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Peptide bond formation by in vitro selected ribozymes

Abstract

An attractive solution to the problem of the origin of protein synthesis in an evolving ‘RNA world’ involves catalysis by nucleic acid without assistance from proteins1,2. Indeed, even the modern ribosome has been considered to be fundamentally an RNA machine3, and the large ribosomal subunit can carry out peptidyl transfer in the absence of most of its protein subunits4. Successive cycles of in vitro selection and amplification5,6,7 have been used to find RNAs that perform many biochemical reactions8,9,10,11,12,13,14,15,16, including transfer of an RNA-linked amino acid to their own 5′-amino-modified terminus15. Here we demonstrate the in vitro selection of ribozymes (196 nucleotides) that perform the same peptidyl transferase reaction as the ribosome: that is, they can join amino acids by a peptide bond. Like ribosome substrates, one amino acid (N-blocked methionine) is esterified to the 3′(2′)-O of adenosine, whereas the acceptor amino acid (phenylalanine) has a free amino group. Our best characterized ribozyme recognizes the amino-acid ester substrate by binding its adenosine moiety, and is therefore capable of utilizing Leu- and Phe- as well as Met-derived substrates. Such lack of specificity with respect to the amino acid is a feature necessary for a generalized protein-synthesizing enzyme.

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Figure 1: Peptide bond formation by a ribosome (left) and by a ribozyme (right).
Figure 2: Activity of the selected RNA pool and transcripts from individual clones from generation 19.
Figure 3: Formation of biotinylated dipeptide catalysed by clone-25 RNA.
Figure 4: Validation of the peptide bond formed by catalysis of clone-25 RNA by HPLC-ESI/MS analysis of the dipeptide product.
Figure 5: Amino-acid specificity of peptide bond formation catalysed by clone-25 ribozyme.

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Acknowledgements

We thank K. Goodrich and E. Podell for oligonucleotide synthesis; R. Barkley and O. Averin for HPLC-MS spectra; and B. Golden, R. Gottlieb, G. Joyce, S. Seiwert and O. Uhlenbeck for comments on the manuscript. B.Z. is supported by a postdoctoral fellowship from the National Institute of General Medical Science, NIH. T.R.C. is an investigator of the Howard Hughes Medical Institute and an American Cancer Society Professor.

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Zhang, B., Cech, T. Peptide bond formation by in vitro selected ribozymes. Nature 390, 96–100 (1997). https://doi.org/10.1038/36375

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