Arg2 tips

BLI measurements were performed on the Octet system (ForteBio). Briefly, amine reactive group 2 (ARG2) tips (ForteBio) were pre-soaked in buffer FPO (PBS + 12.5 mM MgCl₂) before activation with equal parts 20 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) for 5 min, followed by a 5 min incubation with buffer FPO, purified fluorescent proteins or vesicles, quenching in 1 M Tris pH 7.4 for 5 min, and washing with buffer FPO. Example tip loading curves are shown in Figure S2. After loading, tips were stored at 4°C in buffer FPO until use later the same day. SPOT binding was assayed by equilibrating the tips in buffer OS (TE + 12.5 mM MgCl₂ +100 mM NaCl) for 3 min, then measuring association of the appropriate SPOT or origami without aptamer (both at ∼100 nM) for 8-10 min, followed by a 8-10 min dissociation measurement in buffer OS.

The RBDs of SARS-CoV-1, SARS-CoV-2, WIV1, RaTG13, SHC014, ZC45, and ZXC21 were synthesized by GenScript and cloned into vector pCMV with a preceding mu-phosphatase signal peptide and a terminal octa-histidine tag. Plasmids were transfected into 150mL suspension expi293F or HEK293F cells at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm and harvested 3 days later. Clarified supernatants were purified in batch over Talon resin (Takara) prior to buffer exchanging into 20mM Tris pH8 150mM NaCl and flash freezing.
Biolayer interferometry binding assays were performed on an Octet Red instrument at 30°C with shaking at 1,000 RPM. ARG2 biosensors were hydrated in water then activated for 300 s with an NHS-EDC solution (ForteBio) prior to amine coupling. 5–10 μg/mL of each RBD was loaded in a buffer containing 10mM pH5 sodium acetate onto ARG2 tips (ForteBio) for 600 seconds and then quenched into 1M ethanolamine for 600 seconds. A baseline in 10X kinetics buffer (ForteBio) was collected for 120 s prior to immersing the sensors in a 1:3 serial dilution of his-tagged human ACE2 (Sino Biologicals) ranging from 1,000 to 0.83nm in 10X kinetics buffer. Curve fitting was performed using a 1:1 binding model and the ForteBio data analysis software. Mean k_on, and k_off values were determined with a global fit applied to all data.