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Flexizymes for genetic code reprogramming

Nature Protocols volume 6, pages 779–790 (2011)Cite this article

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Abstract

Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation apparatus, referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-methyl amino acids, D-amino acids and physiologically stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions. Preparation of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in ∼1 week. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.

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Figure 1: Schematic overview of genetic code reprogramming and expression of nonstandard peptides using the FIT system.
Figure 2
Figure 3: Acid PAGE analysis of products of flexizyme reaction with microhelix RNA under various reaction conditions.
Figure 4: Typical results of cyclic N-alkyl peptide syntheses by genetic code reprogramming.

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Acknowledgements

We thank Y. Yamagishi for the discussion and proofreading. We thank H. Murakami and T. Kawakami for the contributions to the development of the methods presented in this study. We also thank P.C. Reid and C.J. Hipolito for proofreading. This work was supported by grants from the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (S) (16101007), Specially Promoted Research (21000005), a research and development projects of the Industrial Science and Technology Program in the New Energy and Industrial Technology Development Organization, and the World Class University project of the MEST and the NRF (R31-2008-000-10103-0) to H.S.; and grants from the Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) (22750145) to Y.G. and (B) (22710210) to T.K.

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Author notes

  1. Yuki Goto and Takayuki Katoh: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.,

    Yuki Goto, Takayuki Katoh & Hiroaki Suga

  2. Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo, Japan.,

    Hiroaki Suga

  3. Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and College of Pharmacy, Seoul National University, Seoul, Korea

    Hiroaki Suga

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  1. Yuki Goto
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Contributions

Y.G. developed the methods presented in this study. Y.G. and T.K. validated the protocol, wrote the article and prepared the figures. H.S. supervised all the work and prepared the final version of the manuscript.

Corresponding author

Correspondence to Hiroaki Suga.

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Competing interests

We disclosed a patent related to this technology when this manuscript was submitted. Note that the patent includes some technical advancements to the FIT system that are not yet ready for publication.

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Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat Protoc 6, 779–790 (2011). https://doi.org/10.1038/nprot.2011.331

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