Fig. 1: Design and in silico modelling of de novo quenchbodies.

A Schematic representation of the working principle of a quenchbody. In the absence of the antigen (left), the fluorophore is quenched by tryptophans in the antibody. In the antigen-bound state (right,) the fluorophore is displaced, resulting in dequenching and an increase in fluorescence intensity. B Proposed mechanism of the MBP-binding quenchbody (blue) modelled from PDB ID: 5M14, with N-terminal covalently conjugated fluorophore (green) undergoing quenching due to interaction with the intrinsic CDR-based tryptophans (red spheres). Upon binding to the MBP antigen (grey surface model), the fluorophore is sterically occluded from tryptophans (W101, W110 and W115), which is associated with increased fluorescence intensity. C Average normalised distribution histogram of the distance of the fluorophore to any of the three tryptophans (W101, W110 and W115) derived from MD simulations in the absence (blue) or presence (green) of antigen for the MBP-binding nanobody. The fluorophore is considered quenched by tryptophan at distances ≤10 Å (hatched zone). Individual plots for W101, W110 and W115 are provided (Fig. S1A). D Average normalised distribution histogram of the distance of the fluorophore to either of the two tryptophans (W103 and W115) derived from MD simulations in the absence (blue) or presence (green) of antigen for the lysozyme-binding nanobody. Individual plots for W103 and W115 are provided (Fig. S1B).