cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2',3' cy clic p hosphates and 5' hy droxyl ends

RNA Biol. 2021 Nov 12;18(sup2):818-831. doi: 10.1080/15476286.2021.1999105. Epub 2021 Dec 14.


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].

Keywords: 5ʹ-3ʹ exonuclease Xrn1; RNA pyrophosphohydrolase (RppH); RtcB ligase; T4 RNA ligase truncated KQ; next-generation sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Computational Biology
  • Escherichia coli / genetics
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Expression Profiling* / methods
  • Gene Library
  • Genomics / methods
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / genetics*
  • RNA-Seq / methods*


  • Fungal Proteins
  • RNA, Catalytic
  • hammerhead ribozyme

Grants and funding

This research was supported by a grant of the German Research Foundation(DFG) to C.E.W. (Deutsche ForschungsgemeinschaftLU1889/4) . V.J.O. was supported by a Predoc Award from Leipzig University (jointly given to V.J.O. and C.E.W.) and C.E.W. is also supported by a fellowship of the Peter and Traudl Engelhorn Foundation. Research was also funded in part by the German Federal Ministry for Education and Research (BMBF Bundesministerium für Bildung und Forschung 031A538B, de.NBI/RBC) to P.F.S