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Exploring the Minimal RNA Substrate of Flexizymes
Author(s) -
Fujino Tomoshige,
Kondo Taishi,
Suga Hiroaki,
Murakami Hiroshi
Publication year - 2019
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900150
Subject(s) - acylation , rna , substrate (aquarium) , transfer rna , ribozyme , chemistry , biochemistry , stereochemistry , combinatorial chemistry , biology , catalysis , gene , ecology
Flexizymes are tRNA acylation ribozymes that have been successfully used to facilitate genetic code reprogramming. They are capable of charging acid substrates onto various tRNAs and tRNA analogues. However, their minimal RNA substrate has not been investigated. Here we have designed fluorescently labeled short RNAs corresponding to the four, three, and two bases (4bRNA, 3bRNA, 2bRNA) at the tRNA 3′‐end and explored the minimal RNA substrate of flexizymes, dFx and eFx. 3bRNA was the observed minimal RNA substrate of the flexizymes, but the efficiency of acylation of this short RNA was two to three times lower than that of 4bRNA. The efficiency of acylation of 4bRNA was comparable with that of the microhelix, a 22‐base RNA conventionally used as a tRNA analogue for analyzing acylation efficiency. We also compared the efficiencies of acylation of the microhelix and 4bRNA with various acid substrates. Thanks to the short length of 4bRNA, its acyl‐4bRNA products exhibited larger mobility shifts in gel electrophoresis than those exhibited by acyl‐microhelix products with every substrate tested. This indicated that 4bRNA was an ideal RNA substrate for analyzing the efficiency of acylation by flexizymes.