Predicting Accessible Splice Sites for Trans-Splicing Ribozymes

The Tetrahymena group I intron is a ribozyme that catalyzes its own self-excision from 26S pre-ribosomal RNA. It has been shown that this ribozyme can be modified to operate in trans on a separate mRNA molecule. In this scenario, the ribozyme effectively “edits” the target mRNA by replacing the 3'- portion of the latter with a new sequence. This kind of ribozyme may be used to repair mutated, disease-related mRNAs before they are translated into protein. However, the target mRNA will fold into a complex structure and thereby hide many splice sites. Therefore, a key problem in the engineering of such trans-splicing ribozymes is the identification of accessible splice sites on the given target mRNA. Here we present a computational model, based on changes in the free energies of mRNA and ribozyme binding, to predict accessible splice sites for trans-splicing ribozymes. Based on these computational predictions we engineered several trans-splicing ribozymes. Their splicing efficiency was determined experimentally and compared with the computational predictions. We believe that computational modeling will enable us to design efficient ribozymes for therapeutic applications.