Efficient Bacterial Transcription of DNA Nanocircle Vectors with Optimized Single-Stranded Promoters Tarsuo Ohmichi; Angéle Maki; Eric T. Kool A modular and extensible RNA-based gene- regulatory platform for engineering cellular function Maung Nyan Win and Christina D. Smolke

Goals: Investigate whether there are specific sequences and structures of single-stranded DNA that will act as promoters for a bacterial RNA polymerase Find a motif that is formed in only 40 nucleotides of contiguous sequence that can be transplanted from one context to the next and still direct efficient transcription by the bacterial RNA polymerase. Create a promoter that can be used in a DNA nanocircle vector to direct transcription of an active hammerhead ribozyme in E. coli cells

A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Since the discovery of ribozymes that exist in living organisms, there has been interest in the study of new synthetic ribozymes made in the laboratory. For example, artificially-produced self-cleaving RNAs that have good enzymatic activity have been produced. This approach takes advantage of RNA's dual nature as both a catalyst and an informational polymer, making it easy for an investigator to produce vast populations of RNA catalysts using polymerase enzymes. The ribozymes are mutated by reverse transcibing them with reverse transcriptase into various cDNA and amplified with mutagenic PCR.

Fig. 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.

Copyright ©2002 by the National Academy of Sciences Ohmichi, Tatsuo et al. (2002) Proc. Natl. Acad. Sci. USA 99, Fig. 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 2

Copyright ©2002 by the National Academy of Sciences Ohmichi, Tatsuo et al. (2002) Proc. Natl. Acad. Sci. USA 99, Figure 3 Fig. 3. Sequences of clones obtained following the fifteenth round of in vitro selection. Boxes indicate regions of high sequence similarity.

Copyright ©2002 by the National Academy of Sciences Ohmichi, Tatsuo et al. (2002) Proc. Natl. Acad. Sci. USA 99, Fig. 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 ().

Copyright ©2002 by the National Academy of Sciences Ohmichi, Tatsuo et al. (2002) Proc. Natl. Acad. Sci. USA 99, Fig. 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.

Copyright ©2002 by the National Academy of Sciences Ohmichi, Tatsuo et al. (2002) Proc. Natl. Acad. Sci. USA 99, Figure 6 Fig. 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.

Supplementary Material Figure 10

Copyright ©2002 by the National Academy of Sciences Ohmichi, Tatsuo et al. (2002) Proc. Natl. Acad. Sci. USA 99, Fig. 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.

Copyright ©2002 by the National Academy of Sciences Ohmichi, Tatsuo et al. (2002) Proc. Natl. Acad. Sci. USA 99, Figure 8 Fig. 8. (A) Sequences and predicted secondary structures of the monomer ribozymes: active and inactive marA, and short marA. The inactivating A to 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.