minimal tRNA acceptor-stem mimic for flexizyme acylation
Microhelix is a 22-nucleotide RNA that mimics the acceptor stem of a transfer RNA in miniature: a 7-base-pair helix closed by a stable UUCG tetraloop, followed by a discriminator base and the 3′-terminal CCA. Flexizymes recognize only the conserved 3′-CCA region and do not read the body of the tRNA, so a microhelix is charged essentially as efficiently as full-length tRNA despite lacking the anticodon arm, D-arm and T-arm; Katoh and Suga reported closely comparable acylation yields for microhelix and full-length tRNA under identical conditions. It is therefore the standard substrate for assaying flexizyme activity and optimizing acylation conditions, and works with both eFx and dFx. Its small size is a practical advantage: aminoacylation can be read out directly by acid-denaturing PAGE or MALDI-TOF mass spectrometry, without the RNase digestion step that full-length tRNA requires to bring the analyte into a usable mass range. Minihelix and microhelix substrates of this type were also central to establishing that aminoacyl-tRNA synthetase specificity can reside in the acceptor stem alone — the “operational RNA code” for amino acids.
Applications:
1) Francklyn, C., & Schimmel, P. (1989). Aminoacylation of RNA minihelices with alanine. Nature, 337(6206), 478–481
2) Schimmel, P., Giege, R., Moras, D., & Yokoyama, S. (1993). An operational RNA code for amino acids and possible relationship to genetic code. Proceedings of the National Academy of Sciences, 90(19), 8763-8768
3) 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
4) 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
5) Goto, Y., Katoh, T., & Suga, H. (2011). Flexizymes for genetic code reprogramming. Nature Protocols, 6(6), 779-790
6) Katoh, T., & Suga, H. (2019). Flexizyme-catalyzed synthesis of 3’-aminoacyl-NH-tRNAs. Nucleic Acids Research, 47(9), e54
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