Fig. 1 | Nature Communications

Fig. 1

From: A mixed antagonistic/synergistic miRNA repression model enables accurate predictions of multi-input miRNA sensor activity

Fig. 1

A biochemical model explains miRNA repression measured using miRNA sensors. a A schematic of the single-input miRNA low sensors used in this study. b Fluorescence microscopy in HEK293FT cells for a control sensor with miRNA target sites with no activity (FF4) compared to a sensor with target sites for a high activity miRNA (miR-106a-3p). Both images are merged blue/red channels and the scale bar indicates 400 μm. c Corresponding flow cytometry data in HEK293FT cells for no activity (red) and high activity (purple) sensors. Data for a positive control using exogenous siRNA to repress mKate2 is shown for reference (blue). Light scatter points correspond to data; dark points represent median values for data binned by EBFP2 fluorescence; lines indicate fits to the data using a biochemical model; dashed line shows modeling results with absolutely zero miRNA activity. Fluorescence is shown in terms of arbitrary units and with logicle scaling for the axes46. d The single-input repression model annotated with relevant species and rate constants. Each cell is transfected with some number of plasmids (N) which are transcribed (at rate ktrs) and translated (ktln,EBFP2 and ktln,mKate2) to yield fluorescent proteins. mKate2 transcripts are reversibly bound by a miRNA-containing complex (miRfree) forming a bound species (mmKate2,miR) which is catalytically degraded at some rate (kcat). Two parameters, effective miRNA concentration (M) and Michaelis constant (Km) fully characterize miRNA activity. RNA and protein species are non-specifically degraded at some rate. Fixed rate constants are approximated from literature values (Supplementary Table 3). e Model predictions for decreasing Km while holding M constant. As Km decreases, repression increases while switching threshold is maintained. f Experimental perturbation of Km. HEK293FT cells, which exhibit low endogenous miR-21-5p activity, were transfected with sensors containing varying numbers of target sites for miR-21-5p and varying amounts of exogenous miR-21-5p mimic. As the number of target sites was increased (decreasing Km), repression increased as predicted. g Model predictions for increasing M while holding Km constant. As M increases, the switching threshold increases and repression increases as a result. h Experimental perturbation of M. As miR-21-5p mimic concentration is increased, repression increased as predicted

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