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. 2023 Jan 16;51(4):e24. doi: 10.1093/nar/gkac1252

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — circRNA-based gene circuits that activate translation by detecting miRNA. (A) Schematic illustration of miRNA-responsive ON circuit. MS2CP or U1A is encoded on the first miRNA-responsive circRNA switch. The reporter gene (EGFP or MetLuc2) is encoded on the second protein-responsive circRNA switch. (B) Fluorescent images, scatter plots of a flow cytometer, and titration results of ON circuits with miR-302a-5p mimic in HEK293FT cells. The scale bar at the fluorescent images indicates 200 μm. 0.3 pmol of RBP (MS2CP or U1A)-coding mRNA, reporter mRNA and transfection control mRNA were transfected. 0.25, 0.5 or 1 pmol of hsa-miR-302a-5p mimic was co-transfected for evaluation. The plots shown are representative data from three biological replicates. The vertical axis of the scatter plot shows the fluorescence intensity of EGFP, and the horizontal axis shows the fluorescence intensity of iRFP670. (C) Evaluation of circRNA circuit persistence with a miR-302a-5p mimic in HEK293FT cells. 45 fmol of RBP (MS2CP or U1A)-coding mRNA and reporter mRNA were transfected. 1 pmol of hsa-miR-302a-5p mimic was co-transfected to activate the circuits. (D) Evaluation of circRNA circuit persistence with endogenous miR-21–5p in A549 cells. 45 fmol of RBP (MS2CP or U1A)-coding mRNA and reporter mRNA were transfected. Levels of significance are denoted as *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired Student's or Welch's t-test determined by F-test). N.S. means non-significant (P >0.05). All data in this figure are presented as mean ± SD, n = 3.