Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals1,2,3 and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution4,5. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction6,7,8,9,10. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA11, to select for functional proteins from an in vitro translated protein library of >1012independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand12,13,14,15,16,17,18, from both random-sequence libraries12,14,15,16 and libraries based on a known protein fold17,18. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold18,19, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.
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We thank G. S. Cho for providing the RXR library before its publication along with valuable advice; A. D. Keefe for critical input during the initiation of this project; C. Mueller-Vahl for help with protein expression and purification; G.-P. Zhou and J. J. Chou for NMR measurements; A. D. Keefe, G. S. Cho, G. F. Short, R. Larralde, J. M. Carothers, J. K. Ichida, F. P. Seebeck, S. S. Mansy, C. Del Bianco, D. A. Treco, D. S. Wilson, A. J. Bell, A. Luptak, R. Bruckner and Z. Sachs for helpful discussions. This work was supported by a grant from the NASA Astrobiology Institute. B.S. was supported in part by the Emmy Noether-Programm of the Deutsche Forschungsgemeinschaft. J.W.S. is an Investigator of the Howard Hughes Medical Institute.
Author Contributions All experiments were performed by B.S. Both authors designed the experiments, discussed the results and wrote the paper.
The DNA sequences encoding the ligase enzymes 1-7 have been deposited in GenBank under the accession numbers EU019543 to EU019549, respectively. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
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Seelig, B., Szostak, J. Selection and evolution of enzymes from a partially randomized non-catalytic scaffold. Nature 448, 828–831 (2007). https://doi.org/10.1038/nature06032
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