Joongoo Lee^1,4, Kevin J. Schwarz^2,4, Do Soon Kim¹, Jeffrey S. Moore^2,3,* & Michael C. Jewett ^1,*

¹Department of Chemical and Biological Engineering and Center for Synthetic Biology, Northwestern University, Evanston, IL 60208, USA. ²Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. ³The Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. ⁴These authors contributed equally: Joongoo Lee, Kevin J. Schwarz.

^*Corresponding author

Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials.