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

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 i...

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Veröffentlicht in:Nucleic acids research Jg. 43; H. 13; S. e85
Hauptverfasser: Kobori, Shungo, Nomura, Yoko, Miu, Anh, Yokobayashi, Yohei
Format: Journal Article
Sprache:Englisch
Veröffentlicht: England Oxford University Press 27.07.2015
Schlagworte:
Allosteric Regulation
Gene Library
Genetic Engineering - methods
HEK293 Cells
High-Throughput Nucleotide Sequencing
Humans
Methods Online
RNA Cleavage
RNA, Catalytic - chemistry
RNA, Catalytic - metabolism
Sequence Analysis, DNA
ISSN:0305-1048, 1362-4962
Online-Zugang:Volltext
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Zusammenfassung: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.
Bibliographie:ObjectType-Article-1
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ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkv265