Fig. 5: Assessment of the effect of the scaffold configuration on the performance of the FNAP assemblies.
a, A diagram depicting the base multimerization scaffold (2T), its linear (Lin) variant and a variant with more flexible vertex regions achieved by substituting one of the T nucleotides with a hexaethylene glycol spacer (Flex). FNAPs with extended spacers separate the binding region from the assembly’s core. Corresponding coarse-grained models of the scaffold structures are given below. b, Close-to-average oxDNA models of the designed scaffolds. RMSF, root mean square fluctuation. c, Distribution of end-to-end distances between FNAP attachment points for various assemblies, based on coarse-grained simulations. The average distribution of all three distances is plotted for each particle. d, Competition BLI sensorgrams for FNAP assemblies with various scaffold and binding unit compositions; spike protein without assemblies is plotted with a dashed line. All measurements were performed in duplicate (n = 2, technical replicates), and average signals are plotted with s.d. ranges highlighted. e, A heatmap summarizing the mean areas under the curve (AUC) calculated from the BLI data for each scaffold and FNAP combination. f, Schematic of sMEDUSA, which features two conformational states in dynamic equilibrium, with the closed conformation being stabilized upon multivalent cis-interaction with the spike protein (shown in grey). g, Conformational state diagram for m1 and m2 devices as a function of temperature and Mg2+ concentration. The condition used in the spike protein binding assays is indicated by an asterisk. h, Fluorescence intensity spectral scans for m1 sMEDUSA. i, Fluorescence intensity spectral scans for m2 sMEDUSA. The respective spectral scans of sMEDUSAs in the presence of 250 mM MgCl2 are included as a reference for maximal FRET. j, Change in 670 nm/560 nm fluorescence ratio relative to sMEDUSA in buffer for m1 and m2 sMEDUSAs in the presence of increasing concentrations of spike protein, and for scrambled non-modified (snm) sMEDUSAs at 0.25 μM spike concentration. Dashed lines represent 670 nm/560 nm ratio change for m1 and m2 sMEDUSAs in the presence of 100 μg ml−1 BSA. All FRET assays were performed in triplicate (n = 3, technical replicates), with the average values plotted and s.d. ranges highlighted.
