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. 2003 May 27;100(11):6353-7.
doi: 10.1073/pnas.1132122100. Epub 2003 May 16.

Programming peptidomimetic syntheses by translating genetic codes designed de novo

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Programming peptidomimetic syntheses by translating genetic codes designed de novo

Anthony C Forster et al. Proc Natl Acad Sci U S A. .

Abstract

Although the universal genetic code exhibits only minor variations in nature, Francis Crick proposed in 1955 that "the adaptor hypothesis allows one to construct, in theory, codes of bewildering variety." The existing code has been expanded to enable incorporation of a variety of unnatural amino acids at one or two nonadjacent sites within a protein by using nonsense or frameshift suppressor aminoacyl-tRNAs (aa-tRNAs) as adaptors. However, the suppressor strategy is inherently limited by compatibility with only a small subset of codons, by the ways such codons can be combined, and by variation in the efficiency of incorporation. Here, by preventing competing reactions with aa-tRNA synthetases, aa-tRNAs, and release factors during translation and by using nonsuppressor aa-tRNA substrates, we realize a potentially generalizable approach for template-encoded polymer synthesis that unmasks the substantially broader versatility of the core translation apparatus as a catalyst. We show that several adjacent, arbitrarily chosen sense codons can be completely reassigned to various unnatural amino acids according to de novo genetic codes by translating mRNAs into specific peptide analog polymers (peptidomimetics). Unnatural aa-tRNA substrates do not uniformly function as well as natural substrates, revealing important recognition elements for the translation apparatus. Genetic programming of peptidomimetic synthesis should facilitate mechanistic studies of translation and may ultimately enable the directed evolution of small molecules with desirable catalytic or pharmacological properties.

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Figures

Fig. 3.
Fig. 3.
Translations incorporating three adjacent different unnatural amino acids by reassigning arbitrarily chosen codons. (a) Reassignment of NAAC,TACC, and VGUU codons of the universal genetic code to encode unnatural amino acids (U1–3) of our choosing. Two additional adaptors, termed formula image and formula image, were constructed to give the group of three chemoenzymatically synthesized tRNAs shown, differing only in their anticodon sequences (blue). The template (black), potentially encoding the natural translation product fMNTVE (green), was synthesized to test for the adjacent incorporation of three different unnatural amino acids by using these synthetic adaptors. (b) Rudimentary new genetic codes. Translation of the five-codon mRNA illustrated in a according to the blue code (Upper) would give the product fM-yU-mS-eU-E, whereas the purple code (Lower) would give fM-yU-eU-mS-E. (c) Dependence on each unnatural aa-tRNA for synthesis of fM-yU-mS-eU-E and fM-yU-eU-mS-E. All translations contained purified ribosomes and factors, formula image, and formula image. The positive control translation (data not shown) was supplemented with mRNA MVE (22) and unlabeled formula image, and yielded 8,600 dpm of product. The fM-yU-mS-eU-E translation (blue open triangle) was supplemented instead with mRNA MNTVE (a) and substrates formula image, formula image, and formula image (charged according to the blue genetic code), each at 1 μM, whereas control translations (red open triangles) omitted the individual unnatural aa-tRNAs listed below the x axis. The fM-yU-eU-mS-E translation (purple filled triangle) was also supplemented with mRNA MNTVE and formula image but differed in containing formula image and formula image (see purple genetic code), whereas control translations (red filled triangles) omitted the aa-tRNAs listed. Background dpm obtained in a translation without mRNA was subtracted. (d) HPLC analysis of a replicate of the complete translation performed in c by using the blue code. Radiolabeled translation reaction was treated with alkali, mixed with authentic unlabeled marker peptide (fM-yU-mS-eU-E), and analyzed by reversed-phase HPLC on a C18 column using a 4–32% acetonitrile/water gradient (large plot), or mixed with two closely related markers of identical amino acid composition and analyzed by a shallower 9–14% gradient to maximize resolution (Inset). The elution positions of the marker peptides are indicated above the chromatograms. (e) HPLC analysis (as in d Inset) of a replicate of the complete translation performed in c by using the purple code, demonstrating synthesis of fM-yU-eU-mS-E.
Fig. 2.
Fig. 2.
Translations incorporating five adjacent unnatural amino acids site-specifically. (a) mRNA sequence, encoded natural translation product without Glu-tRNAGlu (green), and encoded unnatural translation product when formula image is replaced with formula image (blue). (b) Incorporation of five adjacent eU amino acids. Positive control translations (green) contained the purified ribosomes and factors, mRNA MT5V, formula image, ≈3 μM formula image, and formula image. In other translations, natural formula image was omitted (negative controls in red) or replaced with ≈3 μM formula image (blue). Product values (dpm after subtraction of background dpm obtained in control translations lacking mRNA) represent three experiments performed on three different occasions with three different preparations of formula image. Bars indicate standard deviations. X, amino acid variable. (c) HPLC analysis of a replicate of the translations performed with formula image in b. Radiolabeled translation reaction was treated with alkali, mixed with authentic unlabeled marker peptide [fM(eU)5V dissolved in 88% formic acid], and analyzed by reversed-phase HPLC on a C18 column. The chromatogram shows a 27–71% acetonitrile/water linear gradient in the presence of 0.1% trifluoroacetic acid. The elution position of the marker peptide is indicated above the chromatogram. Peptide products were not detectable on a 2–32% acetonitrile/water gradient used for resolving less hydrophobic peptides such as fMT5V (ref. ; data not shown).
Fig. 1.
Fig. 1.
Our purified substrate-based translation system lacking RS activities. (a) The core translation machinery (blue) is depicted incorporating multiple unnatural amino acids (U1,U2,...) into peptidomimetic product. Escherichia coli served as the source for our natural components. IF1, IF2, IF3, His-tagged initiation factors; EF-Tu, EF-Ts, EF-G, His-tagged elongation factors. An mRNA template containing a Shine and Dalgarno ribosome binding site (SD) is colored purple, substrates are green, and products are red. Regeneration of GTP from GDP is catalyzed by pyruvate kinase using phosphoenolpyruvate substrate (data not shown). After translation, peptide products are released from the peptidyl-tRNAs by base-catalyzed hydrolysis [termination factors were omitted from the system for simplicity and because rapid product release would be undesirable for ribosome display experiments (29)]. (b) Natural E. coli tRNAAsn (ref. ; black; the anticodon is purple) and our synthetic ligated derivative, tRNAAsnB (differences from the natural tRNA in blue).

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