Novel Method for Selection of tRNA-Driven Ribozymes with Enhanced Stability in Mammalian Cells

Abstract

Intracellular stability is a critical determinant of the activity of a ribozyme in vivo. In previous studies, we succeeded in constructing an effective system for the expression of ribozymes using the promoter of a human gene for tRNA^Val. The resultant tRNA^Val-driven ribozymes (tRNA-ribozymes) had a half-life of approximately 100 minutes. In the present study, we established a novel system for the selection of tRNA-ribozymes that were more stable than a previously generated optimally designed tRNA-ribozyme, and we confirmed that the newly selected tRNA-ribozymes worked well. Selective pressure was applied by treating cells that expressed tRNA-ribozymes with actinomycin D, and the system yielded tRNA-ribozymes with enhanced stability. The sequences isolated after selection exhibited some similarities. Furthermore, some selected tRNA-ribozymes had almost the same activity as or higher activity than that of the optimally designed tRNA-ribozyme despite the fact that the selective pressure was not aimed at enhancing the cleavage activity. Our approach might be very useful for selection not only of ribozymes with enhanced stability but also of other functional nucleic acids in vivo.