FRET assays for RNase L activity

RNase L activity was determined using fluorescence resonance energy transfer (FRET) assays as described previously [12, 44]. In Fig 7F, the recombinant murine RNase L (100 nM) was pre-incubated with purified L* (10 μM) for 30 min on ice followed by addition of increasing concentration of p3(2–5)A3. In Fig 7G, mouse RNase L was pre-incubated with varying concentration of L* followed by addition of 3nM of p3(2–5)A3. Alternatively, RNase L was pre-incubated with 3 nM p3(2–5)A3 followed by addition of varying concentration of L*. RNase L activity was monitored with cleavage of dual labeled probe [12, 44]. Best-fit curves and associated kinetic parameters were established using a nonlinear regression model with GraphPad Prism software.