Abstract
Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. We here describe a protocol that facilitates the 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 non-proteinogenic 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-acyl groups, d-amino acids, N-methyl amino acids, and physiologically stable macrocyclic scaffolds. Facility of the protocol allows for a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions.
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Acknowledgments
We thank Dr. Hiroshi Murakami for the contributions to the development of the methods presented in this study. We thank Dr. Takayuki Katoh for assistance with manuscript preparation. We also thank Mr. Dai Shimomai for proofreading. This work was supported by grants of Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (S) (16101007), Specially Promoted Research (21000005), and a research and development projects of the Industrial Science and Technology Program in the New Energy and Industrial Technology Development Organization (NEDO) to H.S., and grants of Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) (22750145) and PREST, Japan Science and Technology Agency to Y.G..
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Goto, Y., Suga, H. (2012). Flexizymes as a tRNA Acylation Tool Facilitating Genetic Code Reprogramming. In: Hartig, J. (eds) Ribozymes. Methods in Molecular Biology, vol 848. Humana Press. https://doi.org/10.1007/978-1-61779-545-9_29
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DOI: https://doi.org/10.1007/978-1-61779-545-9_29
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