Figure 3.

Compensatory mutagenesis of the H-H kissing loop interaction in the RNase L ciRNA. (A) Diagram of RNA FRET probe. (B) Illustration of FRET in RNase L activity assays. (C) Three complementary bases in the loop of stem-loop 1 and stem-loop 4 of the ciRNA were changed to their complementary sequences, individually and simultaneously. Mutations highlighted in grey. (D) RNase L activity measured by FRET assay, as previously established.⁶ Reactions containing 20 nM RNase L, 20 nM 2–5A, 100 nM FRET probe and 100 nM of the indicated viral RNAs; Poliovirus ciRNA (yellow line), loop 1 mutant ciRNA (L1M, purple line), loop 4 mutant ciRNA (L4M, brown line), PV ciRNA containing the compensatory loop 1 and loop 4 mutations (CM, azure line). A negative control reaction incubated without RNase L (probe alone, grey line). A positive control reaction containing FRET probe, RNase L and 2–5A without poliovirus ciRNA (probe + RNase L + 2–5A, black line). A control reaction containing 100 nM of an alternate fragment of the poliovirus ORF (2123, pale blue line, poliovirus nucleotides 5889–6181). RFU plotted versus time of incubation.