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

Abstract

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 depsipeptides1–3. 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 substrates4–6. 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. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several β-amino acids, α,α-disubstituted-amino acids and β-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of β-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

Incorporation of Multiple β2-Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase

“…Such next-generation molecules provide strategies for improved biologic therapies, tools for bioremediation, and plastic-like materials that biodegrade. But progress toward sequence-defined non-protein materials l has been exceptionally slow. − Although most elements of the translational machinery tolerate even wildly divergent α-amino acid side chains, altered backbones are tolerated predominantly in vitro , at small scale, under nonphysiological conditions, and with efficiencies and fidelities that have not been rigorously evaluated.…”

“…Despite enormous interest in sequence-defined biomaterials containing non-α-amino acid monomers, progress towards their in vivo biosynthesis has been exceptionally slow. − There exists one report in which a single β 2 -ester has been introduced into a protein using the wild-type E. coli ribosome in vitro , but there are no examples in which any β 2 -hydroxy acid has been introduced into a ribosomal product in vivo .…”

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification

“…A recent study by Schepartz et al demonstrated the ability to expand substrate scope to encode for a variety of backbone modifications, such as α-hydroxy, α-thio, and N -formyl amino acids . Also, Chin et al have devloped a novel tRNA display system to identify aminoacyl tRNA synthetases for β-amino acids, α,α-disubstituted-amino acids, and β-hydroxy acids . Subsequent integration of these suppression systems into phage display will allow for increased diversity of the displayed peptides and enhanced stability of the identified peptide ligands.…”

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

“…222,223 Recently, a report by Dunkelmann and co-workers in 2024 demonstrated in vivo site-selective incorporation of three β 3 -AAs [β 3 -pBrAla, (S)-β 3 -(m-bromophenyl)alanine , (S)-β 3 -(m-trifluoromethyl)-L-phenylalanine] and one α,α-disubstituted amino acid [(S)-αmethyl-4-iodo-L-phenylalanine] into GFP(150UAG) using E. coli. 494 Compared to in vitro methodologies, there remains much room for improvement for in vivo peptide display platforms utilizing npMs, as many hit peptides have weak, micromolar activities and backbone-modifying npMs typically cannot be included. 495 As all current platforms only use a single npM during affinity maturation, the most likely area that could be significantly improved is the use of mutually orthogonal ARS/ tRNA pairs to incorporate multiple npMs into peptide libraries.…”

“…174 Briefly, this specialized tRNA display selects for PylRS variants that acylate their cognate tRNAs with npMs, independent of ribosomal translation efficiency. 494 As such, there is significant potential for the rapid expansion of available npMs for in vivo genetic code manipulation.…”