Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs

Abstract

Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/^PyltRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/Mm^PyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/Mm^PyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/^PyltRNA_CUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/^PyltRNA pairs that are mutually orthogonal to the MmPylRS/Mm^PyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/^PyltRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.

Figure 1. The structures of the amino acids used in this work.

1, Nε-((tertbutoxy)carbonyl)-L-lysine; 2 Nε-(((1R,8S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)-L-lysine, exo isomer shown, 4:1 exo/endo mixture used; 3, Nε-benzyloxycarbonyl-L-lysine; 4, Nε-(((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl)-L-lysine.

Figure 2. Identifying ΔNPylRS/^PyltRNA pairs are active and orthogonal in E. coli

(a) Classification of identified PylRS sequences according to the presence of a domain homologous to D. hafniense PylSn either within the same gene (pylSn-pylSc fusion class), present within a separate gene in the same genome (pylSn class), or absent entirely (ΔpylSn class). (b) In vivo amber suppression activity assay using E. coli DH10B bearing pBAD GFP(150TAG)His₆ and the corresponding pKW PylRS/^PyltRNA_CUA plasmid in the presence and absence of BocK (1) demonstrates the activity of ΔNPylRS/^PyltRNA_CUA pairs and the orthogonality of several ^PyltRNAs with respect to E. coli aminoacyl-tRNA sythetases. Each data point shows the mean of three technical replicates that form one biological replicate; the error bars show the mean and SEM of three independent biological replicates. (c), (d), (e) Confirmation of specific BocK incorporation into purified GFPHis₆ by MmPylRS/Mm^PyltRNA_CUA, MaPylRS/Ma^PyltRNA_CUA and G1PylRS/G1^PyltRNA_CUA analysed by electrospray ionisation mass spectrometry (ESI-MS, predicted mass 27,942 Da, observed mass 27,942 Da) reveals the functional orthogonality of each PylRS with respect to E. coli tRNAs. The ESI-MS experiments in (c), (d), (e) were performed once. Raw ESI-MS spectra are provided in Supplementary Fig. 4.

Figure 3. Mutual orthogonality amongst natural PylRS/^PyltRNA pairs.

In vivo amber suppression activity assay using E. coli DH10B bearing pBAD GFP(150TAG)His₆ and the corresponding pKW PylRS/^PyltRNA_CUA plasmid in the presence and absence of BocK (1) demonstrates the activity of the indicated PylRS with cognate and non cognate ^PyltRNA_CUA. Each data point shows the mean of three technical replicates that form one biological replicate; the error bars show the mean and SEM of three independent biological replicates.

Figure 4. Evolution of Ma^PyltRNA to create mutually orthogonal PylRS/^PyltRNA pairs.

(a) Evolution Ma^PyltRNA to abolish its function with MmPylRS while preserving its function with MaPylRS, via positive selection in the presence of MaPylRS followed by a negative screen in the presence of MmPylRS. (b) Libraries of Ma^PyltRNA created by randomising or expanding the length of the variable loop to 4, 5 or 6 randomised nucleotides. (c) Variable loop sequences for the Ma^PyltRNA hits identified. Hits are grouped according to the library from which they were identified. Hits 6, 8, 10, 12, 15, 16, 17, 18 and 20 also contained a G55A mutation; hits 14, 19, 22, 23 and 24 also contained a G55T mutation; hits 11, 13 and 21 also contained a G55C mutation; hit 7 also contained a C15T mutation. (d), (e) In vivo amber suppression activity assay (libraries N = 3 and 4 (d) and libraries N = 5 and 6 (e)). Each data point shows the mean of three technical replicates that form one biological replicate; the error bars show the mean and SEM of three independent biological replicates. wt, wild-type

Figure 5. Engineering the active site of MaPylRS for selective ncAA incorporation.

(a) In vivo amber suppression activity assay using E. coli DH10B bearing pBAD GFP(150TAG)His₆ and the corresponding pKW PylRS/^PyltRNA_CUA plasmid in the presence and absence of BCNK (2) demonstrates the transferability of the MmPylRS-AF mutations into MaPylRS to facilitate improved incorporation of BCNK using MaPylRS-AF. Each data point shows the mean of three technical replicates that form one biological replicate; the error bars show the mean and SEM of three independent biological replicates. (b) In vivo amber suppression activity assay using E. coli DH10B bearing pBAD GFP(150TAG)His₆ and the corresponding pKW PylRS/^PyltRNA_CUA plasmid in the presence and absence of CbzK (3) and CypK (4) demonstrates the selective incorporation of CypK by MmPylRS and the selective incorporation of CbzK by MaPylRS-MutRS1. Each data point shows the mean of three technical replicates that form one biological replicate; the error bars show the mean and SEM of three independent biological replicates.

Figure 6. Encoding distinct ncAAs using mutually orthogonal PylRS/^PyltRNA pairs.

(a) GFPHis₆ purified from E. coli containing GFP(150TAG)His₆, MmPylRS/Mm^PyltRNA_UCCU and MaPylRS-MutRS1/Ma^PyltRNA(6)_CUA and analyzed by SDS-PAGE. Two independent experiments were performed with similar results. (b) Electrospray ionization mass spectrometry of GFPHis₆ purified from E. coli containing GFP(150TAG)His₆, MmPylRS/Mm^PyltRNA_UCCU, MaPylRS-MutRS1/Ma^PyltRNA(6)_CUA in the presence of both CbzK and CypK. The peak corresponds to CbzK incorporation (predicted mass 27,976 Da, observed mass 27,975 Da). ESI-MS was performed once. (c) GFPHis₆ purified from E. coli containing GFP(150TAG)His₆, MmPylRS/Mm^PyltRNA_CUA, MaPylRS-MutRS1/Ma^PyltRNA(6)_UACU and analyzed by SDS-PAGE. Two independent experiments were performed with similar results. (d) Electrospray ionization mass spectrometry of GFPHis₆ purified from E. coli containing GFP(150TAG)His₆, MmPylRS/Mm^PyltRNA_CUA, MaPylRS-MutRS1/Ma^PyltRNA(6)_UACU in the presence of both CbzK and CypK. The peak corresponds to CypK incorporation (predicted mass 27,952 Da, observed mass 27,951 Da). ESI-MS was performed once. (e) GST-CaM purifications from E. coli containing ribo-Q1, o-GST-CaM(1TAG, 40AGGA), MmPylRS/Mm^PyltRNA_UCCU, MaPylRS-MutRS1/Ma^PyltRNA(6)_CUA grown in presence and absence of the indicated ncAAs. Samples were analyzed by SDS-PAGE. Two independent experiments were performed with similar results.