Evolution of Spliceozymes in E.coli
Group I intron ribozymes are a class of catalytic RNA that can excise themselves from a pre-mRNA sequence without the aid of the RNA/protein complex, spliceosome. Group I introns from Tetrahymena thermophila have been modified to catalyze the same reaction but on a substrate in trans. One such example is the “spliceozyme”. These are tetrahymena based trans-splicing ribozymes that can remove 100 nucleotides long sequences from a substrate mRNA. Theoretically, these spliceozymes could be used to treat genetic diseases caused due to intron retention. However certain limitations such as delivery of the ribozymes, trans-splicing efficiency, binding and splicing at the correct splice site, control of expression levels and off-target effects need to be overcome. A previous study tried to improve the trans-splicing efficiency of a spliceozyme by evolving it for 10 rounds in bacterial cells. This resulted in a clone called W11 that enhanced the trans-splicing efficiency by decreasing the formation of side products. However, the expression levels of these spliceozymes were too high (~10,000 molecules per cell) to be used in clinical settings. Additionally, the spliceozyme was optimized to remove the intron from a single splice site. This study focused on addressing these limitations by evolving spliceozyme W11 in E.coli cells under low expression levels and challenging it to remove different intron sequences from two different splice sites. Twelve rounds of evolution resulted in two spliceozyme sequences that showed better growth in E.coli than W11. It currently unclear, how the mutations observed in the winning spliceozymes improve their trans-splicing efficiency in E.coli cells. Future experiments like invitro assays and site directed mutagenesis need to be conducted in order to understand how these mutations benefit the spliceozymes.