An expanded set of amino acid analogs for the ribosomal translation of unnatural peptides.
BACKGROUND
The application of in vitro translation to the synthesis of unnatural peptides may allow the production of extremely large libraries of highly modified peptides, which are a potential source of lead compounds in the search for new pharmaceutical agents. The specificity of the translation apparatus, however, limits the diversity of unnatural amino acids that can be incorporated into peptides by ribosomal translation. We have previously shown that over 90 unnatural amino acids can be enzymatically loaded onto tRNA.


RESULTS
We have now used a competition assay to assess the efficiency of tRNA-aminoacylation of these analogs. We have also used a series of peptide translation assays to measure the efficiency with which these analogs are incorporated into peptides. The translation apparatus tolerates most side chain derivatives, a few alpha,alpha disubstituted, N-methyl and alpha-hydroxy derivatives, but no beta-amino acids. We show that over 50 unnatural amino acids can be incorporated into peptides by ribosomal translation. Using a set of analogs that are efficiently charged and translated we were able to prepare individual peptides containing up to 13 different unnatural amino acids.


CONCLUSIONS
Our results demonstrate that a diverse array of unnatural building blocks can be translationally incorporated into peptides. These building blocks provide new opportunities for in vitro selections with highly modified drug-like peptides.
Hartman
Matthew C T
MC
Howard Hughes Medical Institute, Department of Molecular Biology, Simches Research Center, Massachusetts General Hospital, Boston, Massachusetts, United States of America.
Josephson
Kristopher
K
Lin
Chi-Wang
CW
Szostak
Jack W
JW
D000596:Amino Acids / Q000737:chemistry*; D002264:Carboxylic Acids / Q000737:chemistry; D002474:Cell-Free System; D005821:Genetic Techniques; D007700:Kinetics; D008954:Models, Biological; D010442:Peptide Chain Initiation, Translational; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D012270:Ribosomes / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; D002264:Carboxylic Acids; D019151:Peptide Library; D010455:Peptides; D012335:RNA, Ribosomal; D012343:RNA, Transfer