4.3. RNA-PPRH Binding Analyses

The capacity of the modified PPRHs to bind to their target sequence in the survivin promoter was analyzed using electrophoretic mobility shift assays (EMSA). The dsDNA probe corresponding to the target sequence of survivin was obtained by mixing equal amounts of each single-stranded oligodeoxynucleotide in a 150 mM NaCl solution (Forward strand, 5′-[FAM]CTGCACTCCATCCCTCCCCT-3′; Reverse strand, 5′-AGGGGAGGGATGGAGTGCAG-3′). The forward strand was labeled with FAM (6-Carboxyfluorescein) at its 5′-end. The solution was incubated at 90 °C for 5 min and then allowed to cool down slowly to room temperature (about 1 h). The duplex was resolved in a nondenaturing 20% polyacrylamide gel, visualized using UV shadowing (254 nm), and gel-purified. DNA concentration was determined by measuring its absorbance at 260 nm using a NanoDrop ND-1000 spectrophotometer (ThermoFisher, Barcelona, Spain).

The binding of PPRHs to their target sequence was analyzed using two approaches: (I) by incubation of the PPRHs with a ssDNA probe or (II) by incubation of the PPRH with a dsDNA probe. The ssDNA or the dsDNA probes were incubated with the different modified PPRHs in 20 µL reaction mixtures. In both cases, a buffer containing 10 mM MgCl₂, 100 mM NaCl, 5% glycerol, 20 units of RNAse inhibitor, and 50 mM HEPES, pH 7.2 in H₂O DEPC was used (Merck, Madrid, Spain). For ssDNA binding reactions, tRNA was added as an unspecific competitor, while for dsDNA binding reactions, Poly(dI:dC) was used (1:2 ratio for both cases, ng probe: ng unspecific competitor). ssDNA binding reactions were incubated for 30 min at 37 °C, whereas dsDNA binding reactions were incubated for 30 min at 65 °C. HpSC6 was used as a negative control in both cases. The products of the binding reactions were resolved by electrophoresis in nondenaturing 8% polyacrylamide gels (PAGE) containing 10 mM MgCl₂, 5% glycerol, and 50 mM HEPES, pH 7.2 (Merck, Madrid, Spain) at a fixed voltage of 220 V and 4 °C. The ImageQuant software v5.2 (GE Healthcare, Barcelona, Spain) was used to visualize the results.