Exploring the Substrate Range of Wild-type Aminoacyl-tRNA Synthetases

Experimental scheme

We used the E. coli wild-type AARS enzymes with their native amber or opal suppressor tRNAs^[6]. They were supU (tRNA^Trp_CUA), trpT (tRNA^TrpTyr_UCA), supF (tRNA), supP (tRNA^Leu_{CUA CUA}) and supD (tRNA^Ser_CUA). The TyrRS/supF pair was used to optimize the fluorescence signal arising from read-through of the sfGFP-UAG2 mRNA in a tyrA⁻ auxotrophic stain (Figure S1). The auxotrophic strains (Table S1) transformed with the genes for the AARS/suppressor tRNA pair (Figure S2 and Table S2) were grown at 37°C for 16 h in minimal medium with 1% glycerol supplemented with the 20 canonical amino acids (1 mM). After washing to remove the amino acids and transfer to minimal medium, the strains were cultured in a 384-well plate format where each well contained one member of our nsAA library at 1 mM concentration. The nsAAs library contained 313 members, of which 272 were previously reported ^[5b] (Tables S3-S5). The fluorescence readout, which was contingent on suppression of the sfGFP-UAG2 mRNA, was used as a measure of the ability of the corresponding endogenous AARS (LeuRS, SerRS, TrpRS and TyrRS) to recognize the nsAA substrates. This allowed a measurement of the range of amino acid substrate specificity for the AARS.

The wild-type protein sequence of sfGFP contains a single Trp (1) residue (W57). Under our incorporation conditions with wild-type tRNA^Trp_CCA this W57 could also be replaced by Trp analogs (2-4), Cys analog 5 and Phe analog 6 (Scheme 1; Figure 1A). The fluorescence intensities generated by nsAAs 4 and 5 were comparable to that of Trp. A similar substrate preference was observed with the TrpRS/supU pair (Figure 1B), although the pattern of suppression efficiencies was different. This change in substrate range stemmed purely from the different anticodon sequence, since the supU tRNA differs only by a single base change from the endogenous tRNA^Trp_CCA isoacceptor.^[6] We also tested the TrpRS substrate range with the natural opal suppressor tRNA^Trp_UCA encoded by trpT. As expected, the TrpRS/trpT pair incorporated Trp (1) into sfGFP but at a lower efficiency (Figure 1C). Interestingly, a Trp lactic acid analog (2) (Figure 1C) was a better substrate than Trp for TrpRS/trpT pair. Thus, different tRNAs gave somewhat different suppression profiles suggesting that the nature of the anticodon may affect the amino acid recognition, a property that was also seen with glutaminyl-tRNA synthetase^[8]. This suggests that in the strategy to develop orthogonal translation systems for genetic code expansion, modulations in amino acid recognition should be considered when tRNA anticodons are altered.

The substrate specificity profile of TyrRS/supF pair indicated that eight aromatic ring-containing nsAAs (3-4, 7-12) were substrates with appreciable signals but had low suppression efficiencies, albeit comparable to that of Tyr (13) (Figure 2A). The LeuRS/supP pair revealed three major fluorescence signals that indicated efficient incorporation of Leu (14) and two related nsAAs (15-16) that contained γ-vinyl and α-hydroxyl functional groups (Figure 2B). Previous studies showed γ-trifluoromethyl-leucine (17) to be a substrate for LeuRS [9]; our nsAA library screening also gave a small positive signal.

Our auxotrophic strain strategy provided sufficient amounts of Trp, Tyr, and Leu, but a much smaller concentration of Ser. Serine is involved in multiple cellular pathways, and is subject to high metabolic flux^[10], and the higher conversion rate of serine may have led to a lower amino acid level. Thus, the SerRS/supD pair gave a low fluorescence signal with Ser (Figure 2C; 18), but high signals with two Ser analogs (Figure 2C; 19 and 20). Compared to the wider substrate range of the TyrRS (Figure 2A) and TrpRS (Figure 1B), the smaller substrate range of LeuRS (Figure 2B) and SerRS (Figure 2C) may be explained that the latter two synthetases have an editing function, while the former two do not^{[1a, 11]}.

LC-MS/MS analysis of amino acids incorporated at positions 2 and 57 of sfGFP-UAG2

We performed liquid chromatography with tandem mass spectrometry (LC-MS/MS) to confirm the results by fluorescence screening mentioned above. For the TrpRS/supU pair, LC-MS/MS analysis identified nsAA 6 incorporation at position 2 and Trp at position 57 of sfGFP-6 protein produced from adding 1 mM nsAA 6 (Figure 3). Cross-talk events, i.e. Trp incorporation at position 2 and nsAA 6 at position 57, were not detected from the LC-MS/MS analysis. New peaks that appear may be caused by recognition of the introduced tRNA suppressor by AARSs other than the intended AARS (e.g. 6 in Figure 1A and 1B). Such unintended cross-talk may remain undetected; Trp incorporation was not detected at position 2 in the LC-MS/MS analysis of the sfGFP. Therefore, we are not able to rule out the possibility of cross-talk from other AARSs although the amino acid purity had been established.

The active sites of TrpRS, TyrRS, LeuRS and SerRS

While the nsAA library contained analogs of the cognate substrates of all four tested AARSs, TrpRS and TyrRS displayed a much larger productive substrate range than did LeuRS and SerRS. The four AARSs subjected to the nsAA library screening displayed diverse substrate specificities. TyrRS incorporated two Trp analogs (3 and 4) and Tyr analogs whose phenyl groups were substituted at the para and meta positions (7-13), although the nsAA incorporation efficiency was low (Figure 2A). TyrRS accommodated Tyr analogs as well as larger compounds like Nⁱⁿ-formyl-Trp (3) and Nⁱⁿ-boc-Trp (4). This implies that the TyrRS amino acid binding pocket is wider and deeper than necessary for accommodating Tyr. Sequence and structure based evolutionary analyses show the relationship of TrpRS and TyrRS.^[12] There is high backbone conservation between TrpRS and TyrRS suggesting that side chain variations in the amino acid binding pocket are the main source of discrimination between Trp and Tyr.^[12a] Thus the use of nsAAs containing aromatic side chains (3 and 4) by both enzymes is plausible. AARSs with narrow amino acid substrate range (SerRS and LeuRS) tend to recognize nsAAs similar to their cognate amino acid. Intrinsic editing mechanism that prevent formation of misacylated tRNA by SerRS and LeuRS may be responsible for the limited substrate response.^[1a]

tRNA sequence and amino acid selection

The tRNA anticodon affects the amino acid substrate range of its cognate AARS. Given this differential aminoacylation property, select tRNA anticodon variants may permit specific incorporation of nsAAs at targeted codons. For instance, nsAA 6 was efficiently charged onto tRNA^Trp_CUA (supU) but not tRNA^Trp_CCA (Figure 1A and 1B); this observation was further corroborated by LC-MS/MS analyses that revealed incorporations of nsAA 6 at position 2 and Trp at W57 position, respectively. Since wild-type AARSs differentially aminoacylate their tRNA substrates based on their anticodon sequences, synonymous codons may encode the incorporation of distinct palettes of nsAAs. Superposition of the substrate range profile of tRNA anticodon variants can reveal outliers that may be candidates for specific nsAA incorporation. This approach may allow incorporation of more nsAAs by reducing the degeneracy of the canonical genetic code in vivo.

System optimization and screening of incorporation of canonical or non-standard amino acids by sfGFP production

E. coli strain BW25113 or its auxotrophic strain JW2581-1 (tyrA⁻) was co-transformed with pBAD-sfGFP-UAG2 and pTech-supF (Figure S1, Table S1 and S2). BW25113 cells were cultured in 10 mL M9 medium (M9 salt supplemented with 1% glycerol, 2 mM MgSO₄, 0.1 mM CaCl₂) supplemented with 1 mM of the 20 canonical amino acids, 34 μg/mL chloramphenicol (Cm) and 100 μg/mL ampicillin (Amp) at 37 ^oC. JW2581-1 cells were cultured in 10 mL M9 medium supplemented with 1 mM of the 20 canonical amino acids, 50 μg/mL kanamycin (Kan), 34 μg/mL Cm and 100 μg/mL Amp at 37 °C. When cell A₆₀₀ reached 0.7 to 1.0, the cells were pelleted and washed thrice with M9 medium, and resuspended in M9 medium (to an A₆₀₀ of 1.0) supplemented with 1 mM of the appropriate nsAAs without 20 natural amino acids or 1mM Tyr, and arabinose (0.2 %) was added to induce sfGFP protein production for 12 h at 37 °C. Cells were collected by centrifugation and re-suspended in 1× phosphate buffered saline (PBS) with same culture volume. The fluorescence emission intensity of cells was measured at excitation wavelength 485/20 nm and emission wavelength 528/20 nm. The suppression experiments on the five natural tRNA suppressors (pTech-supU, pTech-trpT, pTech-supD and pTech-supP) were performed with the same conditions but in different auxotrophic strains (Figure S2 and Table S1).

For the substrate screening, we used a library that contained 313 nsAAs (containing 41 new entry nsAAs) (Table S3-S5), and its details were previously reported^[5b]. E. coli auxotrophic stains were transformed with either pBAD-sfGFP (JW2580 trpC⁻); pBAD-sfGFP-UGA2 in conjunction with pTech-trpT (JW2580 trpC⁻); or pBAD-sfGFP-UAG2 in conjunction with pTech-supD (JW2880 serA⁻), pTech-supP (JW5807 leuB⁻), pTech-supF (JW2581 tyrA⁻) or pTech-supU (JW2580 trpC⁻) plasmids (Table S1 and S2). Cells were cultured in 100 mL M9 medium supplemented with 1 mM of the 20 natural amino acids, 34 μg/mL Cm, 50 μg/mL Kan, and 100 μg/mL Amp at 37 ^oC. The JW2580 trpC⁻ cell strain containing pBAD-sfGFP was similarly cultured but without Cm in the medium. The cells were pelleted and washed twice with M9 medium, and resuspended in M9 medium (to an A₆₀₀ of 1.0) supplemented with 0.2% arabinose was added. Aliquots (50 μL) of the cell suspension were transferred into the wells of a 384-well plate containing a different nsAA (1 mM) in each well. Cells were continuous shaken for 12 h at 37 °C, with monitoring of fluorescence intensity (excitation wavelength 485/20 nm; emission wavelength 528/20 nm) and optical density (A₆₀₀ value) every 10 minutes. Four wells (A1, A2, I1 and I2) without added nsAAs were used as controls to measure the background signals. The nsAA screening experiments were performed in triplicate. For the annotated peaks generated by 1-20 the standard deviations of fluorescence intensities are listed in Table S8.

LC-MS/MS analyses of digested peptides of sfGFP proteins

The sfGFP sample purified from cells expressing supU was analyzed via LC-MS/MS. The full-length protein was trypsin digested by a standard in-gel digestion protocol, and analyzed via LC-MS/MS performed on an LTQ Orbitrap XL equipped with a Waters nanoACQUITY™ UPLC™ system. A Waters Symmetry® C18 180 μm × 20 mm trap column and a 1.7 μm, 100 μm × 250 mm nanoAcquity™ UPLC™ column (35°) were utilized for peptide separation. Trapping was done at 15 μL/min, 99% Buffer A (100% water, 0.1% formic acid) for 1 min. Peptide separation was performed at 300 nL/min with Buffer A and Buffer B (100% CH₃CN containing 0.1% formic acid). A linear gradient (51 min) was run with 5% buffer B at initial conditions, 50% B at 50 min, and 85% B at 51 min. MS was acquired in the Orbitrap using 1 microscan, and a maximum inject time of 900 ms followed by data dependent MS/MS acquisitions in the ion trap (via Collision Induced Dissociation, CID). The Mascot search algorithm was used to search for the appropriate non-canonical substitution (Matrix Science).