Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells.

Synthetic RNA formulations and viral vectors are the two main approaches for delivering small therapeutic RNA to human cells. Here we report findings supporting an alternative strategy in which an endogenous human RNA polymerase (RNAP) is harnessed to make RNA hairpin-containing small RNA from synthetic single-stranded DNA oligonucleotides. We report that circularizing a DNA template strand encoding a pre-microRNA hairpin mimic can trigger its circumtranscription by human RNAP III in vitro and in human cells. Sequence and secondary structure preferences that appear to promote productive transcription are described. The circular topology of the template is required for productive transcription, at least in part, to stabilize the template against exonucleases. In contrast to bacteriophage and Escherichia coli RNAPs, human RNAPs do not carry out rolling circle transcription on circularized templates. While transfected DNA circles distribute between the nucleus and cytosol, their transcripts are found mainly in the cytosol. Circularized oligonucleotides are synthetic, free of the hazards of viral vectors and maintain small RNA information in a stable form that RNAP III can access in a cellular context with, in some cases, near promoter-like precision and biologically relevant efficiency.