In vitro genetic code reprogramming and expansion to study protein function and discover macrocyclic peptide ligands

Abstract

The ability to introduce non-canonical amino acids into peptides and proteins is facilitated by working within in vitro translation systems. Non-canonical amino acids can be introduced into these systems using sense codon reprogramming, stop codon suppression, and by breaking codon degeneracy. Here, we review how these techniques have been used to create proteins with novel properties and how they facilitate sophisticated studies of protein function. We also discuss how researchers are using in vitro translation experiments with non-canonical amino acids to explore the tolerance of the translation apparatus to artificial building blocks. Finally, we give several examples of how non-canonical amino acids can be combined with mRNA-displayed peptide libraries for the creation of protease-stable, macrocyclic peptide libraries for ligand discovery.

Suppression of Formylation Provides an Alternative Approach to Vacant Codon Creation in Bacterial In Vitro Translation

“…Genetic code reprogramming provides many opportunities in the generation of biomolecules with novel and useful properties. Examples include antibody‐drug conjugates, conjugated vaccines, mechanistic studies, and generating new catalysts [1–3] . Reprogramming of the genetic code requires a tRNA charged with an unnatural amino acid and a vacant codon.…”

Genetically Encoded Fragment-Based Discovery from Phage-Displayed Macrocyclic Libraries with Genetically Encoded Unnatural Pharmacophores

“…[1][2][3][4][5] Such functionalization expands existing genetically encoded chemical space to incorporate unnatural pharmacophores not present in the original peptide libraries, allowing the discovery of value-added molecules with properties not offered by peptides alone. [6][7][8][9] Numerous reports demonstrated the power of discovery of potent ligands from phage-and mRNA-displayed libraries in which unnatural pharmacophores were grafted onto the peptides in million-to-trillion-scale genetically-encoded library [10][11][12] . Genetically encoded fragment-based discovery (GE-FBD) 10 from such libraries is conceptually similar to canonical fragment-based design (FBD), which is a powerful method for the development of ligands, drug leads and three FDA-approved drugs to date.…”

Genetically Encoded Fragment-Based Discovery (GE-FBD) from Phage-Displayed Macrocyclic Libraries with Genetically-Encoded Unnatural Pharmacophores

“…[1][2][3][4][5] Such functionalization expands existing genetically encoded chemical space to incorporate unnatural pharmacophores not present in the original peptide libraries, allowing the discovery of value-added molecules with properties not offered by peptides alone. [6][7][8][9] Numerous reports demonstrated the power of discovery of potent ligands from phage-and mRNA-displayed libraries in which unnatural pharmacophores were grafted onto the peptides in million-to-trillion-scale genetically-encoded library [10][11][12] . Genetically encoded fragment-based discovery (GE-FBD) 10 from such libraries is conceptually similar to canonical fragment-based design (FBD), which is a powerful method for the development of ligands, drug leads and three FDA-approved drugs to date.…”