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High-Throughput Mutational Analysis of a Twister Ribozyme
- Source :
- Angewandte Chemie (International Ed. in English)
- Publication Year :
- 2016
- Publisher :
- Wiley, 2016.
-
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.
- Subjects :
- 0301 basic medicine
DNA Mutational Analysis
Mutant
010402 general chemistry
01 natural sciences
Catalysis
Deep sequencing
deep sequencing
03 medical and health sciences
RNA, Catalytic
Ribozymes
Ligase ribozyme
biology
Chemistry
Communication
Ribozyme
High-Throughput Nucleotide Sequencing
RNA
high-throughput assays
General Medicine
General Chemistry
Molecular biology
Communications
0104 chemical sciences
030104 developmental biology
Biochemistry
biology.protein
Mammalian CPEB3 ribozyme
VS ribozyme
Subjects
Details
- ISSN :
- 14337851
- Volume :
- 55
- Database :
- OpenAIRE
- Journal :
- Angewandte Chemie International Edition
- Accession number :
- edsair.doi.dedup.....482c41f3e072137b56f3cb037e787348