Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism
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The paper describes a new method called "tRNA display" to engineer orthogonal aminoacyl-tRNA synthetases that can attach non-canonical monomers (ncMs) to tRNAs, independent of whether the ncMs can be polymerized by the ribosome. Current methods rely on the ncM being a ribosomal substrate, creating an evolutionary deadlock between engineering the synthetase and engineering the ribosome. tRNA display breaks this deadlock.
tRNA display involves fusing the synthetase gene to a split tRNA gene to create an stmRNA construct. The activity of the synthetase in acylating the tRNA can then be directly linked to the mRNA sequence encoding the synthetase. Active stmRNAs are selectively isolated and their sequences identified. This enables rapid discovery of synthetases for ncMs without requiring ribosomal translation.
Using tRNA display, the authors evolved synthetases selective for several ncMs, including β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids. These ncM-specific synthetase-tRNA pairs were used to site-specifically incorporate the ncMs into proteins in E. coli, including a β-amino acid incorporated into GFP. Inventing a translation independent aaRS evolution platform. Centered around evolved PylRS variants for the 3 new classes of monomers. Establishing a new, general in vitro selection for aminoacyl-tRNA synthetase enzymes that charge tRNAs with a wide variety of small molecules. Inclusive of tRNA not yet compatible with known ribosome variants Enable site specific incorporation of beta-amino acids and alpha,alpha-disubstituted amino acids. With PoC data in GFP. tRNA display expands the scope of genetically encodable monomers. Future work could use it to evolve ribosomes capable of polymerizing problematic ncMs.
https://www.nature.com/articles/s41586-023-06897-6
