Direct testing of twister ribozyme a from over a thousand organisms reveals a broad tolerance for structural imperfections

Twister ribozymes are an extensively studied class of nucleolytic RNAs that specifically and rapidly cleave their backbone. Thousands of unique twisters have been proposed using sequence homology and structural descriptors. Yet, most of these candidates have not been validated. To address this gap, we developed CHiTA (Cleavage High-Throughput Assay), a high-throughput pipeline to test putative ribozymes en masse utilizing massively parallel oligonucleotide synthesis and next-generation sequencing. As proof of principle, we applied CHiTA to a small set of known active and inactive ribozymes. We then used CHiTA to test two large sets of twister ribozymes: over 1,600 reported twister candidates and ~1,000 new twister candidates. We identified the new candidates computationally in ~1,000 organisms, representing a massive increase in the number of twister ribozyme candidates and ribozyme-harboring organisms. Approximately 94% of these candidates were active and cleaved specifically. Analysis of their structural features revealed that many substitutions and helical imperfections can be tolerated. We repeated our computational search with structural descriptors updated from our analysis, whereupon we identified and confirmed the first active twister ribozyme in mammals. CHiTA broadly expands the number of active twister ribozymes and provides a powerful method for functional analyses of other RNA classes.