Generation of Ribozymes by Rolling Circle Transcription of Promoterless Single-Stranded DNA Circles in Mammalian Cells.

Abstract

Self-processing hairpin ribozymes have been synthesized from promoterless single-stranded DNA circles (73 nt) within mammalian cells. Following lipid-mediated transient transfection, DNA circles were efficiently internalized by mouse L cells (OST7-1) that stably express T7 RNA polymerase confining it to the cytoplasm. Cellular uptake of circular DNA templates and intracellular accumulation of ribozyme RNA transcripts from these DNA circles were progressive, both peaking at 24 h after transfection. Intracellular transcription generated RNA concatemers accumulating to a level of ~100 copies per cell. Transcription appears to be independent of specific promoter sequences but depends on T7 RNA polymerase. The data presented here may support the hypothesis that single stranded bubble regions within duplex DNA can serve as de novo initiation sites for RNA transcription not only in vitro but also in the cytoplasm of mammalian cells. These results may provide a model for the rolling circle transcription of small circular nucleic acids in mammalian cells.