Fig. 3.

Ratiometric sensors for microRNA activity. (A) A bidirectional promoter (Bi-CMV) simultaneously drives expression of AcGFP whose 3′ UTR contains three MREs. DsRedEx1 is expressed in the antisense direction as an internal control. Lentiviral packaging elements include psi and self-inactivating (SIN) LTRs. (B) SH-SY5Y neuroblastoma cells expressing the 132-MRE sensor were transfected with increasing amounts (up to 10 nM total) of miR-132 mimic and a miR-Scrm mimic (pink = 0 nM; cyan = 0.1 nM; orange = 1.0 nM; green = 10 nM). The G/R ratio in 1 × 10⁴ individual cells per condition was assessed using flow cytometry and graphed as a cumulative frequency distribution. Black and blue lines represent the response of the negative control No-MRE sensor to the same concentrations of miR-132 mimic. (C) The 132-MRE sensor responded to the exogenous (0.1 nM) miR-132 mimic (blue) but not to the highly related miR-212 mimic (red). (D) 132-MRE and 212-MRE sensors were expressed in SH-SY5Y cells under growth (white bars) and differentiation (black bar) conditions (1% serum + 10 ng/mL retinoic acid for 2 d). The G/R ratios were normalized to that of the Scrm-MRE negative control. (E) Addition of 2′O-methyl inhibitors specific to miR-132 confirmed that the 132-MRE sensor detected endogenous miR-132 in SH-SY5Y cells. (F) The same concentrations of 2′O-methyl inhibitors did not affect expression of the No-MRE control vector.