Figure 7. Extended Data Figure 7.

Plasmid-encoded cis-active ribozyme. a. Schematic depiction of F30-Broccoli-cis (FBC)-MTR1 construct. b. Dot plot for FBC-MTR1 generated by Vienna RNAfold (http://rna.tbi.univie.ac.at/), indicates high probability of folding into the designed structure. c. LC-MS analysis of SVPD/BAP-digested FBC-MTR1 in vitro transcript after reaction with m⁶G. Extracted ion chromatogram (EIC) for detection of MH⁺ 282.11±0.05 (methylated adenosines) shows production of m¹A, and m⁶A to a small extent (due to partial Dimroth rearrangement during digestion). Bottom trace for synthetic references m¹A and m⁶A (50 nM each), is same as shown in Fig 3d. d. Primer extension stop assays also confirm activity of FBC-MTR1 transcribed in vitro and in vivo, in the presence of total E.coli RNA. Left: full gel image shown in Fig. 4a for in vitro transcribed FBC-MTR1. Right: primer extension on total E.coli RNA, isolated 1 h after IPTG induction, and incubated with indicated m⁶G or BG concentration in vitro. These experiments were independently repeated two times with similar results.