Expression and purification of MaPylRS, MaFRS1, MaFRS2 and MaFRSA for biochemical analysis

The plasmids used to express WT MaPylRS (pET32a-MaPylRS) and MaFRS1 (pET32a-MaFRS1) were constructed by inserting synthetic double-stranded DNA (dsDNA) fragments (Supplementary Table 1) into the NdeI-NdeI cut sites of a pET32a vector using the Gibson method75. pET32a-MaFRS2 and pET32a-MaFRSA were constructed from pET32a-MaFRS1 using a Q5 Site-Directed Mutagenesis Kit (NEB). Primers RF31 and RF32, and RF32 and RF33 (Supplementary Table 1) were used to construct pET32a-MaFRS2 and pET32a-MaFRSA, respectively. The sequences of the plasmids spanning the inserted regions were confirmed via Sanger sequencing at the University of California Berkeley DNA Sequencing Facility using T7 forward and reverse (T7 F and T7 R) primers (Supplementary Table 1), and the complete sequence of each plasmid was confirmed by the Massachusetts General Hospital Center for Computational and Integrative Biology DNA Core.

Chemically competent cells were prepared by following a modified published protocol76. Briefly, 5 ml Luria-Bertani (LB) medium was inoculated using a freezer stock of BL21-Gold (DE3)pLysS cells. The following day, 50 ml LB was inoculated with 0.5 ml of the culture from the previous day and incubated at 37 °C with shaking at 200 r.p.m. until the culture reached an optical density at 600 nm (OD600) in the range of 0.3–0.4. The cells were collected by centrifugation at 4,303g for 20 min at 4 °C. The cell pellet was resuspended in 5 ml of sterile filtered TSS solution (10% (wt/v) polyethylene glycol 8,000, 30 mM MgCl2, 5% (v/v) dimethylsulfoxide in 25 g l−1 LB). The chemically competent cells were portioned into 100 µl aliquots in 1.5 ml microcentrifuge tubes, flash frozen in liquid N2 and stored at −80 °C until use. The following protocol was used to transform plasmids into chemically competent cells: 20 µl KCM solution (500 mM KCl, 150 mM CaCl2 and 250 mM MgCl2) was added to a 100 µl aliquot of cells held on ice along with approximately 200 ng of the requisite plasmid and water to a final volume of 200 µl. The cells were incubated on ice for 30 min and then heat-shocked by placing them for 90 s in a water bath heated to 42 °C. Immediately after heat shock, the cells were placed on ice for 2 min, after which 800 µl LB was added. The cells were then incubated at 37 °C with shaking at 200 r.p.m. for 60 min. The cells were plated onto LB agar plates with the appropriate antibiotic and incubated overnight at 37 °C.

The plasmids used to express WT MaPylRS, MaFRS1, MaFRS2, and MaFRSA were transformed into BL21-Gold (DE3)pLysS chemically competent cells and plated onto LB agar plates supplemented with 100 µg ml−1 carbenicillin. Colonies were picked the following day and used to inoculate 10 ml LB supplemented with 100 µg ml−1 carbenicillin. The cultures were incubated overnight at 37 °C with shaking at 200 r.p.m. The following day, the 10 ml cultures were used to inoculate 1 l LB supplemented with 100 µg ml−1 carbenicillin in 4-l baffled Erlenmeyer flasks. The cultures were incubated at 37 °C with shaking at 200 r.p.m. for 3 h until they reached an OD600 of 0.6–0.8. At this point, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the incubation was continued for 6 h at 30 °C with shaking at 200 r.p.m. The cells were collected by centrifugation at 4,303g for 20 min at 4 °C and the cell pellets stored at −80 °C until the expressed protein was purified as described below.

The following buffers were used for protein purification: wash buffer: 50 mM sodium phosphate (pH 7.4), 500 mM NaCl, 20 mM β-mercaptoethanol, 25 mM imidazole; elution buffer: 50 mM sodium phosphate (pH 7.4), 500 mM NaCl, 20 mM β-mercaptoethanol, 100 mM imidazole; storage buffer: 100 mM HEPES-K (pH 7.2), 100 mM NaCl, 10 mM MgCl2, 4 mM dithiothreitol (DTT), 20% (v/v) glycerol. One cOmplete Mini EDTA-free protease inhibitor tablet was added to the wash and elution buffers immediately before use. To isolate protein, cell pellets were resuspended in wash buffer (5 ml per g cells). The resultant cell paste was lysed at 4 °C by sonication (Branson Sonifier 250) over 10 cycles of 30 s sonication followed by 30 s manual swirling. The lysate was centrifuged at 4,303g for 10 min at 4 °C to separate the soluble and insoluble fractions. The soluble lysate was incubated at 4 °C with 1 ml Ni nitrilotriacetic acid (Ni-NTA) agarose resin (washed with water and equilibrated with wash buffer) for 2 h. The lysate–resin mixture was added to a 65 g RediSep disposable sample load cartridge (Teledyne ISCO) and allowed to drain at room temperature. The protein-bound Ni-NTA agarose resin was then washed three times with 10 ml aliquots of wash buffer. The protein was eluted from the Ni-NTA agarose resin by rinsing the resin three times with 10 ml elution buffer. The elution fractions were pooled and concentrated using a 10 kDa molecular weight cut-off (MWCO) Amicon Ultra-15 centrifugal filter unit (4,303g, 4 °C). The protein was then buffer-exchanged into the storage buffer using the same centrifugal filter unit until the imidazole concentration was less than 5 µM. The protein was dispensed into 20 µl single-use aliquots and stored at −80 °C for up to 8 months. The protein concentration was measured using the Bradford assay77. The yields were between 8 and 12 mg l−1. Proteins were analysed by SDS–PAGE (Supplementary Fig. 2) using Any kD Mini-PROTEAN TGX precast protein gels (BioRad). The gels were run at 200 V for 30 min.

Proteins were analysed by LC–MS to confirm their identity (Supplementary Fig. 2). The samples analysed by MS were resolved using a Poroshell StableBond 300 C8 column (2.1 mm × 75 mm, 5 µm; Agilent, 660750-906) with a 1290 Infinity II ultra-high-performance liquid chromatograph (UHPLC; Agilent, G7120AR). The mobile phases used for separation were 0.1% formic acid in water (mobile phase A) and 100% acetonitrile (mobile phase B), and the flow rate was 0.4 ml min−1. After an initial hold at 5% B for 0.5 min, the proteins were eluted using a linear gradient from 5% to 75% B for 9.5 min, a linear gradient from 75% to 100% B for 1 min, a hold at 100% B for 1 min, a linear gradient from 100% to 5% B for 3.5 min and finally a hold at 5% B for 4.5 min. The protein masses were analysed by LC–HRMS using a 6530 Q-TOF AJS-ESI (Agilent, G6530BAR) instrument. The following parameters were used: gas temperature = 300 °C, drying gas flow = 12 l min−1, nebulizer pressure = 35 psi, sheath gas temperature = 350 °C, sheath gas flow = 11 l min−1, fragmentor voltage = 175 V, skimmer voltage = 65 V, peak-to-peak voltage (Vpp) = 750 V, capillary voltage (Vcap) = 3,500 V, nozzle voltage = 1,000 V and collection rate = 3 spectra s−1.

Analytical size exclusion chromatography (SEC; Supplementary Fig. 2) was performed on an ÄKTA Pure 25 instrument. A flow rate of 0.5 ml min−1 was used for all steps. A Superdex 75 Increase 10/300 GL column (stored and operated at 4 °C) was washed with 1.5 column volumes of degassed, sterile, filtered MilliQ water. The column was equilibrated in 1.5 column volumes of SEC buffer: 100 mM HEPES (pH 7.2), 100 mM NaCl, 10 mM MgCl2, 4 mM DTT. Approximately 800 µg of protein in 250 µl SEC buffer was loaded into a 500 µl capillary loop. The sample loop was washed with 2.0 ml SEC buffer as the sample was injected onto the column. The sample was eluted in 1.5 column volumes of SEC buffer and analysed by UV spectrophotometry at 280 nm.

Transcription and purification of tRNAs

The DNA template used for transcribing Ma-tRNAPyl (ref. 39) was prepared by annealing and extending the single-stranded DNA oligonucleotides Ma-PylT-F and Ma-PylT-R (2 mM; Supplementary Table 1) using OneTaq 2x Master Mix (NEB). The annealing and extension process was performed using the following protocol on a thermocycler (BioRad, C1000 Touch): 94 °C for 30 s, 30 cycles of 94 °C for 20 s, 53 °C for 30 s and 68 °C for 60 s, and finally 68 °C for 300 s. Following the extension, the reaction mixture was supplemented with sodium acetate (pH 5.2) to a final concentration of 300 mM, washed once with 1:1 (v/v) acid phenol–chloroform, twice with chloroform and the dsDNA product precipitated by addition of ethanol to a final concentration of 71%. The pellet was resuspended in water and the concentration of dsDNA determined using a NanoDrop ND-1000 device (Thermo Scientific). The template starts with a single C preceding the T7 promoter, which increases the yields of T7 transcripts78. The penultimate residue of Ma-PylT-R carries a 2′-methoxy modification, which reduces non-templated nucleotide addition by T7 RNA polymerase during in vitro transcription79.

Ma-tRNAPyl was transcribed in vitro using a modified version of a published procedure80. Transcription reactions (25 µl) contained the following components: 40 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 mM DTT, 2 mM spermidine, 5 mM ATP, 5 mM cytidine triphosphate, 5 mM guanosine triphosphate, 5 mM uridine triphosphate, 20 mM guanosine monophosphate, 0.2 mg ml−1 bovine serum albumin, 20 mM MgCl2, 12.5 ng µl−1 DNA template and 0.025 mg ml−1 T7 RNA polymerase. The reaction mixtures were incubated at 37 °C in a thermocycler for 3 h. Four 25 µl reactions were pooled, and then sodium acetate (pH 5.2) was added to a final concentration of 300 mM in a volume of 200 µl. The transcription reaction mixtures were extracted once with 1:1 (v/v) acid phenol–chloroform, washed twice with chloroform and the tRNA product precipitated by adding ethanol to a final concentration of 71%. After precipitation, the tRNA pellet was resuspended in water and incubated with 8 U of RQ1 RNAse-free DNAse (Promega) at 37 °C for 30 min according to the manufacturer’s protocol. The tRNA was then washed with acid phenol–chloroform and chloroform as described above, precipitated and resuspended in water. To remove small molecules, the tRNA was further purified using a Micro Bio-Spin P-30 gel column, Tris buffer (RNase-free; BioRad) after first exchanging the column buffer with water according to the manufacturer’s protocol. The tRNA was precipitated once more, resuspended in water, quantified using a NanoDrop ND-1000 device, aliquoted and stored at −20 °C.

tRNA was analysed by urea–PAGE (Supplementary Fig. 2) using 10% Mini-PROTEAN Tris-borate-ethylenediaminetetraacetic acid-urea gel (BioRad). The gels were run at 120 V for 30 min and then stained with SYBR Safe gel stain (Thermo Fisher) for 5 min before imaging. Ma-tRNAPyl was analysed by LC–MS to confirm its identity. Samples were resolved on an ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 50 mm, 60 °C; Waters, 186002350) using an ACQUITY UPLC I-Class PLUS instrument (Waters, 186015082). The mobile phases used were 8 mM triethylamine, 80 mM hexafluoroisopropanol and 5 µM EDTA (free acid) in 100% MilliQ water (mobile phase A) and 4 mM triethylamine, 40 mM hexafluoroisopropanol and 5 µM EDTA (free acid) in 50% MilliQ water–50% methanol (mobile phase B). The analysis was performed at a flow rate of 0.3 ml min−1 and began with mobile phase B at 22%, increasing linearly to 40% B over 10 min, followed by a linear gradient from 40% to 60% B for 1 min, a hold at 60% B for 1 min, a linear gradient from 60% to 22% B over 0.1 min and then a hold at 22% B for 2.9 min. The mass of the RNA was analysed by LC–HRMS with a Xevo G2-XS Tof instrument (Waters, 186010532) in negative ion mode with the following parameters: capillary voltage = 2,000 V, sampling cone = 40, source off-set = 40, source temperature = 140 °C, desolvation temperature = 20 °C, cone gas flow = 10 l h−1, desolvation gas flow = 800 l h−1 and collection rate = 1 spectrum s−1. The expected masses of the oligonucleotide products were calculated using the AAT Bioquest RNA Molecular Weight Calculator. Deconvoluted mass spectra were obtained using the MaxEnt software (Waters).

Procedure for RNAse A assays

The reaction mixtures (25 µl) used to acylate tRNA contained the following components: 100 mM HEPES-K (pH 7.5), 4 mM DTT, 10 mM MgCl2, 10 mM ATP, 0–10 mM substrate, 0.1 U E. coli inorganic pyrophosphatase (NEB), 25 µM Ma-tRNAPyl and 2.5 µM enzyme (MaPylRS, MaFRS1, MaFRS2 or MaFRSA). The reaction mixtures were incubated at 37 °C in a dry air incubator for 2 h. The tRNA samples from the enzymatic acylation reactions were quenched with 27.5 µl RNAse A solution (1.5 U µl−1 RNAse A (MilliporeSigma) and 200 mM sodium acetate, pH 5.2) and incubated for 5 min at room temperature. The proteins were then precipitated by the addition of 50% trichloroacetic acid (Sigma-Aldrich) to a final concentration of 5%. After precipitating protein at −80 °C for 30 min, insoluble material was removed by centrifugation at 21,300g for 10 min at 4 °C. The soluble fraction was then transferred to autosampler vials, kept on ice until immediately before LC–MS analysis and returned to ice immediately afterwards.

The samples analysed by MS were resolved using a Zorbax Eclipse XDB-C18 RRHD column (2.1 mm × 50 mm, 1.8 μm, room temperature; Agilent, 981757-902) fitted with a guard column (Zorbax Eclipse XDB-C18, 2.1 mm ×5 mm, 1.8 µm; Agilent, 821725-903) and a 1290 Infinity II UHPLC (Agilent, G7120AR). The mobile phases used were 0.1% formic acid in water (mobile phase A) and 100% acetonitrile (mobile phase B). The analysis was performed at a flow rate of 0.7 ml min−1 and began with mobile phase B held at 4% for 1.35 min, followed by a linear gradient from 4% to 40% B over 1.25 min, a linear gradient from 40% to 100% B over 0.4 min, a linear gradient from 100% to 4% B over 0.7 min and then finally B held at 4% for 0.8 min. Acylation was confirmed by correctly identifying the exact masses of the 2′- and 3′-acyl-adenosine products corresponding to the substrate tested in the EICs recorded by LC–HRMS using a 6530 Q-TOF AJS-ESI instrument (Agilent, G6530BAR). The following parameters were used: fragmentor voltage = 175 V, gas temperature = 300 °C, gas flow = 12 l min−1, sheath gas temperature = 350 °C, sheath gas flow = 12 l min−1, nebulizer pressure = 35 psi, skimmer voltage =65 V, Vcap = 3,500 V and collection rate = 3 spectra s−1. The expected exact masses of the acyl-adenosine nucleosides (Supplementary Table 2) were calculated using ChemDraw 19.0 and extracted from the TICs (±100 ppm).

Procedure for determining aminoacylation yields using intact tRNA MS

Enzymatic tRNA acylation reactions (25 µl) were performed as described in Procedure for RNAse A assays section. Sodium acetate (pH 5.2) was added to the acylation reactions to a final concentration of 300 mM in a volume of 200 µl. The reaction mixtures were then extracted once with a 1:1 (v/v) mixture of acidic phenol (pH 4.5) and chloroform and washed twice with chloroform. After extraction, the acylated tRNA was precipitated by adding ethanol to a final concentration of 71% and incubation at −80 °C for 30 min, followed by centrifugation at 21,300g for 30 min at 4 °C. After removing the supernatant, acylated tRNA was resuspended in water and kept on ice for analysis.

The tRNA samples from the enzymatic acylation reactions were analysed by LC–MS as described in Transcription and purification of tRNAs section. Because the unacylated tRNA peak in each TIC contained tRNA species that could not be enzymatically acylated (primarily tRNAs that lack the 3′-terminal adenosine81), simple integration of the acylated and non-acylated peaks in the absorbance at 260 nm (A260) chromatogram could not accurately quantify the acylation yield. To accurately quantify the acylation yield, we used the following procedure. For each sample, mass data were collected between m/z = 500 and 2,000. A subset of the mass data collected defined as the raw MS deconvolution range (Supplementary Figs. 3–35) was used to produce the deconvoluted mass spectra (Supplementary Figs. 3–35). The raw MS deconvolution range of each macromolecule species contained multiple peaks corresponding to different charge states of that macromolecule. Within the raw mass spectrum deconvolution range we identified the most abundant charge state peak in the raw mass spectrum of each tRNA species (unacylated, monoacylated and diacylated tRNA), which is identified as the major ion by an asterisk in Supplementary Figs. 3c–35c. To quantify the relative abundance of each species, the exact mass of the major ions (±0.3000 Da) was extracted from the TIC to produce the EICs (Supplementary Figs. 3–35). The EICs were integrated and the areas of the peaks that aligned with the correct peaks in the TIC (as determined from the deconvoluted mass spectrum) were used to quantify the yields (Supplementary Table 3). For the malonic acid substrates, the integrated peak areas of the EICs of both the malonic acid and decarboxylation products were added together to determine the overall acylation yield. Each sample was injected three times; the chromatograms and spectra in Supplementary Figs. 3–35 are representative, and the yields shown in Supplementary Table 3 are averages of the three injections. The expected masses of the oligonucleotide products were calculated using the AAT Bioquest RNA Molecular Weight Calculator, and the molecular masses of the small molecules added to them were calculated using ChemDraw 19.0. All the masses identified in the mass spectra are summarized in Supplementary Data 1.

Malachite green assay to monitor adenylation

Enzymatic adenylation reactions were monitored using malachite green following a previous protocol with modifications56. Each adenylation reaction (60 µl) contained the following components: 200 mM HEPES-K (pH 7.5), 4 mM DTT, 10 mM MgCl2, 0.2 mM ATP, 0–10 mM substrate, 4 U m−1E. coli inorganic pyrophosphatase (NEB) and 2.5 µM enzyme (MaFRS1 or MaFRSA). The adenylation reactions were incubated at 37 °C in a dry air incubator. Aliquots (10 µl) were withdrawn after 0, 5, 10, 20 and 30 min and quenched by addition to an equal volume of 20 mM EDTA (pH 8.0) on ice. Once all aliquots had been withdrawn, 80 µl malachite green solution (Echelon Biosciences) was added to each aliquot and the mixture incubated at room temperature for 30 min. After shaking for 30 s to remove bubbles, the absorbance at 620 nm was measured on a Synergy HTX plate reader (BioTek). The absorbance was then converted to phosphate concentration using a phosphate calibration curve (0–100 µM) and plotted against time to determine the turnover number.

Structure determination

The following synthetic dsDNA sequence was cloned upstream of MaFRSA (MaPylRS N166A, V168A) into pET32a-MaFRSA by Gibson assembly75 and used for subsequent crystallographic studies: GSS linker-6xHis-SSG linker-thrombin site-MaFRSA (Supplementary Table 1). The sequence of the pET32a-6xHis-thrombin-MaFRSA plasmid was confirmed by Sanger sequencing at Genewiz using the primers T7 F and T7 R (Supplementary Table 1). The procedure used to express and purify MaFRSA for crystallography using pET32a-6xHis-thrombin-MaFRSA was adapted from a protocol used to express and purify WT MaPylRS (ref. 60). BL21-Gold (DE3)pLysS competent cells (Agilent) were transformed with pET32a-6xHis-thrombin-MaFRSA and grown in TB media at 37 °C. Protein expression was induced at an OD600 of 1.2 with 1 mM IPTG. The temperature was lowered to 20 °C and growth was allowed to continue overnight. The cells were pelleted for 1 h at 4,300g and then resuspended in lysis buffer (50 mM potassium phosphate (pH 7.4), 25 mM imidazole, 500 mM sodium chloride, 5 mM β-mercaptoethanol and 1 cOmplete Mini EDTA-free protease inhibitor tablet). The cells were then lysed by homogenization (Avestin Emulsiflex C3). After centrifugation for 1 h at 10,000g, the clarified lysate was bound to TALON metal affinity resin (Takara Bio) for 1 h at 4 °C, washed with additional lysis buffer and eluted with elution buffer (50 mM potassium phosphate (pH 7.4), 500 mM imidazole, 500 mM sodium chloride and 5 mM β-mercaptoethanol). The eluate was dialysed overnight at 4 °C into cleavage buffer (40 mM potassium phosphate (pH 7.4), 100 mM NaCl and 1 mM DTT) and then incubated overnight at room temperature with thrombin protease on a solid agarose support (MilliporeSigma). Following cleavage, the protein was passed over additional TALON resin to remove the 6xHis tag and dialysed overnight at 4 °C into sizing buffer (30 mM potassium phosphate (pH 7.4), 200 mM NaCl and 1 mM DTT). The protein was concentrated and loaded onto a HiLoad 16/600 Superdex 200 pg column (Cytiva Life Sciences) equilibrated with sizing buffer on an ÄKTA Pure 25 fast-liquid chromatograph. Purified MaFRSA was dialysed into storage buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2 and 10 mM β-mercaptoethanol), concentrated to 20 mg ml−1, aliquoted and flash-frozen for crystallography (Supplementary Fig. 2).

The initial crystallization screening conditions were adapted from Seki et al.60. Crystals were grown by hanging-drop vapour diffusion in 24-well plates. Then, 25 µl of 100 mM m-CF3-2-BMA (pH ≈ 7) was added to 1.5 ml microcentrifuge tubes and the water was removed by evaporation. The dried aliquots were then resuspended at a concentration of 100 mM with MaFRSA in storage buffer at three concentrations (6.9, 12.3 and 19.2 mg ml−1) and 10 mM AMP-PNP lithium salt hydrate. The protein–substrate solution (1 µl) was mixed in a 1:1 ratio with the reservoir solution (1 µl) containing 10 mM Tris-HCl (pH 7.4) and 26% polyethylene glycol 3,350, and then incubated above 1 ml of reservoir solution at 18 °C. Crystals with an octahedral shape appeared within 1 week. The crystals were plunged into liquid nitrogen to freeze with no cryoprotectant.

Data were collected at the Advanced Light Source beamline 8.3.1 at 100 K using a wavelength of 1.11583 Å. Data collection and refinement statistics are presented in Supplementary Table 4. The diffraction data were indexed and integrated with XDS82, and then merged and scaled with Pointless83 and Aimless84. The crystals were formed in the space group I4 with unit cell dimensions 108.958, 108.958 and 112.26 Å. The structure was solved by molecular replacement with Phaser85 using a single chain of the WT apo structure of MaPylRS (PDB code: 6JP2)60 as the search model. Two copies of MaFRSA were present in the asymmetric unit. The model was improved with iterative cycles of manual model building in Coot (ref. 86) alternating with refinement in Phenix87,88 using data with a resolution of up to 1.8 Å. Structural analysis and figures were generated using Pymol (version 2.4.2)89.

In vitro translation initiation

The Ma-tRNAPyl-ACC dsDNA template was prepared as described in Transcription and purification of tRNAs section using the primers Ma-PylT-ACC F and Ma-PylT-ACC R (Supplementary Table 1). Ma-tRNAPyl-ACC was also transcribed, purified and analysed as described previously. The enzymatic tRNA acylation reactions (150 µl) were performed as described in Procedure for RNAse A assays section with slight modifications. The enzyme concentration was increased to 12.5 µM (monomers 7, 14 and 15) or 25 µM (monomer 13) and the incubation time was increased to 3 h at 37 °C. Sodium acetate (pH 5.2) was added to the acylation reactions to a final concentration of 300 mM in a volume of 200 µl. The reactions were then extracted once with a 1:1 (v/v) mixture of acidic phenol (pH 4.5) and chloroform and washed twice with chloroform. After extraction, the acylated tRNA was precipitated by adding ethanol to a final concentration of 71% and incubation at −80 °C for 30 min, followed by centrifugation at 21,300g for 30 min at 4 °C. The acylated tRNAs were resuspended in water to a concentration of 307 µM immediately before in vitro translation.

Templates for the expression of MGVDYKDDDDK were prepared by annealing and extending the oligonucleotides MGVflag-1 and MGVflag-2 using Q5 High-Fidelity 2x Master Mix (NEB; Supplementary Table 1). The annealing and extension procedure was performed using the following protocol on a thermocycler (BioRad C1000 Touch): 98 °C for 30 s, 10 cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 45 s, 10 cycles of 98 °C for 10 s, 67 °C for 30 s and 72 °C for 45 s, and finally 72 °C for 300 s. Following the extension, the reaction mixture was supplemented with sodium acetate (pH 5.2) to a final concentration of 300 mM, extracted once with a 1:1 (v/v) mixture of basic phenol (pH 8.0) and chloroform, and washed twice with chloroform. The dsDNA product was precipitated by the addition of ethanol to a final concentration of 71% and incubation at −80 °C for 30 min, followed by centrifugation at 21,300g for 30 min at 4 °C. The dsDNA pellets were washed once with 70% (v/v) ethanol, resuspended in 10 mM Tris-HCl (pH 8.0) to a concentration of 500 ng µl−1 and stored at −20 °C until use in translation.

In vitro transcription/translation by codon skipping of the short FLAG tag-containing peptides X-Val-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (XV-Flag, where X = 7, 13, 14 or 15) was carried out using the PURExpress Δ (aa, tRNA) Kit (NEB, E6840S) according to a previous protocol with slight modifications17. The XV-Flag peptides were produced from the following reaction mixtures (12.5 µl): solution A (∆tRNA and ∆aa; 2.5 µl), amino acid stock mix (33 mM l-valine, 33 mM l-aspartic acid, 33 mM l-tyrosine and 33 mM l-lysine; 1.25 µl), tRNA solution (1.25 µl), solution B (3.75 µl), 250 ng dsDNA MGVDYKDDDDK template (0.5 µl) and Ma-tRNAPyl-ACC acylated with 7, 13, 14 or 15 (3.25 µl). The reactions were incubated in a thermocycler (BioRad C1000 Touch) at 37 °C for 2 h and quenched on ice.

The translated peptides were purified from the in vitro translation reaction mixtures by enrichment using anti-FLAG M2 magnetic beads (MilliporeSigma) according to the manufacturer’s protocol with slight modifications. For each peptide, 10 µl of a 50% (v/v) suspension of magnetic beads was used. The supernatant was pipetted from the beads on a magnetic manifold. The beads were then washed twice by incubating with 100 µl TBS buffer (150 mM NaCl and 50 mM Tris-HCl (pH 7.6)) for 10 min at room temperature and then removing the supernatant each time using a magnetic manifold. The in vitro translation reaction mixtures were added to the beads and incubated at room temperature for 30 min with periodic agitation. The beads were washed again three times with 100 µl TBS as described above. The peptides were eluted upon incubation with 12.5 µl of 0.1 M glycine·HCl (pH 2.8) for 10 min. The supernatant was transferred to vials and kept on ice for analysis.

The purified peptides were analysed according to a previously reported protocol17. The supernatant was analysed on an ZORBAX Eclipse XDB-C18 column (1.8 μm, 2.1 mm × 50 mm, room temperature; Agilent) using a 1290 Infinity II UHPLC (Agilent, G7120AR). The following protocol was used for separation: an initial hold at 95% solvent A (0.1% formic acid in water) and 5% solvent B (acetonitrile) for 0.5 min, followed by a linear gradient from 5% to 50% solvent B for 4.5 min at a flow rate of 0.7 ml min−1. The peptides were identified by LC–HRMS using a 6530 Q-TOF AJS-ESI instrument (Agilent, G6230BAR). The following parameters were used: fragmentor voltage = 175 V, gas temperature = 300 °C, gas flow rate = 12 l min−1, sheath gas temperature = 350 °C, sheath gas flow rate = 11 l min−1, nebulizer pressure = 35 psi, skimmer voltage = 5 V, Vcap = 3,500 V and collection rate = 3 spectra s−1. The expected exact masses of the major charge state of each peptide were calculated using ChemDraw 19.0 and extracted from the TICs (±100 ppm).

Plasmids used for in vivo studies

The plasmids used to express WT sfGFP (pET22b-T5/lac-sfGFP) and 151TAG-sfGFP (pET22b-T5/lac-sfGFP-151TAG) in E. coli have been described previously90. pET22b-T5/lac-sfGFP-200TAG was constructed from pET22b-T5/lac-sfGFP using a Q5 Site-Directed Mutagenesis Kit (NEB) with primers CS43 and CS44 (Supplementary Table 1). The synthetase/tRNA plasmid for WT MaPylRS (pMega-MaPylRS) was constructed by inserting a synthetic dsDNA fragment (pMega-MaPylRS) (Supplementary Table 1) into the NotI-XhoI cut sites of a pUltra vector65 by Gibson assembly75 using NEBuilder HiFi DNA Assembly Master Mix (NEB). pMega-MaFRSA was constructed by inserting a synthetic dsDNA fragment (made by annealing primers RF48 and RF49) after inverse PCR of pMega-MaPylRS with primers RF61 and RF62 (Supplementary Table 1) using Gibson assembly75. The sequences of the plasmids spanning the inserted regions were confirmed via Sanger sequencing at the University of California Berkeley DNA Sequencing Facility using primers T7 F and T7 R (Supplementary Table 1), and the complete sequence of each plasmid was confirmed by full-plasmid sequencing at Primordium Labs.

Plate reader analysis of sfGFP expression

E. coli DH10B chemically competent cells were transformed with pET22b-T5/lac-sfGFP-200TAG and either pMega-MaPylRS or pMega-MaFRSA. Colonies were picked and grown overnight in LB with the appropriate antibiotics. The following day, the OD600 of the overnight culture was measured, and all cultures were diluted with LB to an OD600 of 0.10 to generate a seed culture. A monomer cocktail was prepared in LB supplemented with 2 mM IPTG, 2 mM monomer 1, 2, 20 or 21, and the appropriate antibiotics at two times the final concentration (200 µg ml−1 carbenicillin and 100 µg ml−1 spectinomycin). In a 96-well plate (Corning 3904), 100 µl of the seed culture was combined with 100 µl of each monomer cocktail to bring the starting OD600 to 0.05 and half the concentration of the monomer cocktail. The 96-well plate was sealed with a Breathe-Easy sealing membrane (Diversified Biotech) and loaded into a Synergy HTX plate reader (BioTek). The plate was incubated at 37 °C for 24 h with continuous shaking. Two readings were made at 10 min intervals, that is, the absorbance at 600 nm, to measure cell density, and sfGFP fluorescence with excitation at 485 nm and emission at 528 nm.

Expression and purification of sfGFP variants

The plasmids used to express WT sfGFP and sfGFP-200TAG were co-transformed with pMega-MaPylRS or pMega-MaFRSA into DH10B or DH10B ΔaspC ΔtyrB chemically competent cells and plated onto LB agar plates supplemented with 100 µg ml−1 carbenicillin and 100 µg ml−1 spectinomycin. Colonies were picked the following day and used to inoculate 10 ml LB supplemented with 100 µg ml−1 carbenicillin and 100 µg ml−1 spectinomycin. The cultures were incubated overnight at 37 °C with shaking at 200 r.p.m. The following day, 1 ml of each culture was used to inoculate 100 ml TB or defined media (adapted from a published protocol55 with glutamate excluded and 19 other amino acids at 200 µg ml−1) supplemented with 100 µg ml−1 carbenicillin and 100 µg ml−1 spectinomycin in 250-ml baffled Erlenmeyer flasks. The cultures were incubated at 37 °C with shaking at 200 r.p.m. for ~4 h until they reached an OD600 of 1.0–1.2. At this point, IPTG was added to a final concentration of 1 mM and incubation was continued overnight at 37 °C with shaking at 200 r.p.m. The cells were collected by centrifugation at 4,303g for 20 min at 4 °C.

sfGFP variants were purified according to a previously published protocol71. The following buffers were used for protein purification: lysis/wash buffer: 50 mM sodium phosphate (pH 8), 300 mM NaCl and 20 mM imidazole; elution buffer: 50 mM sodium phosphate (pH 8) and 250 mM imidazole; storage buffer: 50 mM sodium phosphate (pH 7), 250 mM NaCl and 1 mM DTT. One cOmplete Mini EDTA-free protease inhibitor tablet was added to the wash and elution buffers immediately before use. To isolate protein, cell pellets were resuspended in 10 ml wash buffer. The resultant cell paste was lysed at 4 °C by homogenization (Avestin Emulsiflex C3) for 5 min at 15,000–20,000 psi. The lysate was centrifuged at 4,303g for 15 min at 4 °C to separate the soluble and insoluble fractions. The soluble lysate was incubated at 4 °C with 1 ml TALON resin (washed with water and equilibrated with wash buffer) for 1 h. The lysate–resin mixture was centrifuged at 4,303g for 5 min to pellet. The supernatant was removed and the protein-bound TALON resin was then washed three times with 5 ml lysis/wash buffer, centrifuging between washes to pellet. The protein was eluted from TALON resin by rinsing the resin five times with 1 ml elution buffer. The elution fractions were pooled and dialysed overnight at 4 °C into storage buffer using 12,000–14,000 MWCO dialysis tubing. The protein concentration was measured using the Pierce 660 nm assay91. Protein samples were concentrated as needed with a 10 kDa MWCO Amicon Ultra-15 centrifugal filter unit (4,303g, 4 °C) to reach a concentration of ≥0.22 mg ml−1 (Supplementary Fig. 37). The protein was stored at 4 °C for later analysis. Yields were between 24 and 324 mg l−1 when expressed in TB, and between 3.6 and 3.7 mg l−1 when expressed in the defined media described above. Proteins were analysed by LC–MS as described above.

Protease digestion and fragment identification by MS

Each isolated sfGFP sample (~10–25 µg) was denatured with 6 M guanidine in 0.15 M Tris buffer (pH 7.5), followed by disulfide reduction with 8 mM DTT at 37 °C for 30 min. The reduced sfGFP was alkylated in the presence of 14 mM iodoacetamide at 25 °C for 25 min, followed by quenching using 6 mM DTT. The reduced and alkylated protein was exchanged into ~40 µl of 0.1 M Tris buffer (pH 7.5) using a Microcon 10 kDa membrane, and then 2.5 µg endoproteinase GluC (in a 0.25 µg µl−1 solution) was added directly to the membrane to achieve an enzyme/substrate ratio of at least 1:10. After 3 h at 37 °C, the digestion was quenched with an equal volume of 0.25 M acetate buffer (pH 4.8) containing 6 M guanidine. The peptide fragments were collected by spinning down through the membrane and subjected to LC–MS/MS analysis.

LC–MS/MS analysis was performed on an Agilent 1290-II HPLC device directly connected to a Thermo Fisher Q Exactive HF high-resolution mass spectrometer. Peptides were separated on a Waters HSS T3 reversed-phase column (2.1 mm × 150 mm) at 50 °C with a 70 min acetonitrile gradient (0.5–35%) in water containing 0.1% formic acid in the mobile phase at a total flow rate of 0.25 ml min−1. The MS data were collected at a resolution of 120,000, followed by data-dependent higher-energy collision dissociation MS/MS at a normalized collision energy of 25%.

Proteolytic peptides were identified and quantified using MassAnalyzer, a program92 developed in house (available in Biopharma Finder, Thermo Fisher). The program performs feature extraction, peptide identification, retention time alignment93 and peak integration in an automated fashion.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.