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Angew Chem Int Ed Engl. 2016 Aug 22;55(35):10354-7. doi: 10.1002/anie.201605470. Epub 2016 Jul 27.

High-Throughput Mutational Analysis of a Twister Ribozyme.

Author information

1
Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904 0495, Japan.
2
Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904 0495, Japan. yohei.yokobayashi@oist.jp.

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.

KEYWORDS:

RNA; deep sequencing; high-throughput assays; ribozymes

PMID:
27461281
PMCID:
PMC5113685
DOI:
10.1002/anie.201605470
[Indexed for MEDLINE]
Free PMC Article

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