Expanding the genetic code with diverse backbone structures across diverse sequence contexts

Piedrafita, C.; Dickson, A.; Richter, D.; Weber, C.; Elliott, T. S.; Liu, Z.; Zhang, F.; Li, Y.; Dunkelmann, D. L.; Morgan, T.; Liu, K. C.; Chin, J. W.

doi:10.64898/2026.04.16.718949

bioRxiv · 10.64898/2026.04.16.718949

Expanding the genetic code with diverse backbone structures across diverse sequence contexts

Piedrafita, C.·Dickson, A.·Richter, D.·Weber, C.·Elliott, T. S.·Liu, Z.·Zhang, F.·Li, Y.·Dunkelmann, D. L.·Morgan, T.·Liu, K. C.·Chin, J. W.

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Abstract

Expanding the genetic code to enable the selective and specific incorporation of non-canonical monomers (ncMs), beyond -L amino acids with variant sidechains, is a key outstanding challenge. Here we discover orthogonal aminoacyl-tRNA synthetases that selectively and specifically acylate their cognate orthogonal tRNA in vivo with eleven new ncMs spanning five different chemical classes: ,-disubstituted-amino acids, malonic acids, carboxylic acids, {beta}2-amino acids and N-cyclic amino acids. We demonstrate that co-translational incorporation of ,-disubstituted-amino acids, {beta}2-amino acids, {beta}3-amino acids and N-cyclic amino acids is strongly dependent on the codons either side of the codon used to direct ncM incorporation, with several ncMs incorporated at less than 1% of sequence contexts. We evolve orthogonal tRNAs that enable the incorporation of previously unincorporated ncMs, enable the incorporation of ncMs at >95% of sequence contexts and, increase the incorporation efficiency at challenging sequence contexts up to 40-fold. We demonstrate the encoded cellular synthesis of proteins and macrocycles containing ncMs and, explicitly demonstrate that our evolved tRNAs provide direct access to a wider range of genetically encoded macrocyclic sequences containing ncMs. Our results provide a foundation for composing, discovering and manufacturing proteins and peptides with functions augmented by ncMs.

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Piedrafita, C., Dickson, A., Richter, D., Weber, C., Elliott, T. S., Liu, Z., Zhang, F., Li, Y., Dunkelmann, D. L., Morgan, T., Liu, K. C., Chin, J. W.. 2026-04-17. Expanding the genetic code with diverse backbone structures across diverse sequence contexts. https://doi.org/10.64898/2026.04.16.718949

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