Figure 2. Molecular titration model for miRNA-mediated regulation can be exploited for UTA fluorescent reporters.

(A) The titration model adapted from (ref. ²⁵ and methods section) describes the steady-state levels of free mRNA (r) and miRNA associated mRNA (r*). The steady state solution for r contains two parameters that define the shape of the function: λ and θ, λ proportional to the effective dissociation constant of miRNA-mRNA (k_off) and inverse-proportional to the on-rate (k_on) constant of miRNA-mRNA complex formation while θ is proportional to miRNA concentration. (B) Several solutions were simulated using custom R scripts for r as a function of r₀. Increasing values of θ (left) and λ (right) were utilized for simulations. Only values within the experimentally determined range of YFP intensities were used for r₀. Control (non-human targeted) siRNA GL-2 reproduced model predictions; three different GL2 target UTA reporters were used to evaluate λ, i.e. bulged with 19 matching complementary base pairs, and 3 unpaired bases (Triangles), one 21 base perfectly complementary target (circles), and three copies of 21 exact complementary bases separated by 4 unpaired bases (diamonds). To mimic effector expression differences (θ) synthetic Gl-2 siRNA was co-transfected at different concentration (C) 20 nM (D) 1.5 nM and (E) 0.5 nM. The transfer functions for cells transfected with only the dual reporter (negative control) are depicted in black. (F) Simulation of data-derived parameters describes three functional miRNA classes. The GL-2 (siRNA) experimental transfer functions were fitted using non-linear regression methods to define θ and λ range; steady state solutions for nine combinations of θ (Colors) and 1/λ (lines) are depicted.