A Tryptophanyl-tRNA Synthetase/tRNA Pair for Unnatural Amino Acid Mutagenesis in E. coli**

The genetic incorporation of unnatural amino acids (UAAs) with novel structures and properties into proteins in living cells has provided a powerful new tool to both probe and modify protein structure and function. The desired UAA is encoded by a nonsense or a frameshift codon using an engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pair that is orthogonal to the host cell (i.e., not cross-reactive with endogenous host aaRSs, tRNAs, or amino acids). While several orthogonal aaRS/tRNA pairs have been reported for UAA mutagenesis in E. coli, only the archaebacteria-derived tyrosyl and pyrrolysyl aaRS/tRNA pairs have been successfully used to genetically encode a large number of UAAs. To alter the specificity of the aaRS, a library of active-site mutants is subjected to rounds of positive and negative selection on the basis of their ability to incorporate amino acids into amber mutants of essential or toxic proteins. Development of new orthogonal aaRS/tRNA pairs with structurally distinct active sites should further expand the chemical diversity of genetically encoded UAAs. A tryptophanyl-tRNA synthetase (TrpRS)/tRNA pair is particularly attractive owing to the large tryptophan binding site of TrpRS that can potentially accommodate novel UAAs with large side chains. A modified TrpRS/tRNA pair from Saccharomyces cerevisiae was recently shown to be an orthogonal amber suppressor in E. coli. Here we report significant improvements to the amber suppression efficiency of the ScTrpRS/tRNACUA Trp pair by modifications to the acceptor stem of tRNACUA . Furthermore, we evolved active-site variants of TrpRS which are able to charge 3-(1-naphthyl)alanine, 1-methyl-tryptophan, 3-benzothienyl-alanine, and 6methyl-tryptophan with high fidelity and efficiency. Replacement of Trp66 in enhanced cyan fluorescent protein (ECFP) by these UAAs yielded ECFP variants with altered spectral features. To explore the possibility of using the ScTrpRS/ tRNACUA Trp pair to encode newUAAs, the wild-type ScTrpRS was inserted into the pBK plasmid, where it is expressed constitutively under the glnS promoter. Two reported variants (AS3.4 and AS3.5) of tRNACUA ScTrp driven by constitutive lpp promoters were cloned into two separate pREP vectors to generate the pREP-tRNACUA ScTrp plasmids. The pREP plasmid also encodes a chloramphenicol acetyl transferase (CAT) gene with an amber stop codon at a permissive site (Asp112TAG). To evaluate the amber suppression efficiency of the ScTrpRS/tRNACUA ScTrp pair, the pREP-tRNACUA ScTrp plasmids (encoding either tRNACUA AS3.4 or -AS3.5) were transformed into E. coli DH10B with or without the pBK-ScTrpRS plasmid. The chloramphenicol resistance (ChlorR) of strains expressing tRNACUA ScTrp alone or in combination with ScTrpRS is indicative of the crossreactivity of the tRNACUA ScTrp with host aminoacyl-tRNA synthetases and the amber suppression efficiency of this heterologous pair, respectively. While both variants of the tRNACUA ScTrp were found to be orthogonal to the endogenous aaRSs (ChlorR 200 mgmL ). We next explored the possibility of further improving the efficiency of the ScTrpRS/tRNACUA -AS3.5 pair (Figure S1 in the Supporting Information). There are two wobble G:U pairs in the stem regions of tRNA, G49:U65 and G4:U69 (Figure 1A). Mutation of such G:U pairs to G:C was previously shown to enhance the translational efficiency of a tRNA. Two tRNACUA -AS3.5 variants were generated, either with a U65C mutation alone (tRNACUA -AS3.51) or in combination with a U69Cmutation (tRNACUA -AS3.52). The mutant tRNACUA -AS3.51 exhibited a modest increase in activity (ChlorR ca. 35 mgmL ) and remained orthogonal to E. coli (Figure S1), while the double mutant tRNACUA AS3.52 had a significantly higher activity (ChlorR ca. 150 mgmL ), but diminished orthogonality (Figure S1). We next attempted to generate orthogonal tRNA variants with enhanced activity by subjecting a large acceptor-stem mutant library of the corresponding tRNA to a double-sieve selection scheme. The first five base pairs in the acceptor stem of tRNACUA -AS3.51 were randomized to all possible combinations to generate a library of approximately 10 variants. This library was subjected to alternating rounds of positive and negative selection to identify active, yet orthogonal variants. In the positive round of selection, tRNA mutants [*] Dr. A. Chatterjee, H. Xiao, Dr. P. Y. Yang, G. Soundararajan, Prof. Dr. P. G. Schultz Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) E-mail: schultz@scripps.edu Homepage: http://schultz.scripps.edu/ [] These authors contributed equally to this work.