vivo. Protein Eng., Des. Sel. 2003;16(9):699–706. doi: 10.1093/protein/gzg084. [ DOI ] [ PubMed ] [ Google Scholar ] Eichler J. F., Cramer J. C., Kirk K. L., Bann J. G.. Biosynthetic Incorporation of Fluorohistidine into Proteins in E. Coli: A New Probe of Macromolecular Structure. ChemBioChem. 2005;6(12):2170–2173. doi: 10.1002/cbic.200500249. [ DOI ] [ PubMed ] [ Google Scholar ] Natter Perdiguero A., Deliz Liang A.. Practical Approaches to Genetic Code Expansion with Aminoacyl-tRNA Synthetase/tRNA Pairs. CHIMIA. 2024;78(1–2):22–31. doi: 10.2533/chimia.2024.22. [ DOI ] [ PubMed ] [ Google Scholar ] Sharma V., Wang Y.-S., Liu W. R.. Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics. ACS Chem. Biol. 2016;11(12):3305–3309. doi: 10.1021/acschembio.6b00940. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ortmayer M., Fisher K., Basran J., Wolde-Michael E. M., Heyes D. J., Levy C., Lovelock S. L., Anderson J. L. R., Raven E. L., Hay S., Rigby S. E. J., Green A. P.. Rewiring the “Push-Pull” Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code. ACS Catal. 2020;10(4):2735–2746. doi: 10.1021/acscatal.9b05129. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ortmayer M., Hardy F. J., Quesne M. G., Fisher K., Levy C., Heyes D. J., Catlow C. R. A., de Visser S. P., Rigby S. E. J., Hay S., Green A. P.. A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase. JACS Au. 2021;1(7):913–918. doi: 10.1021/jacsau.1c00145. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Drienovská I., Rioz-Martínez A., Draksharapu A., Roelfes G.. Novel Artificial Metalloenzymes by in Vivo Incorporation of Metal-Binding Unnatural Amino Acids. Chem. Sci. 2015;6(1):770–776. doi: 10.1039/C4SC01525H. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Mayer C., Dulson C., Reddem E., Thunnissen A.- M. W. H., Roelfes G.. Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid. Angew. Chem., Int. Ed. 2019;58(7):2083–2087. doi: 10.1002/anie.201813499. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Burke A. J., Lovelock S. L., Frese A., Crawshaw R., Ortmayer M., Dunstan M., Levy C., Green A. P.. Design and Evolution of an Enzyme with a Non-Canonical Organocatalytic Mechanism. Nature. 2019;570(7760):219–223. doi: 10.1038/s41586-019-1262-8. [ DOI ] [ PubMed ] [ Google Scholar ] Trimble J. S., Crawshaw R., Hardy F. J., Levy C. W., Brown M. J. B., Fuerst D. E., Heyes D. J., Obexer R., Green A. P.. A Designed Photoenzyme for Enantioselective [2 + 2] Cycloadditions. Nature. 2022;611(7937):709–714. doi: 10.1038/s41586-022-05335-3. [ DOI ] [ PubMed ] [ Google Scholar ] Longwitz L., Leveson-Gower R. B., Rozeboom H. J., Thunnissen A.- M. W. H., Roelfes G.. Boron Catalysis in a Designer Enzyme. Nature. 2024;629(8013):824–829. doi: 10.1038/s41586-024-07391-3. [ DOI ] [ PubMed ] [ Google Scholar ] Jackson J. C., Duffy S. P., Hess K. R., Mehl R. A.. Improving Nature’s Enzyme Active Site with Genetically Encoded Unnatural Amino Acids. J. Am. Chem. Soc. 2006;128(34):11124–11127. doi: 10.1021/ja061099y. [ DOI ] [ PubMed ] [ Google Scholar ] Ugwumba I. N., Ozawa K., Xu Z.-Q., Ely F., Foo J.-L., Herlt A. J., Coppin C., Brown S., Taylor M. C., Ollis D. L., Mander L. N., Schenk G., Dixon N. E., Otting G., Oakeshott J. G., Jackson C. J.. Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids. J. Am. Chem. Soc. 2011;133(2):326–333. doi: 10.1021/ja106416g. [ DOI ] [ PubMed ] [ Google Scholar ] Green A. P., Hayashi T., Mittl P. R. E., Hilvert D.. A Chemically Programmed Proximal Ligand Enhances the Catalytic Properties of a Heme Enzyme. J. Am. Chem. Soc. 2016;138(35):11344–11352. doi: 10.1021/jacs.6b07029. [ DOI ] [ PubMed ] [ Google Scholar ] Pott M., Hayashi T., Mori T., Mittl P. R. E., Green A. P., Hilvert D.. A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold. J. Am. Chem. Soc. 2018;140(4):1535–1543. doi: 10.1021/jacs.7b12621. [ DOI ] [ PubMed ] [ Google Scholar ] Huang H., Yan T., Liu C., Lu Y., Wu Z., Wang X., Wang J.. Genetically Encoded Nδ-Vinyl Histidine for the Evolution of Enzyme Catalytic Center. Nat. Commun. 2024;15(1):5714. doi: 10.1038/s41467-024-50005-9. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Fischer, S. ; Natter Perdiguero, A. ; Lau, K. ; Deliz Liang, A. . Hydrophobic Tuning with Non-Canonical Amino Acids in a Copper Metalloenzyme bioRxiv 2025. 10.1101/2025.02.09.636911. [ DOI ] Dunkelmann D. L., Piedrafita C., Dickson A., Liu K. C., Elliott T. S., Fiedler M., Bellini D., Zhou A., Cervettini D., Chin J. W.. Adding α,α-Disubstituted and β-Linked Monomers to the Genetic Code of an Organism. Nature. 2024;625:603–610. doi: 10.1038/s41586-023-06897-6. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Xiao H., Peters F. B., Yang P.-Y., Reed S., Chittuluru J. R., Schultz P. G.. Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase. ACS Chem. Biol. 2014;9(5):1092–1096. doi: 10.1021/cb500032c. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Beránek V., Willis J. C. W., Chin J. W.. An Evolved Methanomethylophilus Alvus Pyrrolysyl-tRNA Synthetase/tRNA Pair Is Highly Active and Orthogonal in Mammalian Cells. Biochemistry. 2019;58(5):387–390. doi: 10.1021/acs.biochem.8b00808. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Dunkelmann D. L., Willis J. C. W., Beattie A. T., Chin J. W.. Engineered Triply Orthogonal Pyrrolysyl–tRNA Synthetase/tRNA Pairs Enable the Genetic Encoding of Three Distinct Non-Canonical Amino Acids. Nat. Chem. 2020;12(6):535–544. doi: 10.1038/s41557-020-0472-x. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Pott M., Tinzl M., Hayashi T., Ota Y., Dunkelmann D., Mittl P. R. E., Hilvert D.. Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme. Angew. Chem., Int. Ed. 2021;60(27):15063–15068. doi: 10.1002/anie.202103437. [ DOI ] [ PubMed ] [ Google Scholar ] Cheung J. W., Kinney W. D., Wesalo J. S., Reed M., Nicholson E. M., Deiters A., Cropp T. A.. Genetic Encoding of a Photocaged Histidine for Light-Control of Protein Activity. ChemBioChem. 2023;24(7):e202200721. doi: 10.1002/cbic.202200721. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Taylor C. J., Hardy F. J., Burke A. J., Bednar R. M., Mehl R. A., Green A. P., Lovelock S. L.. Engineering Mutually Orthogonal PylRS/tRNA Pairs for Dual Encoding of Functional Histidine Analogues. Protein Sci. 2023;32(5):e4640. doi: 10.1002/pro.4640. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Hutton A. E., Foster J., Sanders J. E. J., Taylor C. J., Hoffmann S. A., Cai Y., Lovelock S. L., Green A.. An Efficient Pyrrolysyl-tRNA Synthetase for Economical Production of MeHis-Containing Enzymes. Faraday Discuss. 2024;252:295. doi: 10.1039/D4FD00019F. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Icking L.-S., Riedlberger A. M., Krause F., Widder J., Frederiksen A. S., Stockert F., Spädt M., Edel N., Armbruster D., Forlani G., Franchini S., Kaas P., Konak B. M. K., Krier F., Lefebvre M., Mazraeh D., Ranniger J., Gerstenecker J., Gescher P., Voigt K., Salavei P., Gensch N., Di Ventura B., Öztürk M. A.. iNClusive: A Database Collecting Useful Information on Non-Canonical Amino Acids and Their Incorporation into Proteins for Easier Genetic Code Expansion Implementation. Nucleic Acids Res. 2024;52(D1):D476–D482. doi: 10.1093/nar/gkad1090. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Tseng H.-W., Baumann T., Sun H., Wang Y.-S., Ignatova Z., Budisa N.. Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids. Molecules. 2020;25(19):4418. doi: 10.3390/molecules25194418. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Hutton A. E., Foster J., Crawshaw R., Hardy F. J., Johannissen L. O., Lister T. M., Gérard E. F., Birch-Price Z., Obexer R., Hay S., Green A. P.. A Non-Canonical Nucleophile Unlocks a New Mechanistic Pathway in a Designed Enzyme. Nat. Commun. 2024;15(1):1956. doi: 10.1038/s41467-024-46123-z. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Isom D. G., Castañeda C. A., Cannon B. R., García-Moreno E.. B. Large Shifts in pKa Values of Lysine Residues Buried inside a Protein. Proc. Natl. Acad. Sci. U.S.A. 2011;108(13):5260–5265. doi: 10.1073/pnas.1010750108. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ancona N., Bastola A., Alexov E.. PKAD-2: New Entries and Expansion of Functionalities of the Database of Experimentally Measured pKa’s of Proteins. J. Comput. Biophys Chem. 2023;22(5):515–524. doi: 10.1142/S2737416523500230. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Yokoyama K., Uhlin U., Stubbe J.. Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pKa Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase. J. Am. Chem. Soc. 2010;132(24):8385–8397. doi: 10.1021/ja101097p. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Bryson D. I., Fan C., Guo L.-T., Miller C., Söll D., Liu D. R.. Continuous Directed Evolution of Aminoacyl-tRNA Synthetases. Nat. Chem. Biol. 2017;13(12):1253–1260. doi: 10.1038/nchembio.2474. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Bryson D. I., Fan C., Guo L. T., Miller C., Söll D., Liu D. R.. Continuous Directed Evolution of Aminoacyl-tRNA Synthetases. Nat. Chem. Biol. 2017;13(12):1253–1260. doi: 10.1038/nchembio.2474. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Zhang Q., Jiang L., Niu Y., Li Y., Chen W., Cheng J., Ding H., Chen B., Liu K., Cao J., Wang J., Ye S., Yang L., Wu J., Xu G., Lin J., Yu H.. Machine Learning-Guided Evolution of Pyrrolysyl-tRNA Synthetase for Improved Incorporation Efficiency of Diverse Noncanonical Amino Acids. Nat. Commun. 2025;16(1):6648. doi: 10.1038/s41467-025-61952-2. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ding W., Zhao H., Chen Y., Zhang B., Yang Y., Zang J., Wu J., Lin S.. Chimeric Design of Pyrrolysyl-tRNA Synthetase/tRNA Pairs and Canonical Synthetase/tRNA Pairs for Genetic Code Expansion. Nat. Commun. 2020;11(1):3154. doi: 10.1038/s41467-020-16898-y. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Zhao H., Ding W., Zang J., Yang Y., Liu C., Hu L., Chen Y., Liu G., Fang Y., Yuan Y., Lin S.. Directed-Evolution of Translation System for Efficient Unnatural Amino Acids Incorporation and Generalizable Synthetic Auxotroph Construction. Nat. Commun. 2021;12(1):7039. doi: 10.1038/s41467-021-27399-x. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Costello A., Peterson A. A., Lanster D. L., Li Z., Carver G. D., Badran A. H.. Efficient Genetic Code Expansion without Host Genome Modifications. Nat. Biotechnol. 2024;43:1116–1127. doi: 10.1038/s41587-024-02385-y. [ DOI ] [ PubMed ] [ Google Scholar ] Wang Y.-S., Fang X., Wallace A. L., Wu B., Liu W. R.. A Rationally Designed Pyrrolysyl-tRNA Synthetase Mutant Has a Broad Substrate Spectrum. J. Am. Chem. Soc. 2012;134(6):2950–2953. doi: 10.1021/ja211972x. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Vatansever E. C., Yang K. S., Geng Z. Z., Qiao Y., Li P., Xu S., Liu W. R.. A Designed, Highly Efficient Pyrrolysyl-tRNA Synthetase Mutant Binds o-Chlorophenylalanine Using Two Halogen Bonds. J. Mol. Biol. 2022;434(8):167534. doi: 10.1016/j.jmb.2022.167534. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Guo L.-T., Wang Y.-S., Nakamura A., Eiler D., Kavran J. M., Wong M., Kiessling L. L., Steitz T. A., O’Donoghue P., Söll D.. Polyspecific Pyrrolysyl-tRNA Synthetases from Directed Evolution. Proc. Natl. Acad. Sci. U.S.A. 2014;111(47):16724–16729. doi: 10.1073/pnas.1419737111. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Hohl A., Karan R., Akal A., Renn D., Liu X., Ghorpade S., Groll M., Rueping M., Eppinger J.. Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen. Sci. Rep. 2019;9(1):11971. doi: 10.1038/s41598-019-48357-0. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Young D. D., Young T. S., Jahnz M., Ahmad I., Spraggon G., Schultz P. G.. An Evolved Aminoacyl-tRNA Synthetase with Atypical Polysubstrate Specificity. Biochemistry. 2011;50(11):1894–1900. doi: 10.1021/bi101929e. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Tawfik O. K. A. D. S.. Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective. Annu. Rev. Biochem. 2010;79(1):471–505. doi: 10.1146/annurev-biochem-030409-143718. [ DOI ] [ PubMed ] [ Google Scholar ] Abramson J., Adler J., Dunger J., Evans R., Green T., Pritzel A., Ronneberger O., Willmore L., Ballard A. J., Bambrick J., Bodenstein S. W., Evans D. A., Hung C.-C., O’Neill M., Reiman D., Tunyasuvunakool K., Wu Z., Žemgulytė A., Arvaniti E., Beattie C., Bertolli O., Bridgland A., Cherepanov A., Congreve M., Cowen-Rivers A. I., Cowie A., Figurnov M., Fuchs F. B., Gladman H., Jain R., Khan Y. A., Low C. M. R., Perlin K., Potapenko A., Savy P., Singh S., Stecula A., Thillaisundaram A., Tong C., Yakneen S., Zhong E. D., Zielinski M., Žídek A., Bapst V., Kohli P., Jaderberg M., Hassabis D., Jumper J. M.. Accurate Structure Prediction of Biomolecular Interactions with AlphaFold 3. Nature. 2024;630(8016):493–500. doi: 10.1038/s41586-024-07487-w. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Liu D. R., Schultz P. G.. Progress toward the Evolution of an Organism with an Expanded Genetic Code. Proc. Natl. Acad. Sci. U.S.A. 1999;96(9):4780–4785. doi: 10.1073/pnas.96.9.4780. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Seo D., Koh B., Eom G., Kim H. W., Kim S.. A Dual Gene-Specific Mutator System Installs All Transition Mutations at Similar Frequencies in Vivo. Nucleic Acids Res. 2023;51(10):e59. doi: 10.1093/nar/gkad266. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Nozawa K., O’Donoghue P., Gundllapalli S., Araiso Y., Ishitani R., Umehara T., Söll D., Nureki O.. Pyrrolysyl-tRNA Synthetase–tRNAPyl Structure Reveals the Molecular Basis of Orthogonality. Nature. 2009;457(7233):1163–1167. doi: 10.1038/nature07611. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Seki E., Yanagisawa T., Kuratani M., Sakamoto K., Yokoyama S.. Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus Alvus Pyrrolysyl-tRNA Synthetase. ACS Synth. Biol. 2020;9(4):718–732. doi: 10.1021/acssynbio.9b00288. [ DOI ] [ PubMed ] [ Google Scholar ] Passaro, S. ; Corso, G. ; Wohlwend, J. ; Reveiz, M. ; Thaler, S. ; Somnath, V. R. ; Getz, N. ; Portnoi, T. ; Roy, J. ; Stark, H. ; Kwabi-Addo, D. ; Beaini, D. ; Jaakkola, T. ; Barzilay, R. . Boltz-2: Towards Accurate and Efficient Binding Affinity Prediction bioRxiv 2025. 10.1101/2025.06.14.659707. [ DOI ] Dunkelmann D. L., Oehm S. B., Beattie A. T., Chin J. W.. A 68-Codon Genetic Code to Incorporate Four Distinct Non-Canonical Amino Acids Enabled by Automated Orthogonal mRNA Design. Nat. Chem. 2021;13(11):1110–1117. doi: 10.1038/s41557-021-00764-5. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Grome M. W., Nguyen M. T. A., Moonan D. W., Mohler K., Gurara K., Wang S., Hemez C., Stenton B. J., Cao Y., Radford F., Kornaj M., Patel J., Prome M., Rogulina S., Sozanski D., Tordoff J., Rinehart J., Isaacs F. J.. Engineering a Genomically Recoded Organism with One Stop Codon. Nature. 2025;639(8054):512–521. doi: 10.1038/s41586-024-08501-x. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Mohler K., Aerni H.-R., Gassaway B., Ling J., Ibba M., Rinehart J.. MS-READ: Quantitative Measurement of Amino Acid Incorporation. Biochim. Biophys. Acta, Gen. Subj. 2017;1861(11):3081–3088. doi: 10.1016/j.bbagen.2017.01.025. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Agostini F., Völler J.-S., Koksch B., Acevedo-Rocha C. G., Kubyshkin V., Budisa N.. Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology. Angew. Chem., Int. Ed. 2017;56(33):9680–9703. doi: 10.1002/anie.201610129. [ DOI ] [ PubMed ] [ Google Scholar ] Beattie A. T., Dunkelmann D. L., Chin J. W.. Quintuply Orthogonal Pyrrolysyl-tRNA Synthetase/tRNAPyl Pairs. Nat. Chem. 2023;15(7):948–959. doi: 10.1038/s41557-023-01232-y. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Abarbanel O. D., Hutchison G. R.. QupKake: Integrating Machine Learning and Quantum Chemistry for Micro-pKa Predictions. J. Chem. Theory Comput. 2024;20(15):6946–6956. doi: 10.1021/acs.jctc.4c00328. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Pan X., Wang H., Li C., Zhang J. Z. H., Ji C.. MolGpka: A Web Server for Small Molecule pKa Prediction Using a Graph-Convolutional Neural Network. J. Chem. Inf. Model. 2021;61(7):3159–3165. doi: 10.1021/acs.jcim.1c00075. [ DOI ] [ PubMed ] [ Google Scholar ] Miyake-Stoner S. J., Refakis C. A., Hammill J. T., Lusic H., Hazen J. L., Deiters A., Mehl R. A.. Generating Permissive Site-Specific Unnatural Aminoacyl-tRNA Synthetases. Biochemistry. 2010;49(8):1667–1677. doi: 10.1021/bi901947r. [ DOI ] [ PubMed ] [ Google Scholar ] Kim, J.-C.

Computational Chemistry for Fun. 2025. McNutt A. T., Li Y., Meli R., Aggarwal R., Koes D. R.. GNINA 1.3: The next Increment in Molecular Docking with Deep Learning. J. Cheminf. 2025;17(1):28. doi: 10.1186/s13321-025-00973-x. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Grasso K. T., Yeo M. J. R., Hillenbrand C. M., Ficaretta E. D., Italia J. S., Huang R. L., Chatterjee A.. Structural Robustness Affects the Engineerability of Aminoacyl-tRNA Synthetases for Genetic Code Expansion. Biochemistry. 2021;60(7):489–493. doi: 10.1021/acs.biochem.1c00056. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Reimer L. C., Carbasse J. S., Koblitz J., Ebeling C., Podstawka A., Overmann J.. BacDive in 2022: The Knowledge Base for Standardized Bacterial and Archaeal Data. Nucleic Acids Res. 2022;50(D1):D741–D746. doi: 10.1093/nar/gkab961. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Sharma V., Zeng Y., Wang W. W., Qiao Y., Kurra Y., Liu W. R.. Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids. ChemBioChem. 2018;19(1):26–30. doi: 10.1002/cbic.201700268. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Natter Perdiguero, A. ; Fischer, S. ; Lischke, A. R. ; Manser, B. P. ; Deliz Liang, A. (2026) Data for “Genetic Incorporation of Diverse Non-Canonical Amino Acids for Histidine Substitution. Zenodo 10.5281/zenodo.18773764 (Accessed on February 25, 2026). [ DOI ] [ PMC free article ] [ PubMed ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials ja5c19599_si_001.pdf (15.2MB, pdf) Data Availability Statement Source and raw data files are available on Zenodo (10.5281/zenodo.18773764). Key plasmids for the application of the aaRS/tRNA pairs reported here are deposited on Addgene. Articles from Journal of the American Chemical Society are provided here courtesy of American Chemical Society ACTIONS View on publisher site PDF (4.6 MB) Cite Collections Permalink PERMALINK Copy RESOURCES Similar articles Cited by other articles Links to NCBI Databases Cite Copy Download .nbib .nbib Format: AMA APA MLA NLM Add to Collections Create a new collection Add to an existing collection Name your collection * Choose a collection Unable to load your collection due to an error Please try again Add Cancel Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top