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. Author manuscript; available in PMC: 2016 Nov 17.
Published in final edited form as: Curr Opin Chem Biol. 2015 Jul 15;28:83–90. doi: 10.1016/j.cbpa.2015.06.008

Figure 1.

Figure 1

Methods for genetic code expansion. (a) Two general paradigms exist for the genetic incorporation of nonstandard amino acids into proteins contrasted with the natural process of encoding the canonical amino acids. The close analog methodology complements a natural amino acid auxotrophy with a close nonstandard analog, enabling global protein labeling by native translational machinery. The orthogonal translation methodology introduces orthogonal translational machinery engineered to charge an orthogonal tRNA with a nonstandard amino acid, enabling site-specific targeted genetic incorporation. Certain nsAAs may require additional mutations in the elongation factor or the ribosome. (b) For targeted genetic incorporation, amber suppression is the most widely used technique. Competition with release factors limits efficiency, and methods are discussed to overcome this. (c) Quadruplet suppression can be performed with appreciable efficiency with the use of an engineered orthogonal 16S ribosomal subunit [27••]. (d) Sense codons can be reassigned by using an orthogonal 23S ribosomal subunit, engineered to accept a synthetic set of tRNAs [60].