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. 2006 Mar 21;103(12):4356-61.
doi: 10.1073/pnas.0509219103. Epub 2006 Mar 13.

Enzymatic aminoacylation of tRNA with unnatural amino acids

Matthew C T Hartman  1 , Kristopher Josephson, Jack W Szostak
Affiliations

Enzymatic aminoacylation of tRNA with unnatural amino acids

Matthew C T Hartman et al. Proc Natl Acad Sci U S A. .

Abstract

The biochemical flexibility of the cellular translation apparatus offers, in principle, a simple route to the synthesis of drug-like modified peptides and novel biopolymers. However, only approximately 75 unnatural building blocks are known to be fully compatible with enzymatic tRNA acylation and subsequent ribosomal synthesis of modified peptides. Although the translation system can reject substrate analogs at several steps along the pathway to peptide synthesis, much of the specificity resides at the level of the aminoacyl-tRNA synthetase (AARS) enzymes that are responsible for charging tRNAs with amino acids. We have developed an AARS assay based on mass spectrometry that can be used to rapidly identify unnatural monomers that can be enzymatically charged onto tRNA. By using this assay, we have found 59 previously unknown AARS substrates. These include numerous side-chain analogs with useful functional properties. Remarkably, many beta-amino acids, N-methyl amino acids, and alpha,alpha-disubstituted amino acids are also AARS substrates. These previously unidentified AARS substrates will be useful in studies of the specificity of subsequent steps in translation and may significantly expand the number of analogs that can be used for the ribosomal synthesis of modified peptides.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
MALDI-MS detection of tRNA charging with natural amino acids. (A) The MALDI-MS AARS assay involves reductive amination of the esterified amino acid with a phosphonium-containing benzaldehyde followed by Nuclease P1 treatment. (B) Assay of aminoacylation of bulk tRNA by all 20 natural amino acids and AARS in a single pot reaction.
Fig. 2.
Fig. 2.
Assay results with various backbone analog classes. (A–C) Assay with N-Me amino acids (A), selected β-amino acids (B), and additional β-amino acids (C).
Fig. 3.
Fig. 3.
Charged analogs activated by AARS.
Fig. 4.
Fig. 4.
Polar, uncharged analogs activated by AARS.
Fig. 5.
Fig. 5.
Aromatic analogs activated by AARS.
Fig. 6.
Fig. 6.
Nonpolar analogs activated by AARS.
Fig. 7.
Fig. 7.
α,α-disubstituted analogs activated by AARS.
See this image and copyright information in PMC

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