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. 2021 Jan 28;11:2462. doi: 10.1038/s41598-021-81679-6

Figure 1.

Figure 1

Scheme of CYP1A2 enzyme activation by theophylline using a theophylline-responsive miRNA switch. An RNA construct with a miRNA containing a theophylline-responsive structure-switching aptamer in the 3′ untranslated region (UTR) of CYP1A2 is depicted in the nucleus of a HEK-293 cell. While the exact mechanism of miRNA processing is unresolved, we assume the primary miRNA is processed by Drosha resulting in the precursor miRNA. Dicer cleavage produces a double-stranded mature miRNA in the cytoplasm35,36. One strand is incorporated into the RNA-induced silencing complex (RISC) and regulates CYP1A2 messenger RNA translation36,37. CYP1A2 enzymatic activity is indirectly measured by the detection of Luciferin, after conversion from externally supplied Luciferin-1A2 representing an easy read-out system. Luciferin-1A2 is converted by CYP1A2 into a luciferin precursor. The luciferin precursor is rapidly converted into luciferin following the addition of D-Cysteine, which is required for the reaction to occur and supplemented in the detection reagent. (a) In the absence of theophylline, CYP1A2 gene silencing occurs via RISC activation and CYP1A2-derived luminescence level is low. (b) In the presence of theophylline, the aptamer is bound. The secondary structure of the miRNA switch is changed, which masks the primary miRNA from processing23. CYP1A2 enzyme activity increases the luminescence levels. If α-naphthoflavone is added, it can selectively inhibit CYP1A2 activity, serving as a control. The mature miRNA sequence (guide strand) is shown in dark green and miRNA* (sense strand) in light green. The theophylline aptamer sequence is shown in light blue, and the corresponding bulge area highlighted by blue, theophylline is depicted in orange in the aptamer region of the theophylline-bound primary miRNA. The 5′ cap is displayed as red circle and AAAA(n) indicates the poly (A) signal of the mature messenger RNA. CYP1A2 enzyme translation status controlled by the miRNA switch is additionally depicted as ‘Enzyme OFF’ (red switch) and ‘Enzyme ON’ (green switch).