Gel strand displacement assay
Gel strand displacement assays were adapted from Doestch et al. (68), with Mn in the buffer instead of Mg (69,70). Strand displacement assays utilize M1, J1 and uM1 RNAs, as described above. RNA strand displacement reactions were performed in 1× MnCl2 FRET buffer (50 mM Tris-HCl, pH 7.5, 1 mM MnCl2, 10 mM NaCl, 1 mM DTT) at 37°C. Two controls were included with each strand displacement assay. Control 1 (C1) is purified J1M1 dsRNA (30 nM purified M1J1, 1× MnCl2 FRET Buffer) and control 2 (C2) is J1uM1 dsRNA (30 nM purified M1J1, 0.3 µM uM1, 1× FRET MnCl2 Buffer). C2 dsRNA J1uM1 was formed by heating purified dsRNA J1M1 and uM1 at 95°C for 2 min and slowly cooling to room temperature. Strand displacement master mix was prepared with 30 nM purified dsRNA in 1× MnCl2 FRET buffer. A zero time point was removed from the master mix, added to 0.3 µM uM1 and 2.5 µl of 5× stop/load buffer, and immediately loaded into a running (100 V) 20% native polyacrylamide gel (20% TBE, 12 well, Novex precast gel). Strand displacement reactions were started by adding uM1 RNA to the strand displacement master mix at 37°C. Time points were withdrawn from the reaction, stopped with 2.5 µl of 5× stop/load buffer. Gels were run at 100 V at 4°C for 3 h. Gels were visualized using Cy3 and Cy5 channels on an Azure Sapphire using the SmartScan feature and overlayed for gel images. Quantification of band intensity was measured using the Cy5 channel with the rectangle and gel plot function in ImageJ. The amount of strand displacement in each lane was quantified by plotting the amount of J1M1 (Cy5) dsRNA divided by the total RNA (J1M1, J1uM1 and M1; Cy5), which was normalized to the amount of dsRNA (J1M1; Cy5) at time point zero and graphed using GraphPad Prism with second order polynomial regression analysis. Differences between the assay endpoints (at 15 min) for each strand displacement reaction were determined using Welch’s t -test (GraphPad Prism).