High-throughput experimental validation of novel hairpin ribozymes

The small self-cleaving hairpin ribozyme has served as a model for RNA structure and function and has been engineered for biotechnology applications. Hairpin ribozymes were thought to be rare with only four known examples, which limited the interpretation of their biological importance and the starting sequences for engineering efforts. Recently, a bioinformatics approach identified hundreds of different RNA sequences in metatranscriptomic data that matched a novel permutation of the hairpin ribozyme. However, the self-cleavage activity of most of these sequences has not been experimentally demonstrated. Here, a high-throughput sequencing-based approach was used to evaluate the cotranscriptional self-cleavage of 855 different hairpin ribozymes in parallel. The results showed that nearly all sequences can catalyze a self-cleavage reaction under these experimental conditions. In addition, we observed that rare nucleotides at highly conserved positions did not prevent observable ribozyme activity. The distribution of observed activity suggests that the metatranscriptomic sequences could contain random mutations from efficient wild-type ribozymes. The results further validate the bioinformatics approach that was used for ribozyme discovery and raise questions about the biological roles of these ribozymes in the diverse environments where they were discovered.