Fig. 3: Engineering miRNA-responsive IRES translation switches to implement logic gates and cell-type classifiers in mammalian cells.

a–f Implementing logic gates by programming MITA/MITR design. The IRES-mediated tagBFP fluorescence intensity was measured in HEK293 cells with miRNA mimics or control mimics co-transfection. a Schematic illustration of OR logic gate implemented by ligation of two TL16 modules for miR-199a and miR-FF4. b Tandem ligation of two different TL16 modules exhibited OR-logic computation function. c Schematic illustration of AND logic gate implemented by programming SL3 design for miR-199a and miR-FF4. d Integration of two different miRBS into SL3 design exhibited AND-logic computation function. e Schematic illustration of NOT logic gate (i.e., MITR) implemented by inserting miRBS fully complementary to miR-FF4. f Insertion of miRBS fully complementary to the input miRNA exhibited NOT-logic computation function. g Construction and optimization of miR-21 responsive MITAs design by variating the number of base-pairing or mismatches in designed structures. TL, SL, and AJ were all designed to sense endogenous miR-21. IRES-mediated tagBFP fluorescence intensity was measured in Huh7 (miR-21 high) and HEK293 (miR-21 low) cells. h Construction and optimization of miR-18a responsive MITAs design by variating the number of base-pairing or mismatches in designed structures. TL, SL, and AJ were all designed to sense endogenous miR-18a. IRES-mediated tagBFP fluorescence intensity was measured in Huh7 (miR-18a low) and HEK293 (miR-18a high) cells. i Schematic illustration of the construction of cell-type classifiers based on the PROMITAR platform. The mKate fluorescence protein was co-transfected as an internal control. j The MITA and MITR designs showed cooperating effects regarding cell-type classification. HEK293 and Huh7 cell lines were used for the transfection experiments. Data are presented as mean values with error bars representing the standard deviation of three independent biological replicates (n = 3 in each group). Statistical analysis of the results was performed by a two-tailed unpaired Welch’s t-test, assuming unequal variances. *p < 0.05. **p < 0.005 (p = 0.0029 in Fig. 3f). miR, microRNA. pCMV, CMV promoter. phEF-1a, EF-1a promoter. Schematic illustration figures were created with BioRender.com with publication licenses. Source data are provided as a Source Data file.