Description
A fast and easy way to identify new binders to human RIG-I. The Retinoic acid-Inducible Gene I (RIG-I) is a cytosolic pattern recognition receptor which is known to recognize RNA pathogens in cells. This protein is involved in the innate immune system and induces a type 1 interferon response. The RIG-I protein recognizes infected cells by detecting viral dsRNA. Once activated by a dsRNA, the N-terminus domain of RIG-I binds mitochondrial antiviral signaling proteins, thereby activating the IFN-1 signaling pathway.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
The HTRF RIG-I binding assay is a competitive assay format which uses a biotinylated 3 phosphate-dsRNA, a streptavidin-d2, a 6His tagged human RIG-I protein, and an anti 6His Cryptate-labeled antibody. The compound being tested competes with the biotinylated 3p-dsRNA, and thereby prevents FRET from occurring.
Assay protocol
The Human RIG-I binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, samples or standards are dispensed directly into the assay plate. The human His-tagged RIG-I protein is then added, followed by the dispensing of the HTRF reagents: The anti 6His antibody labeled with Terbium cryptate and the biotinylated-3p-dsRNA bound to d2-labeled streptavidin. The reagents labeled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.
Effect of 5' triphosphate vs blunt-end dsRNA for RIG-I binding
Two different ds-RNAs were tested in parallel in the HTRF RIG-I binding assay. The first ds-RNA displays a triphosphate in 5', the second does not. Serial dilutions of both RNAs were carried out in diluent, and then RIG-I protein was dispensed. HTRF detection reagents were then added to the well for the final detection step. Results were collected after 1 hour of incubation at room temperature. The affinity of the RIG-I protein was increased by the presence of the triphosphate in 5' of the dsRNA, as expected.
