Engineering Pyrrolysine Systems for Genetic Code Expansion and Reprogramming.

Over the past 16 years, genetic code expansion and reprogramming in living organisms has been transformed by advances that leverage the unique properties of pyrrolysyl-tRNA synthetase (PylRS)/tRNA pairs. Here we summarize the discovery of the pyrrolysine system and describe the unique properties of PylRS/tRNA pairs that provide a foundation for their transformational role in genetic code expansion and reprogramming. We describe the development of genetic code expansion, from to all domains of life, using PylRS/tRNA pairs, and the development of systems that biosynthesize and incorporate ncAAs using pyl systems.

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids.

Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs.

Mutually orthogonal aminoacyl transfer RNA synthetase/transfer RNA pairs provide a foundation for encoding non-canonical amino acids into proteins, and encoded non-canonical polymer and macrocycle synthesis. Here we discover quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/pyrrolysyl-tRNA (tRNA) pairs. We discover empirical sequence identity thresholds for mutual orthogonality and use these for agglomerative clustering of PylRS and tRNA sequences; this defines numerous sequence clusters, spanning five classes of PylRS/tRNA pairs (the existing classes +N, A and B, and newly defined classes C and S).

A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design.

Orthogonal (O) ribosome-mediated translation of O-mRNAs enables the incorporation of up to three distinct non-canonical amino acids (ncAAs) into proteins in Escherichia coli (E. coli). However, the general and efficient incorporation of multiple distinct ncAAs by O-ribosomes requires scalable strategies for both creating efficiently and specifically translated O-mRNAs, and the compact expression of multiple O-aminoacyl-tRNA synthetase (O-aaRS)/O-tRNA pairs.

Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme*.

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with N -methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate.

Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels.

Giant unilamellar vesicles (GUVs) are a widely used model system for a range of applications including membrane biophysics, drug delivery, and the study of actin dynamics. While several protocols have been developed for their generation in recent years, the use of these techniques involving charged lipid types and buffers of physiological ionic strength has not been widely adopted. This protocol describes the generation of large numbers of free-floating GUVs, even for charged lipid types and buffers of higher ionic strength, using a simple approach involving soft polyacrylamide (PAA) gels.

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 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.

Facile generation of giant unilamellar vesicles using polyacrylamide gels.

Giant unilamellar vesicles (GUVs) are model cell-sized systems that have broad applications including drug delivery, analysis of membrane biophysics, and synthetic reconstitution of cellular machineries. Although numerous methods for the generation of free-floating GUVs have been established over the past few decades, only a fraction have successfully produced uniform vesicle populations both from charged lipids and in buffers of physiological ionic strength. In the method described here, we generate large numbers of free-floating GUVs through the rehydration of lipid films deposited on soft polyacrylamide (PAA) gels.

Total synthesis of Escherichia coli with a recoded genome.

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms.

Display Selection of Exotic Macrocyclic Peptides Expressed under a Radically Reprogrammed 23 Amino Acid Genetic Code.

Bioactive naturally occurring macrocyclic peptides often exhibit a strong bias for hydrophobic residues. Recent advances in in vitro display technologies have made possible the identification of potent macrocyclic peptide ligands to protein targets of interest. However, such approaches have so far been restricted to using libraries composed of peptides containing mixtures of hydrophobic and hydrophilic/charged amino acids encoded by the standard genetic code.

Amide-forming chemical ligation via -acyl hydroxamic acids.

The facile rearrangement of "-acyl isopeptides" to native peptide bonds via ,-acyl shift is central to the success of native chemical ligation, the widely used approach for protein total synthesis. Proximity-driven amide bond formation via acyl transfer reactions in other contexts has proven generally less effective. Here, we show that under neutral aqueous conditions, "-acyl isopeptides" derived from hydroxy-asparagine [aspartic acid-β-hydroxamic acid; Asp(β-HA)] rearrange to form native peptide bonds via an ,-acyl shift.