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. 2023 Jun 16;51(15):8102–8114. doi: 10.1093/nar/gkad518

Figure 3.

Figure 3.

RIG-I binds to metabolite-capped RNAs comparably to 5′ PPP RNA. (A) Bar chart comparing KD, app of RIG-I for various dsRNAs, measured using the RNA-dependent ATP hydrolysis assay. 15 nM of RIG-I was incubated with 2 mM ATP spiked with [γ-32P]-ATP, 1× ATPase Buffer, and increasing concentrations of either 5′ PPP ds39 RNA, 5′ NAD+ ds39 RNA, 5′ dephospho-CoA ds39 RNA, 5′ FAD ds39 RNA, or 5′ OH 5′ 2nt overhang RNA. The rate of ATP hydrolysis for each RNA concentration was measured as a time course at 0’, 20’, 40’, 60’ (n = 3) and fit with a linear equation. The average ATP hydrolysis rate was plotted as a function of RNA concentration and fit using a quadratic equation (Equation 1) to estimate the KD,app. The error bars are derived from the error of fit. Because of the high RNA concentrations relative to RIG-I, very likely RIG-I is bound monomerically, as the cartoon shows. (B) Bar chart comparing KD of RIG-I for various dsRNAs, measured using fluorescence polarization assays. 20 nM of RNA (either 5′ PPP ds39 RNA, 5′ NAD+ ds39 RNA, 5′ dephospho-CoA ds39 RNA, 5′ FAD ds39 RNA, or 5′ OH 5′ 2nt overhang RNA) and 0.5 mM of ATP were incubated with increasing concentrations of RIG-I and fluorescence polarization was measured. Each measured point is an average of 3 trials (n = 3). Each reaction was fit using a hyperbolic equation (Equations 2 and 3), and both KD and error are derived from this fit. Because of high RIG-I concentrations relative to RNA, multiple RIG-I molecules are likely bound to RNA under this assay condition. Thus, the measured KD by this assay is a composite one. (C) Electrophoretic mobility shift assay (EMSA) of RIG-I (0 nM in minus lanes, 75 nM in plus lanes) incubated with 25 nM of either 5′ PPP, 5′ alternative cap, or 5′ OVG 5′ OH RNA dsRNAs and 2 mM ATP. The sulfhydryl group of 5′ dephosphoCoA can form a disulfide linkage with another 5′ dephosphoCoA (note the higher band in the RNA alone lane). Monomer, dimer, and trimer RIG-I are denoted on the right of the gel, with cartoons. (D) Fluorescence polarization RNA competition assay confirming that RIG-I does not bind to single-stranded 5′PPP or 5′ NAD+ RNAs. A pre-mixed 50 nM RIG-I and 20 nM fluorescent 5′PPP dsRNA was added into serially diluted unlabeled ssRNA or 5′PPP dsRNA at 25°C in the presence of 500 uM ATP. The dashed line indicates the polarization value of free RNA.