Figure 1.

Firefly luciferase reporter systems for HTS. (A) “Perfect” and “bulged” binding sites for let-7a and miR-30c miRNAs. A “perfect” site yields perfect base-pairing. If miRNA is bound to AGO2, this type of binding will yield endonucleolytic cleavage in the middle of the base-paired sequence. A “bulged” site is a typical natural miRNA binding site. In this case, silencing of a cognate mRNA requires AGO and associated proteins. Mutated sites carry mutations that should prevent miRNA-mediated repression. (B) Schematic structure of let-7 and miR-30 firefly luciferase reporters used for HTS. pA, polyadenylation signal. (C) Reporter activities upon transient co-transfection with plasmids expressing firefly luciferase without specific miRNA binding sites. The experiment was performed three times, data were normalized to co-transfected non-targeted Renilla luciferase, and the control (firefly luciferase reporter without any miRNA binding sites) was set to one. Except for the control and 1xP miR-30 reporters, which utilized the SV40 promoter and 3′ UTR, all other reporters were driven by the PGK promoter and had BGH 3′ UTR. Error bar = standard deviation (SD). (D) Firefly activity test of specific HeLa and 3T3 clones stably expressing PGK-FF-let-7-3xP reporter upon let-7 repression with let-7 LNA inhibitors (50 nM). Clones 13 and A33 were selected for HTS. (E) Firefly activity test of specific HeLa cells stably transfected with PGK-FF-let-7-4xB or PGK-FF-miR-30-4xB reporters upon miRNA repression with LNA inhibitors (50 nM). Firefly luciferase activities are displayed as relative luciferase units (RLU) recorded by the luminometer.