Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate (2',3'-cP) and a 3'-fragment with a 5'-hydroxyl (5'-OH).
More...Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate (2',3'-cP) and a 3'-fragment with a 5'-hydroxyl (5'-OH). Studying self-cleaving ribozymes over the past 30 years has greatly expanded our knowledge of their multi-faceted biochemical capabilities, their intricate structures and diverse biological roles. This continuously expanding knowledge is 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. Here, we present a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs, among other techniques, the specific ligation-based capture of ribozyme 5'-fragments with 2',3'-cP ends using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3'-fragments, they are enriched from total RNA by degrading RNAs with 5'-PPP and 5'-P.
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