Journal: Nature Communications

Article Title: Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression

doi: 10.1038/s41467-025-60392-2

Figure Lengend Snippet: a Schematic illustration of miRNA-responsive RNA OFF and ON switches coding the gene of interest (GOI). The OFF switch (left) inhibits translation of the protein of interest (POI) in the presence of miRNA activity, while the ON switch (right) promotes translation under the same conditions. b Schematic illustration of trans- protein splicing. In this reaction, split-inteins linked to their flanking peptides (exteins) ligate post-translationally, excising themselves and seamlessly joining the exteins to generate a mature protein. c Schematic illustration of “split ON switch,” a pair of ON switches coding protein fragments conjugated with split-inteins.

Article Snippet: MiRNA mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs. mirVana miRNA Mimics (hsa-miR-21-5p, hsa-miR-302a-5p, and Negative Control #1) (Thermo Fisher Scientific, MC10206, MC12557, and 4464058) were used as mimic molecules in HEK293FT cells.

Techniques: Activity Assay

Journal: Nature Communications

Article Title: Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression

doi: 10.1038/s41467-025-60392-2

Figure Lengend Snippet: a Schematic illustration of the strategy to improve the ON/OFF ratio of ON switch systems. Introducing a “leak-canceller”, an OFF switch coding an inactive C-terminal fragment, together with the split ON switch, enables the suppression of leaky protein activity in miRNA-cells and enhances the ON/OFF ratio. b Relative hmAG1 intensity (hmAG1/iRFP670) of HEK293FT cells treated with miR-21-5p or negative control (NC) mimics. a.u., arbitrary units. Error bars represent means ± SD ( n = 3), and data of each biological replicate are shown as a point. Statistical analysis by two-sided Welch’s t test, ** P < 0.01. Each P -value is listed in Supplementary Table . Source data are provided as a Source Data file. c Representative microscopic images of HEK293FT cells transfected in the same conditions as shown in ( b ). Green fluorescence (left) and bright-field (right) images are shown for each condition. Scale bar, 200 µm. d Representative 2D flow cytometry plots. The horizontal axis shows the fluorescence intensity of iRFP670 (reference), and the vertical axis shows the fluorescence intensity of hmAG1 (reporter).

Article Snippet: MiRNA mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs. mirVana miRNA Mimics (hsa-miR-21-5p, hsa-miR-302a-5p, and Negative Control #1) (Thermo Fisher Scientific, MC10206, MC12557, and 4464058) were used as mimic molecules in HEK293FT cells.

Techniques: Activity Assay, Negative Control, Transfection, Fluorescence, Flow Cytometry

Journal: Nature Communications

Article Title: Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression

doi: 10.1038/s41467-025-60392-2

Figure Lengend Snippet: a Schematic illustration of a toggle-like (two-output) system that displays hmAG1 fluorescence in the presence of target miRNA activity and iRFP670 fluorescence in its absence. “Normal toggle-like system” refers to a system composed of an ON switch coding full-length hmAG1 and an OFF switch coding full-length iRFP670, and “split toggle-like system” refers to a system composed of a pair of ON switches coding split hmAG1 fragments and a pair of OFF switches coding split iRFP670 fragments. b Normalised hmAG1 and iRFP670 fluorescence intensity of HEK293FT cells treated with various miR-21-5p mimic concentrations. Normalised hmAG1 intensity was calculated by normalising the intensity at 2 nM mimic concentration, and normalised iRFP670 intensity was calculated by normalising the intensity at 0 nM mimic concentration. Error bars represent means ± SD ( n = 3, biological replicate). Statistical analysis by two-sided Welch’s t test, * P < 0.05, ** P < 0.01. Each P -value is listed in Supplementary Table . Source data are provided as a Source Data file. c Representative 2D flow cytometry plots of HEK293FT cells treated with various miR-21-5p mimic concentrations. The horizontal axis shows the fluorescence intensity of iRFP670 (reference), and the vertical axis shows the fluorescence intensity of hmAG1. d Euclidean distance between the centroid of the 0 nM mimic concentration plot and those of various mimic concentrations in the logarithmic 2D plot in ( c ). Error bars represent means ± SD ( n = 3, biological replicate). Source data are provided as a Source Data file.

Article Snippet: MiRNA mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs. mirVana miRNA Mimics (hsa-miR-21-5p, hsa-miR-302a-5p, and Negative Control #1) (Thermo Fisher Scientific, MC10206, MC12557, and 4464058) were used as mimic molecules in HEK293FT cells.

Techniques: Fluorescence, Activity Assay, Concentration Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression

doi: 10.1038/s41467-025-60392-2

Figure Lengend Snippet: a Schematic illustration of a two-input logic gate using split-intein, exemplifying an AND gate, in which an output protein is activated only in the presence of both of two different target miRNAs. b Four types of two-input logic circuits. Different colours correspond to those in the truth table, representative 2D flow cytometry plots, and bar graphs, respectively. MiR-21-5p and miR-302a-5p mimics were used as inputs. For example, the input pattern [10] coloured in blue means miR-21-5p was present while miR-302a-5p was absent. The representative 2D flow cytometry plot for each circuit is shown as an overlay of scatter plots for all four input patterns. The relative fluorescence intensity of hmAG1 (hmAG1/iRFP670) was normalised by the highest value for each circuit ([00] in NOR,[10] in A AND NOT B,[01] in NOT A AND B, and [11] in AND). Error bars represent means ± SD ( n = 3), and data of each biological replicate are shown as a point. Statistical analysis by two-sided Dunnett’s test for the ON state of each circuit, ***** P < 0.00001. Each P -value is listed in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: MiRNA mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs. mirVana miRNA Mimics (hsa-miR-21-5p, hsa-miR-302a-5p, and Negative Control #1) (Thermo Fisher Scientific, MC10206, MC12557, and 4464058) were used as mimic molecules in HEK293FT cells.

Techniques: Flow Cytometry, Fluorescence

Journal: Nature Communications

Article Title: Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression

doi: 10.1038/s41467-025-60392-2

Figure Lengend Snippet: a Schematic illustration of a two-type-input logic gate using split-intein, exemplifying a protein/miRNA-resonsive NOR gate, in which an output protein is activated only in the absence of both target protein and miRNA. b Two types of two-type-input logic circuits. L7Ae/miR-21-NOR gate exhibits hmAG1 fluorescence only in the absence of both L7Ae and miR-21-5p (upper). LIN28A/miR-302a-NOR gate exhibits hmAG1 fluorescence only in the absence of both LIN28A and miR-302a-5p (lower). Colours correspond to those in the bar graphs and representative 2D flow cytometry plots, respectively. Error bars represent means ± SD ( n = 3), and data of each biological replicate are shown as a point. Statistical analysis by two-sided Dunnett’s test for the ON state of each circuit, ***** P < 0.00001. Each P -value is listed in Supplementary Table . Source data are provided as a Source Data file. c Comparison of fold changes in cell type-dependent protein activity regulation among miR-302a-responsive OFF switch, LIN28A-responsive OFF switch, and LIN28A/miR-302a NOR gate. The fold changes in relative hmAG1 intensity were calculated as hmAG1/iRFP670 of HEK293FT cells divided by that of hiPSC. Error bars represent means ± SD ( n = 3), and data of each biological replicate are shown as a point. Statistical analysis by two-sided Welch’s t test, * P < 0.05, ** P < 0.01. Each P -value is listed in Supplementary Table . Source data are provided as a Source Data file. d Schematic illustration of a three-input logic gate using two orthogonal split-intein, exemplifying a three-input NOR gate, in which an output protein is activated only in the absence of all target miRNAs, and a three-input AND gate, in which an output protein is activated only in the presence of all target miRNAs. e Validation of miR-21/302a/206 NOR gate (left) and miR-21/302a/206 AND gate (right) in HEK293FT cell treated with different combinations of miR-21-5p, miR-302-5p, miR-206 mimics. Error bars represent means ± SD ( n = 3), and data of each biological replicate are shown as a point. Statistical analysis by two-sided Dunnett’s test for the ON state of each circuit, ***** P < 0.00001. Each P -value is listed in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: MiRNA mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs. mirVana miRNA Mimics (hsa-miR-21-5p, hsa-miR-302a-5p, and Negative Control #1) (Thermo Fisher Scientific, MC10206, MC12557, and 4464058) were used as mimic molecules in HEK293FT cells.

Techniques: Fluorescence, Flow Cytometry, Comparison, Activity Assay, Biomarker Discovery