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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Methods. 2021 May 13;196:104–112. doi: 10.1016/j.ymeth.2021.05.009

Figure 4:

Figure 4:

(A) Multiple transcripts were designed with the aim of validating trans RNA ligation into circular Broccoli in cells. Transcripts containing full-length Broccoli and only the stem1 or stem2 ligation site (transcript 1 and 2, respectively) are efficiently circularized. Transcripts 3, 4, 5, and 6 contain Broccoli A (transcripts 3 and 5) or Broccoli B (transcripts 4 and 6) and do not form fluorescent species on their own. Co-transfection of constructs containing Broccoli A and Broccoli B transcripts leads to a circular Broccoli signal only when the stem1 and stem2 ligation sequences match after ribozyme cleavage, according to Figure 3 (transcript 3 matches with 4 and transcript 5 matches with 6). (B) Live cell fluorescence of circular Broccoli expression or trans ligated circular Broccoli. Circular Broccoli is detected in HEK293T cells expressing either circular Broccoli control (both transcript 1 and 2). Trans ligated circular Broccoli is detected in cells co-transfected for expression of transcript 5 and 6. Brightness levels of these images have been adjusted to improve visibility of fluorescence.