Genetically Encoded Libraries of Nonstandard Peptides
Figure 3
Comparison of the aminoacylation methods used for genetic code reprogramming. (a) Chemoenzymatic acylation method. The dinucleotide carrying the nonproteinogenic amino acid (pdCpA-aa) was enzymatically ligated to tRNA lacking 3′-nucleotides (pCpA). Aminoacyl-tRNAs prepared by the chemoenzymatic acylation method were added to an aaRS-free reconstituted translation system to synthesize a non-standard peptide containing three nonproteinogenic residues (1Z, L-propargylglycine; 2Z, L-O-methylserine; 3Z, L-allylglycine). (b) aaRS tRNA-misacylation method. Aminoacyl-tRNAs carrying nonproteinogenic amino acids were enzymatically generated in a reconstituted translation system. A non-standard peptide containing 13 nonproteinogenic residues was synthesized (, an analog of proteinogenic amino acid X). (c) Flexizyme-based acylation method. An activated amino acid was mixed with tRNAs and flexizyme to synthesize aminoacyl-tRNA. Aminoacyl-tRNAs prepared by the flexizyme-based method were added to a reconstituted translation system to synthesize a non-standard peptide containing six nonproteinogenic residues (4Z, L-acetyllysine; 5Z, L-citrulline; 6Z, L-p-iodophenylalanine).
