Programming peptidomimetic syntheses by translating genetic codes designed de novo

Abstract

Although the universal genetic code exhibits only minor variations in nature, Francis Crick proposed in 1955 that ''the adaptor hypothesis allows one to construct, in theory, codes of bewildering variety.'' The existing code has been expanded to enable incorporation of a variety of unnatural amino acids at one or two nonadjacent sites within a protein by using nonsense or frameshift suppressor aminoacyl-tRNAs (aa-tRNAs) as adaptors. However, the suppressor strategy is inherently limited by compatibility with only a small subset of codons, by the ways such codons can be combined, and by variation in the efficiency of incorporation. Here, by preventing competing reactions with aa-tRNA synthetases, aa-tRNAs, and release factors during translation and by using nonsuppressor aa-tRNA substrates, we realize a potentially generalizable approach for template-encoded polymer synthesis that unmasks the substantially broader versatility of the core translation apparatus as a catalyst. We show that several adjacent, arbitrarily chosen sense codons can be completely reassigned to various unnatural amino acids according to de novo genetic codes by translating mRNAs into specific peptide analog polymers (peptidomimetics). Unnatural aa-tRNA substrates do not uniformly function as well as natural substrates, revealing important recognition elements for the translation apparatus. Genetic programming of peptidomimetic synthesis should facilitate mechanistic studies of translation and may ultimately enable the directed evolution of small molecules with desirable catalytic or pharmacological properties.

Synthetic biology projects in vitro

“…"Pure translation display" (Forster et al 2004) can lack all 20 synthetases (and their inherent aminoacyl-tRNA proofreading and tRNA recharging activities) and release factors, freeing up all 64 codons for reassignment. In this way, ribosomes can be programmed to incorporate multiple adjacent unnatural amino acids with high fidelity (Forster et al 2003). One goal is the directed evolution of peptide analog ligands that are heavily substituted for protease resistance using aminoacyl-tRNA substrates prepared by total or partial (Merryman and Green 2004) synthesis.…”

Enzymatic aminoacylation of tRNA with unnatural amino acids

“…he recent development of translation systems composed entirely of purified components (1,2) has enabled the ribosomal synthesis of peptides composed primarily of unnatural amino acids (3,4). The extension of this program to the synthesis of highly modified drug-like peptides and unnatural biopolymers is limited by the number of unnatural building blocks that are compatible with the translation apparatus.…”

Smart polymeric gels: Redefining the limits of biomedical devices

“…For example, self-assembling structural domains found in native proteins, e.g., β-sheets and α-helices, can be outfitted with integrin binding domains (e.g., Arginine-Glycine-Aspartic Acid or, RGD) or with enzymatic domains [67]. Further, the possibility of going beyond the domain of the natural amino acids and incorporating unnatural amino acids in protein or peptide structures instill greater versatility in genetically engineered motifs [68][69][70][71][72][73][74][75][76]. Prompted by protein engineering as well as self-assembling strategies for nanofabrication, two new models for engineering smart hydrogels are evolving-first, the synthesis of hydrogels or their associative building blocks by genetic engineering methods, and second, the approach of spontaneous association of the building blocks by molecular selfassembly.…”