Extended Data Fig. 1: Encoded cellular incorporation of non-canonical monomers into proteins and into non-canonical polymers requires both tRNA acylation and ribosomal polymerization.
From: Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism
The encoded, site specific, incorporation of a non-canonical monomer (ncM, yellow star) via cellular translation requires both the acylation of an orthogonal tRNA with the ncM by an orthogonal synthetase, and ribosomal polymerization of the ncM into a polymer chain; arrow indicates peptide bond formation between A-site monomer and P-site nascent chain. Current methods for engineering aminoacyl-tRNA synthetases that acylate new monomers rely on translational readouts and therefore require the monomers to be ribosomal substrates. For ncMs that are poor ribosomal substrates this co-dependence creates an evolutionary deadlock in cells; an orthogonal synthetase cannot be evolved to acylate an orthogonal tRNA with ncMs that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs. To break this deadlock, we develop direct selections for orthogonal synthetases to aminoacylate their cognate orthogonal tRNAs with ncMs, independent of whether the ncMs are ribosomal substrates.
