High-Throughput Mutational Analysis of a Twister Ribozyme

Shungo Kobori, Yohei Yokobayashi

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

Recent discoveries of new classes of self-cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self-cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations.

Original languageEnglish (US)
Pages (from-to)10354-10357
Number of pages4
JournalAngewandte Chemie - International Edition
Volume55
Issue number35
DOIs
StatePublished - Aug 22 2016
Externally publishedYes

Keywords

  • deep sequencing
  • high-throughput assays
  • ribozymes
  • RNA

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)

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