%0 Journal Article %1 Olzog2021-cd %A Olzog, V Janett %A Gärtner, Christiane %A Stadler, Peter F %A Fallmann, Jörg %A Weinberg, Christina E %D 2021 %I Informa UK Limited %J RNA Biol. %K (RppH); 5ʹ-3ʹ KQ; RNA RtcB T4 Xrn1; exonuclease ligase ligase; next-generation pyrophosphohydrolase sequencing truncated %N sup2 %P 818--831 %T cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2',3' cyclic phosphates and 5' hydroxyl ends %V 18 %X 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. Figure: see text.

@article{Olzog2021-cd, abstract = {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. [Figure: see text].}, added-at = {2024-09-10T11:54:51.000+0200}, author = {Olzog, V Janett and G{\"a}rtner, Christiane and Stadler, Peter F and Fallmann, J{\"o}rg and Weinberg, Christina E}, biburl = {https://puma.scadsai.uni-leipzig.de/bibtex/248d3cbf9c8ff6cd4b9366500b4693332/scadsfct}, interhash = {5a4ec437d61eed039b5e24ede7e634e0}, intrahash = {48d3cbf9c8ff6cd4b9366500b4693332}, journal = {RNA Biol.}, keywords = {(RppH); 5ʹ-3ʹ KQ; RNA RtcB T4 Xrn1; exonuclease ligase ligase; next-generation pyrophosphohydrolase sequencing truncated}, language = {en}, month = nov, number = {sup2}, pages = {818--831}, publisher = {Informa UK Limited}, timestamp = {2024-09-10T11:54:51.000+0200}, title = {{cyPhyRNA-seq}: a genome-scale {RNA-seq} method to detect active self-cleaving ribozymes by capturing {RNAs} with 2',3' cyclic phosphates and 5' hydroxyl ends}, volume = 18, year = 2021 }