Work

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

Year: 2015

Type: article

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.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkv265

Authors: Shungo Kobori, Yoko Nomura, Anh Miu, Yohei Yokobayashi

Institutions: University of California, Davis, Okinawa Institute of Science and Technology Graduate University

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,992

Funders: National Institutes of Health, National Institute of General Medical Sciences