Engineered triply orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids

Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyl-transfer RNA synthetase/tRNA pairs. These pairs must...

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Bibliographic Details
Published in:Nature chemistry 2020-06, Vol.12 (6), p.535-544
Main Authors: Dunkelmann, Daniel L, Willis, Julian C W, Beattie, Adam T, Chin, Jason W
Format: Article
Language:English
Subjects:
Amino acids
Amino Acids - chemistry
Amino Acids - genetics
Amino Acyl-tRNA Synthetases - chemistry
Amino Acyl-tRNA Synthetases - genetics
Biopolymers
Biosynthesis
Clustering
Decoding
Directed Molecular Evolution
Escherichia coli - genetics
Euryarchaeota - genetics
Genetic Code
Green Fluorescent Proteins - genetics
Lysine - analogs & derivatives
Lysine - chemistry
Lysine - genetics
Models, Molecular
Polypeptides
Proteins
Ribonucleic acid
RNA
RNA, Transfer - chemistry
RNA, Transfer - genetics
Sequence Analysis, Protein
Sequences
Substrate Specificity
Transfer RNA
tRNA
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Summary:Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyl-transfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/ tRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/ tRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and tRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with tRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ tRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/ tRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.
ISSN:1755-4330
1755-4349
DOI:10.1038/s41557-020-0472-x