Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles

Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports. We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro. The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters. Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (similar to 7,500 nt). During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length. Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans. In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans. This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.