Data analysis 9

To study the interactions of VapC11^D5A with tRNA-Leu^CAG, we used ForteBio Octet RED 96 (ForteBio, USA) and CM5 sensors (ForteBio, USA). The experiment was conducted at 25 °C in a buffer containing 20 mM HEPES (pH 7.5) and 150 mM NaCl. VapC11^D5A diluted in 10 mM sodium acetate buffer (pH 5.0) was immobilized using EDC/NHS chemistry on CM5 sensor tips, as per the manufacturer’s recommendations. Before use, the sensor tips were hydrated in assay buffer for 20  min. A 200  μl aliquot of either sample or buffer was added to a 96-microwell plate with 1000 rpm rotation. VapC11^D5A at a concentration of 1 nM was immobilized onto the working sensor tip. Another reference sensor without immobilized protein was subjected to the identical procedure for double referencing and to subtract non-specific tRNA binding with the sensor material. Following immobilization with VapC11^D5A, the binding interaction with different concentrations (25, 100 and 200 nM) of tRNA was performed. Briefly, after a stable baseline was observed, association (350 s) and dissociation (350 s) were monitored, followed by cyclic regeneration/neutralization with 5 mM NaOH and assay buffer. The obtained data were analysed, and theK_D for substrate binding was calculated by the curve fit (1:1) model using ForteBio data analysis 9.0 software.

Biolayer interferometry was performed using the BLItz System (SARTORIUS/ForteBio) with HEK293-derived recombinant monomeric CoV2 S-RBD fused with human Fc (hFC). A 1.5 µg/ml concentration of S-RBD from the Parental virus with hFc (SinoBiological, 40592-V02H), Delta S-RBD fused with hFc (L452R and T478K that are characteristic of the B.1.1.617.2 lineage) (SinoBiological, 40592-V02H3) and Omicron S-RBD with hFc (G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H that are characteristic of the B.1.1.529.2/BA.2 lineage) (R&D System, 11057-CV) was immobilized on anti-human IgG-Fc (AHC)-coated biosensors for 90 s. The baseline interference phase was obtained by measurements taken for 30 s in the reaction buffer (RB: 1x PBS and 0.05% Tween-20). The sensors were subjected to the association phase for 180 s in wells containing 3-fold serially diluted wt hACE2 or hACE2 glycan deletants that were generated as above in RB. The sensors were then immersed in RB for 300 s as the dissociation step. The mean Kon, Koff and apparent K_D values of SARS-CoV-2 RBD binding affinities for hACE2 were calculated from all the binding curves based on their global fit to a 1:1 Langmuir binding model (ForteBio Data analysis 9.0 software) with an R² value of >0.95.

The Fortebio Octet system (Fortebio) was utilized to assess the affinity of huNb26/Nb26-Nbh and Nectin-4 NDC for the recombinant human Nectin-4 antigen. Streptavidin A biosensors were equilibrated with PBST buffer for 10 min. Subsequently, the biosensor streptavidin A was exposed to diluted biotinylated Nectin-4 protein, and after mixing, it was immersed in PBST buffer. The biosensor was then subjected to association with a range of diluted huNb26/Nb26-Nbh or Nectin-4 NDC samples. The association rates (K_on), dissociation rates (K_off), and dissociation constants (K_d) were analyzed utilizing ForteBio Data Analysis 9.0 software.

Eleven α-helical peptides were synthesized (Supplementary Data 7). Purified recombinant NLRP6 was biotinylated in assay buffer (PBS with 0.01% Tween-20) at room temperature for 1 h. The interaction between the peptides and NLRP6 was determined by BLI using an Octet Red 96 instrument (FortéBio, CA, USA). Loading of streptavidin biosensors was conducted by exposing pre-equilibrated biosensor tips in PCR tubes containing 15 μl biotin-NLRP6 (50 μg/ml) for 1 h at room temperature. All of the binding data were collected at 30 °C. The experiment was composed of three steps: (1) baseline, (2) association, and (3) dissociation. Responses (nanometre shift) were calculated using data that were double reference subtracted using reference wells and nonspecific binding of the biosensor to the analyte. Global 1:1 fitting of association and dissociation curves with FortéBio data analysis 9.0 software revealed k_on, k_dis, and K_D binding constants. GraphPad Prism 9.0 was used to visualize curves.

The affinity of Nb4 to recombinant human TROP2 were determined by an fortebio octet system (ForteBio, Menlo Park, CA, USA). Briefly, the streptavidin A biosensors were wetted in PBST buffer for about 10 min. The diluted biotinylated TROP2 protein was coupled to the streptavidin A biosensors. Thereafter, it was incubated with serially diluted Nb4 and then dissociated in PBST buffer. The binding curve and the equilibrium dissociation constant (KD) was analyzed by ForteBio Data Analysis 9.0 software.

FGF1 was immobilized on AR2G sensors as described in 4.3. Measurements were performed in the Kinetics Buffer. Amine-coupled FGF1 was used to capture first scFv at 200 nM for 180 s and then the sensor was moved to the well with the second scFv prepared at the same concentration and the following association was measured for further 180 s. The surface was regenerated with 100 mM glycine, pH 3.5, and the assay was repeated for another scFv-scFv pair. The measurements were continued until all available scFv-scFv configurations were examined. For the scFv-FGFR1 epitope binning assay, ECD_FGFR1-Fc was coupled with activated AR2G sensors, as described for FGF1 in 4.3., at 5 μg/mL in 10 mM sodium acetate, pH 5.0. FGF1 at 1 μM was preincubated with scFv fragment at 1:1 molar ratio for 30 min, and then the ability of complexed FGF1 to bind to the immobilized ECD_FGFR1-Fc was assessed. The sensor surface was regenerated with 100 mM glycine, pH 2.5, and the assay was repeated for the remaining FGF1-scFv complexes. Binding of FGF1 only and unspecific binding of scFvs to the immobilized protein was also verified. All measurements were performed in duplicate. The binding curves were analyzed using ForteBio’s Data Analysis 9.0 software.