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Nucleic Acids Res. 2015 Jul 27;43(13):e85. doi: 10.1093/nar/gkv265. Epub 2015 Mar 30.

High-throughput assay and engineering of self-cleaving ribozymes by sequencing.

Author information

1
Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.
2
Department of Biomedical Engineering, University of California, Davis, CA 95616, USA Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904 0495, Japan yoko@ucdavis.edu.

Abstract

Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes.

PMID:
25829176
PMCID:
PMC4513843
DOI:
10.1093/nar/gkv265
[Indexed for MEDLINE]
Free PMC Article

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