RtcB-mediated ligation to monitor self-cleaving ribozyme activity

Self-cleaving ribozymes are highly abundant catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5p fragment with a 2p,3p cyclic phosphate (2p,3p cP) and a 3p fragment with a 5p hydroxyl (5p OH) end. Recently, several strategies to discover self-cleaving ribozymes by targeted biochemical means have been introduced. These methods rely on different means to enrich ribozyme cleavage fragments. Here, we develop an alternative strategy in which 5p OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3p phosphate of the adapter and then ligates it directly to RNAs with 5p OH ends. Our results demonstrated that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the E. coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured spiked 3p twister ribozyme fragment from Escherichia coli total RNA. This RNA-seq based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5p-OH termini.