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master mix at 37°C. Time points were withdrawn from the reaction,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).