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Enhanced-Flexizyme-U (eFx-U)

$ 165.00 USD
Cat#: 
CR133
Nucleotides: 
45
Sequence: 
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pairs with A73 discriminator base of tRNA

‍‍

Enhanced-flexizyme-U (eFx-U) is a 45-nucleotide catalytic RNA belonging to the flexizyme family of in vitro-selected aminoacyl-tRNA synthetase ribozymes. Flexizymes recognize only the conserved 3′-terminal CCA region of their substrate and do not read the body of the tRNA, so eFx-U charges full-length tRNAs, microhelices and any other RNA carrying a 3′-CCA end with comparable efficiency. eFx accepts amino acids pre-activated as cyanomethyl esters (CME) or 4-chlorobenzyl thioesters (CBT), while dFx uses 3,5-dinitrobenzyl esters (DBE); CME activation is typically applied to aromatic amino acids and CBT or DBE to non-aromatic ones, largely reflecting the solubility of the activated substrate in the reaction buffer, so eFx covers both classes. eFx-U is a core component of the Flexible In Vitro Translation (FIT) system, in which flexizymes charge non-proteinogenic amino acids onto tRNAs whose anticodons are reassigned to vacant codons, enabling ribosomal synthesis of sequence-defined nonstandard peptides including those containing N-methyl amino acids, D-amino acids, and backbone-modified residues.

Applications:

  • Genetic code reprogramming for ribosomal incorporation of non-proteinogenic amino acids
  • Charging tRNAs with CME- and CBT-activated amino acid substrates in vitro
  • Aminoacylation of microhelix and minihelix RNA substrates
  • Synthesis of macrocyclic, thioether-cyclized and backbone-modified peptide libraries
  • Cell-free biosynthesis of thiopeptide and natural-product scaffolds
  • Discovery of bioactive peptides through mRNA display and RaPID selection platforms
  • 1) Murakami, H., Ohta, A., Ashigai, H., & Suga, H. (2006). A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nature Methods, 3(5), 357-359
    2) Goto, Y., Katoh, T., & Suga, H. (2011). Flexizymes for genetic code reprogramming. Nature Protocols, 6(6), 779-790
    3) Xiao, H., Murakami, H., Suga, H., & Ferré-D’Amaré, A.R. (2008). Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature, 454(7202), 358-361
    4) Katoh, T., & Suga, H. (2019). Flexizyme-catalyzed synthesis of 3’-aminoacyl-NH-tRNAs. Nucleic Acids Research, 47(9), e54
    5) Passioura, T., & Suga, H. (2014). Reprogramming the genetic code in vitro. Trends in Biochemical Sciences, 39(9), 400-408
    6) Fleming, S.R., Bartges, T.E., Vinogradov, A.A., Kirkpatrick, C.L., Goto, Y., Suga, H., Hicks, L.M., & Bowers, A.A. (2019). Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides. Journal of the American Chemical Society, 141(2), 758-762
    7) Maini, R., Kimura, H., Takatsuji, R., Katoh, T., Goto, Y., & Suga, H. (2019). Ribosomal Formation of Thioamide Bonds in Polypeptide Synthesis. Journal of the American Chemical Society, 141(51), 20004-20008
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