Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters

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Abstract
We describe experiments aimed at establishing whether circular single-stranded DNAs can form promoters for bacterial transcription from small folded motifs. In vitro selection experiments were carried out on circular 103-nt DNA libraries encoding 40-nt randomized sequences as well as self-processing hammerhead ribozymes. Rounds of rolling circle transcription, reverse transcription–PCR, and recyclization were carried out to optimize transcription efficiency. Sequences were identified that are 80-fold more actively transcribed than the initial library by E. coli RNA polymerase (RNAP). The selected motifs were found to be more active than canonical E. coli promoters in the same context. Experiments also demonstrated that a single-stranded pseudopromoter identified by this selection can be transplanted to other circular DNA contexts and retain transcriptional activity. Results suggest that the promoter is localized in a short (≈40 nt) hairpin, which is smaller than canonical E. coli promoters. To test whether this pseudopromoter was active in bacterial cells, a synthetic DNA nanocircle vector encoding a ribozyme targeted to a site in the mar A drug resistance gene was constructed to contain an optimized single-stranded promoter. It is shown that this DNA circle can act as a “Trojan horse” in E. coli , being actively transcribed by the cellular RNAP and producing ribozymes that cleave a sequence in the mar A drug resistance gene. The use of optimized single-stranded promoters in combination with synthetic nanocircle DNA vectors represents a potentially useful way to engender the synthesis of biologically active RNAs in living cells.