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. 2002 Jan 8;99(1):54-9.
doi: 10.1073/pnas.012589099. Epub 2001 Dec 18.

Efficient Bacterial Transcription of DNA Nanocircle Vectors With Optimized Single-Stranded Promoters

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Efficient Bacterial Transcription of DNA Nanocircle Vectors With Optimized Single-Stranded Promoters

Tatsuo Ohmichi et al. Proc Natl Acad Sci U S A. .
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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 ( approximately 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 marA 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 marA 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.

Figures

Figure 1
Figure 1
Sequence of 103-nt single-stranded DNA nanocircle library containing 40 nt of randomized sequence, and 63 nt of fixed sequence encoding a hammerhead ribozyme.
Figure 2
Figure 2
Improvement of transcription activity over successive rounds of in vitro selection. RNA amount was measured for each successive population at 37°C after 1.5 h. Dark and light bars correspond to the relative RNA amounts (>80-nt product) for the successive population with and without ligation, respectively. The reaction conditions are described in Materials and Methods.
Figure 3
Figure 3
Sequences of clones obtained following the fifteenth round of in vitro selection. Boxes indicate regions of high sequence similarity.
Figure 4
Figure 4
Selected circular DNA motifs engender RNA synthesis in vitro with E. coli RNAP. (A) Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, a control 63-nt nanocircle lacking the randomized domain, and selected individual nanocircles E1, E15, and E38 (after 1.5 h). (B) The relative total RNA amounts (all lengths >80 nt) for the 103-nt initial library, 63-nt nanocircle lacking the randomized domain, and E1, E15, and E38. (C) Time course of the production of monomeric ribozyme for the 103-nt initial library (■), 63-nt nanocircle lacking the randomized domain (○), E1 (▴), and E15 (●).
Figure 5
Figure 5
Sequences and predicted secondary structures of E1, E15, and E38. Boxed part indicates selected sequence from original randomized 40-nt domain; unboxed part encodes ribozyme.
Figure 6
Figure 6
Assessment of transplantability of E15 selected motif to a new nanocircle encoding marA ribozyme. Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, nanocircle E15, the new marA nanocircle, marA nanocircle with inactivated ribozyme, and two 63-nt nanocircle controls.
Figure 7
Figure 7
Effect of nanocircle vectors on the inhibition of CAT activity. (A) Thin-layer chromatogram showing levels of CAT expressed in the presence of 10 μM marA vector and E15 vector. The control lane is with no nanocircle vector. (B) Concentration dependence of down-regulation of CAT activity with marA vector.
Figure 8
Figure 8
(A) Sequences and predicted secondary structures of the monomer ribozymes: active and inactive marA, and short marA. The inactivating A → C mutation is boxed in the first ribozyme. (B) Effect of 10 μM various nanocircle vectors on the inhibition of CAT activity. The plotted data were averaged from three independent experiments.

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