cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2ʹ,3ʹ cyclic phosphates and 5ʹ hydroxyl ends

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.

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity

“…If we had not validated the RT-qPCR method on RNA produced by IVT and introduced the blocking oligonucleotide before applying the method to RNA produced in cells, we would have incorrectly observed substantial cleavage for G2, G3, and G4 in cellsan artifact of sample preparation. These findings have implications for other ribozyme cleavage measurements that require RNA purification/extraction and reverse transcription, such as RNA-seq (32,(38)(39)(40)(41)(42)(43). In particular, reports of context-dependent ribozyme cleavage that used different measurement techniques in vitro and in cells and found differences in activity may warrant additional examination (25,45,46).…”

“…Many methods for measuring self-cleaving ribozyme activity exist, such as electrophoretic separation (26)(27)(28), Förster resonance energy transfer (FRET) (29)(30)(31), connecting ribozyme activity to the expression of a reporter gene (32)(33)(34)(35)(36)(37), RNA sequencing (RNA-seq) (32,(38)(39)(40)(41)(42)(43), and reverse-transcription-polymerase chain reaction (RT-PCR)-based techniques (5,(44)(45)(46). Gel electrophoresis and FRET are well suited for IVT studies, but these techniques can be difficult for RNA produced in cells.…”

“…When considering RNA-seq and RT-qPCR, the choice of method often comes down to the necessary measurement scale. RNA-seq is often used for high throughput measurements, such as screening large libraries of candidate ribozyme sequences or genetic contexts, as sequencing allows for a single multiplexed measurement of the entire library (32,(38)(39)(40)(41)(42). RT-qPCR often makes sense for measuring a small set of sequences, e.g., < 20, as it is less costly than sequencing, requires less sample preparation, and can be performed and analyzed quickly (44,45).…”

Making Invisible RNA Visible: Discriminative Sequencing Methods for RNA Molecules with Specific Terminal Formations

“…As an alternative method to selectively capture 2 ,3 -cP-containing sncRNAs, Arabidopsis thaliana tRNA ligase (AtRNL) has been used [72,73]. Unlike the commonly used T4 RNA ligases 1 and 2, which ligate the 5 -P and 3 -OH ends of RNAs, the AtRNL can specifically ligate the 5 -OH and 2 ,3 -cP ends [72].…”

“…The AtRNL ligation forms 2 -P at the ligation site, which should be removed by CIP or 2 -phosphotransferase such as Saccharomyces cerevisiae Tpt1 for efficient RT [72]. The sequencing of AtRNL-ligated RNAs has successfully identified 2 ,3 -cP-containing sncRNAs such as U6 snRNA, tRNA halves, and 5 -cleavage products of self-cleaving ribozymes [72,73].…”