Detection of the engineered circular riboregulator in vitro
. (a) Sequence scheme of the riboregulator encased in the PIE ribozyme, which produces a circular RNA molecule after self-splicing. Left: diagram of RT over a circular RNA template, showing the creation of a cDNA with multiple copies of the riboregulator due to a rolling cycle, until the RNA is cleaved by the RNase H subunit of the transcriptase. Right: diagram of RT over a linear RNA template, showing the creation of a cDNA with a single copy of the riboregulator. The RT is initiated with primer circRT-Rv (labelled B). (b) Diagram of PCR over the nascent cDNA with appropriate primers. On the one side, PCR with divergent primers (circRT-Fw in red, labelled A, and circRT-Rv in black) only yields a product if the RNA template is circular. On the other hand, PCR with convergent primers (circRT-Lin in blue, labelled C, and circRT-Rv in black) only yields a product if the RNA template includes intron 2 (i.e., if the self-splicing has not occurred, and then, the RNA template is linear). Note that primers A and B hybridize in the riboregulator region, whilst primer C does in the intron 2 region. (c) Agarose gel of RT-PCR with primers described above of total RNA extracted from E. coli
cells expressing circRAJ31 or the C873U mutant. The first lane corresponds to Invitrogen 100 bp DNA ladder. Lanes labelled 1 and 2 correspond to duplicates of the RT reaction. The last lane corresponds to PCR without doing RT (control). Top: there is a product in the case of both circRAJ31 and C873U mutant with primers B and C, indicating that there is always a fraction of noncircularized RNA. Bottom: there is a clear product in the case of circRAJ31 with primers A and B, indicating that circularization has taken place. In the case of the C873U mutant, the product is difficult to see. (d) Sequencing chromatogram of the RT-PCR product (with primer B) from circRAJ31 produced in vivo
. This shows the alignment between the theoretical circular RNA sequence and the actual sequence. The sequencing result covering exons 1 and 2 shows that the splicing and ligation happen at the expected site. See also the gel and sequencing result of RT-PCR from RNA produced in vitro
with T7 polymerase in Figure S3
. (e) Urea gel of total RNA extracted from E. coli
cells expressing circRAJ31. The first lane corresponds to Thermo Sci. RiboRuler low-range 100 bp RNA ladder. Other lanes correspond to extractions in triplicate from different colonies. A lane marked with the asterisk was used for subsequent 2D analysis. (f) On the top, 2D urea-PAGE (here, 1D: 5%, 2D: 7.5% polyacrylamide) of total RNA extracted from E. coli
cells expressing circRAJ31. On the bottom, radioactive hybridization with a probe targeting circRAJ31. Linear RNAs show along a diagonal in 2D, whilst circular RNAs show off this diagonal.