Double combinatorial Optimization of trans-splicing Group I Intron Ribozymes
Trans-splicing group I introns are catalytic RNAs (ribozymes) that are based on naturally self-splicing group I introns. They can be designed to splice and reprogram RNAs indicating a disease status, which is of therapeutic interest. A critical factor for trans-splicing efficiency is the sequence of the extended guide sequence (EGS) appended to the Tetrahymena ribozyme 5'-terminus. However, no high-throughput screening methods are available where optimal EGS sequences can be identified from EGS libraries because successful EGSs are not incorporated into the splice products. Here, we introduce RABADOCS, a double combinatorial selection strategy, that indirectly identifies successful EGSs from a library containing tens of millions of unique randomized EGSs. To demonstrate this method, we optimized the EGS of trans-splicing ribozymes targeting the mutation site G1849U in JAK2-V617F mRNA, which is the most common cancer driver mutation in classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs). The RABADOCS procedure selected 23 EGSs, of which seven showed high in vitro splicing activity, and nine had intermediate activity. In contrast, six randomly chosen EGSs showed no detectable splicing activity. These results confirm RABADOCS' ability to identify EGSs mediating improved trans-splicing activity. We expect that RABADOCS-based optimization will significantly expand the utilization of trans-splicing ribozymes for therapeutic purposes.