Sequential mutagenesis of multiple miR-29a target sites within the 3’ UTR of CASP7 by means of SMAP.

<p>Section A: Full length 3’ UTR of CASP7 was generated harbouring both identified target sites of miR-29a. Section B: For individual but also combined target site mutagenesis, the 3’ UTR was theoretically divided in three segments defined by locations of target sites. Sections C-E: Modular assembly of amplicons resulted in sequential mutagenesis of target sites. Section F: Gel image shows five amplicons after mutagenesis as well as assembled products for reporter gene fusion. NC represents the no template control of the assembly PCR. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-29a mimic compared with a nonsense miRNA control together with the mutated target site 1 (pTKGhCASP7m1), mutated target site 2 (pTKGhCASP7m2), mutated target sites 1 and 2 (pTKGhCASP7m1+2) as well as the wild type control (pTKGhCASP7) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P < 0.05; ***: P < 0.001, paired t test).