cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2',3'
| Autor: | V Janett, Olzog, Christiane, Gärtner, Peter F, Stadler, Jörg, Fallmann, Christina E, Weinberg |
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| Rok vydání: | 2021 |
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| Zdroj: | RNA Biology article-version (VoR) Version of Record |
| ISSN: | 1555-8584 |
| Popis: | Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5′-fragment with a 2′,3′-cyclic phosphate and a 3′-fragment with a 5′-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5′-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3′-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms. |
| Databáze: | OpenAIRE |
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