High-Throughput Mutational Analysis of a Twister Ribozyme

• Published in: Angewandte Chemie International Edition 2016-08, Vol.55 (35), p.10354-10357
• Main Authors: Kobori, Shungo, Yokobayashi, Yohei
• Format: Article
• Language: English
• Subjects: Assaying; Biochemistry; Biology; Chemistry; Communication; Communications; deep sequencing; DNA Mutational Analysis; Engineering; Functional analysis; Functional anatomy; high-throughput assays; High-Throughput Nucleotide Sequencing; Mutants; Mutation; Ribozymes; RNA; RNA, Catalytic - chemistry; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; Robustness; Sensitivity; Structural members
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201605470

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. Ribozyme assay by sequencing: Deep sequencing was used to measure the activity of a self‐cleaving ribozyme and all of its single and double mutants (a total of 10 296 mutants), revealing interesting features of the mutational landscape.