Flexizymes for Genetic Code Reprogramming

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2011 Jun 4

PMID 21637198

Citations 189

Authors

Yuki Goto

Takayuki Katoh

Hiroaki Suga

Affiliations

Soon will be listed here.

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 approximately 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.

Citing Articles

Discovery of selective low molecular weight interleukin-36 receptor antagonists by encoded library technologies.

Velcicky J, Cremosnik G, Scheufler C, Meier P, Wirth E, Felber R Nat Commun. 2025; 16(1):1669.

PMID: 39955284 PMC: 11829961. DOI: 10.1038/s41467-025-56601-7.

Beyond , Pharmaceutical Molecule Production in Cell-Free Systems and the Use of Noncanonical Amino Acids Therein.

Casteleijn M, Abendroth U, Zemella A, Walter R, Rashmi R, Haag R Chem Rev. 2025; 125(3):1303-1331.

PMID: 39841856 PMC: 11826901. DOI: 10.1021/acs.chemrev.4c00126.

Engineered initiator tRNAs can effectively start translation at non-AUG start codons and diversify N-terminal amino acids for mRNA Display.

Helmling C, Chan A, Cunningham C Nucleic Acids Res. 2025; 53(2).

PMID: 39831308 PMC: 11744186. DOI: 10.1093/nar/gkaf003.

Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry.

Fricke R, Knudson I, Swenson C, Smaga S, Schepartz A Nat Protoc. 2025; .

PMID: 39762443 DOI: 10.1038/s41596-024-01086-9.

Discovering covalent cyclic peptide inhibitors of peptidyl arginine deiminase 4 (PADI4) using mRNA-display with a genetically encoded electrophilic warhead.

Mathiesen I, Calder E, Kunzelmann S, Walport L Commun Chem. 2024; 7(1):304.

PMID: 39702664 PMC: 11659602. DOI: 10.1038/s42004-024-01388-9.

References

Clemons Jr W, Brodersen D, McCutcheon J, MAY J, Carter A, Morgan-Warren R . Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: purification, crystallization and structure determination. J Mol Biol. 2001; 310(4):827-43. DOI: 10.1006/jmbi.2001.4778. View

Niwa N, Yamagishi Y, Murakami H, Suga H . A flexizyme that selectively charges amino acids activated by a water-friendly leaving group. Bioorg Med Chem Lett. 2009; 19(14):3892-4. DOI: 10.1016/j.bmcl.2009.03.114. View

Murakami H, Ohta A, Ashigai H, Suga H . A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods. 2006; 3(5):357-9. DOI: 10.1038/nmeth877. View

Schagger H . Tricine-SDS-PAGE. Nat Protoc. 2007; 1(1):16-22. DOI: 10.1038/nprot.2006.4. View

Kawakami T, Ohta A, Ohuchi M, Ashigai H, Murakami H, Suga H . Diverse backbone-cyclized peptides via codon reprogramming. Nat Chem Biol. 2009; 5(12):888-90. DOI: 10.1038/nchembio.259. View

Kwon Y, Kodadek T . Quantitative evaluation of the relative cell permeability of peptoids and peptides. J Am Chem Soc. 2007; 129(6):1508-9. PMC: 2530819. DOI: 10.1021/ja0668623. View

Goto Y, Murakami H, Suga H . Initiating translation with D-amino acids. RNA. 2008; 14(7):1390-8. PMC: 2441986. DOI: 10.1261/rna.1020708. View

Sako Y, Goto Y, Murakami H, Suga H . Ribosomal synthesis of peptidase-resistant peptides closed by a nonreducible inter-side-chain bond. ACS Chem Biol. 2008; 3(4):241-9. DOI: 10.1021/cb800010p. View

Josephson K, Hartman M, Szostak J . Ribosomal synthesis of unnatural peptides. J Am Chem Soc. 2005; 127(33):11727-35. DOI: 10.1021/ja0515809. View

10.

Sagan S, Karoyan P, Lequin O, Chassaing G, Lavielle S . N- and Calpha-methylation in biologically active peptides: synthesis, structural and functional aspects. Curr Med Chem. 2004; 11(21):2799-822. DOI: 10.2174/0929867043364108. View

11.

Kawakami T, Murakami H, Suga H . Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids. J Am Chem Soc. 2008; 130(50):16861-3. DOI: 10.1021/ja806998v. View

12.

Kung H, Redfield B, Treadwell B, ESKIN B, Spears C, Weissbach H . DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors. J Biol Chem. 1977; 252(19):6889-94. View

13.

Kawakami T, Murakami H, Suga H . Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. Chem Biol. 2008; 15(1):32-42. DOI: 10.1016/j.chembiol.2007.12.008. View

14.

Wang L, Xie J, Schultz P . Expanding the genetic code. Annu Rev Biophys Biomol Struct. 2006; 35:225-49. DOI: 10.1146/annurev.biophys.35.101105.121507. View

15.

Xiao H, Murakami H, Suga H, Ferre-DAmare A . Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature. 2008; 454(7202):358-61. DOI: 10.1038/nature07033. View

16.

Baggott J, Johanning G, Branham K, Prince C, Morgan S, Eto I . Cofactor role for 10-formyldihydrofolic acid. Biochem J. 1995; 308 ( Pt 3):1031-6. PMC: 1136826. DOI: 10.1042/bj3081031. View

17.

Sako Y, Morimoto J, Murakami H, Suga H . Ribosomal synthesis of bicyclic peptides via two orthogonal inter-side-chain reactions. J Am Chem Soc. 2008; 130(23):7232-4. DOI: 10.1021/ja800953c. View

18.

Saito H, Kourouklis D, Suga H . An in vitro evolved precursor tRNA with aminoacylation activity. EMBO J. 2001; 20(7):1797-806. PMC: 145511. DOI: 10.1093/emboj/20.7.1797. View

19.

Putz J, Wientges J, Sissler M, Giege R, Florentz C, Schwienhorst A . Rapid selection of aminoacyl-tRNAs based on biotinylation of alpha-NH2 group of charged amino acids. Nucleic Acids Res. 1997; 25(9):1862-3. PMC: 146665. DOI: 10.1093/nar/25.9.1862. View

20.

Cropp T, Anderson J, Chin J . Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells. Nat Protoc. 2007; 2(10):2590-600. DOI: 10.1038/nprot.2007.378. View