Selection and validation of orthogonal tRNA/synthetase pairs for the encoding of unnatural amino acids across kingdoms

Methods Enzymol. 2021;654:3-18. doi: 10.1016/bs.mie.2021.03.009. Epub 2021 Apr 9.


As an increasing number of protein structures are resolved at atomic and near-atomic resolution, conventional amino acid mutagenesis may be insufficient to test many mechanistic hypotheses. As a result, the development of new tRNA/aminoacyl-tRNA synthetase (aaRS) pairs has become an important tool for determining intricate molecular interactions and understanding protein structures. This chapter describes in detail the directed evolution of new tRNA/aaRS pairs in Escherichia coli for the incorporation of non-canonical amino acids (ncAA). Section 1 describes the selection of new tRNA/aaRS pairs in E. coli. Section 2 details the use of a synthetase to incorporate an ncAA into a mammalian cell line, and Sections 1 and 2 both include methods on the determination of synthetase efficacy and fidelity.

Keywords: Amino acyl-tRNA synthetase engineering; Directed evolution; Genetic code expansion; Non-canonical amino acids; Synthetic biology.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / genetics
  • Amino Acyl-tRNA Synthetases* / genetics
  • Animals
  • Escherichia coli / genetics
  • RNA, Transfer / genetics


  • Amino Acids
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases