FIGURE 5.
Surface plasmon resonance of hIRE1α-cyto, substrate, and compound binding characteristics. Schematic of the XBP-1 RNA stem-loop with an extended stem used for surface plasmon resonance binding experiments is shown (A). Surface plasmon resonance binding profiles with the immobilized reagent on the solid chip surface are indicated at the bottom and the soluble binding partner indicated at the top (A–F). Active hIRE1α-cyto was linked to the Biacore chip by amine coupling and demonstrated specific and dose-dependent binding to the substrate confirming that hIRE1α-cyto was active on the chip (B). When compound 3-ethoxy-5,6-dibromosalicylaldehyde was passed over hIRE1α-cyto on the chip, a specific dose-dependent binding was observed with fast on-fast off kinetics (C). The disassociation constant (Kd) was calculated to be ≈100 nm. Biotinylated stem-loop RNA was immobilized to a streptavidin-coated chip and a large mass change was observed when hIRE1α-cyto was passed over (D). Repeated exposure to soluble hIRE1α-cyto degraded the signal likely due to site-specific cleavage of the stem-loop RNA on the solid surface, therefore, only a single concentration is shown. Passage of 3-ethoxy-5,6-dibromosalicylaldehyde over a chip immobilized with the XBP-1 stem-loop RNA did not give rise to a detectable binding signal (E). When RNase A was coupled to the chip and compound 3-ethoxy-5,6-dibromosalicylaldehyde was passed over the surface, no detectable binding was observed (F).