bioRxiv preprint doi: https://doi.org/10.1101/2021.05.24.445427; this version posted June 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Mechanistic studies of non-canonical amino acid mutagenesis Rachel C. Fleisher1,2,*, Nina Michael1,*, and Ruben L. Gonzalez, Jr.1,† 1Department of Chemistry, Columbia University, 3000 Broadway, MC3126, New York, NY 10027, USA 2Current Address: Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel *These authors contributed equally to this work †To whom correspondence should be addressed: Ruben L. Gonzalez Jr., Department of Chemistry, Columbia University, 3000 Broadway, MC3126, New York, NY 10027, USA, Tel.: (212) 854-1096; Fax: (212) 932-1289; Email:
[email protected] When citing this paper, please use the following: Fleischer, R.C., Michael, N., and Gonzalez Jr. RL. 2021. Mechanistic studies of non-canonical amino acid mutagenesis. Methods in Enzymology. Vol. 656: Submitted. Abstract Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non- canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.24.445427; this version posted June 3, 2021.