Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase–tRNA pairs

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Abstract

A central challenge in expanding the genetic code of cells to incorporate non-canonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)–tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach – named tRNA Extension (tREX) – to determine the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA–cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expands the number of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of additional orthogonal pairs and a foundation for encoding the cellular synthesis of non-canonical biopolymers.

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Adding new chemistries to the genetic code.

Chang C Liu, Peter Schultz (2010)

The development of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs has led to the addition of approximately 70 unnatural amino acids (UAAs) to the genetic codes of Escherichia coli, yeast, and mammalian cells. These UAAs represent a wide range of structures and functions not found in the canonical 20 amino acids and thus provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function.

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Universal rules and idiosyncratic features in tRNA identity.

R Giege, M Sissler, C Florentz (1998)

Correct expression of the genetic code at translation is directly correlated with tRNA identity. This survey describes the molecular signals in tRNAs that trigger specific aminoacylations. For most tRNAs, determinants are located at the two distal extremities: the anticodon loop and the amino acid accepting stem. In a few tRNAs, however, major identity signals are found in the core of the molecule. Identity elements have different strengths, often depend more on k cat effects than on K m effects and exhibit additive, cooperative or anti-cooperative interplay. Most determinants are in direct contact with cognate synthetases, and chemical groups on bases or ribose moieties that make functional interactions have been identified in several systems. Major determinants are conserved in evolution; however, the mechanisms by which they are expressed are species dependent. Recent studies show that alternate identity sets can be recognized by a single synthetase, and emphasize the importance of tRNA architecture and anti-determinants preventing false recognition. Identity rules apply to tRNA-like molecules and to minimalist tRNAs. Knowledge of these rules allows the manipulation of identity elements and engineering of tRNAs with switched, altered or multiple specificities.

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Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.

Heinz Neumann, Kaihang Wang, Lloyd Davis … (2010)

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.