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
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.
%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
}