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

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Abstract

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 aminoacyltransfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/ Pyl tRNA pairs are the most widely used system for genetic code expansion. Here we reveal that the sequences of ΔNPylRS/ ΔNPyl tRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyl tRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyl tRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ ΔNPyl tRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/ Pyl 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.

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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.

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Genetically encoding N(epsilon)-acetyllysine in recombinant proteins.

Heinz Neumann, Sew Peak-Chew, Jason W Chin (2008)

N(epsilon)-acetylation of lysine (1) is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. No general methods exist to synthesize proteins containing N(epsilon)-acetyllysine (2) at defined sites. Here we demonstrate the site-specific incorporation of N(epsilon)-acetyllysine in recombinant proteins produced in Escherichia coli via the evolution of an orthogonal N(epsilon)-acetyllysyl-tRNA synthetase/tRNA(CUA) pair. This strategy should find wide applications in defining the cellular role of this modification.

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RNAstructure: web servers for RNA secondary structure prediction and analysis

Stanislav Bellaousov, Jessica S. Reuter, Matthew G. Seetin … (2013)

RNAstructure is a software package for RNA secondary structure prediction and analysis. This contribution describes a new set of web servers to provide its functionality. The web server offers RNA secondary structure prediction, including free energy minimization, maximum expected accuracy structure prediction and pseudoknot prediction. Bimolecular secondary structure prediction is also provided. Additionally, the server can predict secondary structures conserved in either two homologs or more than two homologs. Folding free energy changes can be predicted for a given RNA structure using nearest neighbor rules. Secondary structures can be compared using circular plots or the scoring methods, sensitivity and positive predictive value. Additionally, structure drawings can be rendered as SVG, postscript, jpeg or pdf. The web server is freely available for public use at: http://rna.urmc.rochester.edu/RNAstructureWeb.