Extended Data Fig. 2: In contrast to L12, the ‘no linker’ L0 nanoantenna does not allow FAM to bind to unoccupied biotin-binding site on streptavidin nor reach the surface of bAP.

(a) Effect of free biotin binding on the nanoantenna-SA platform for the L0 (left) and L12 (right) nanoantennas. (b) Effect of free biotin binding on the nanoantenna-SA-bAP complex for the L0 and L12 nanoantennas. Discussion: (a) When the L0 or L12 nanoantennas bind to SA, their FAM fluorescence is quenched. Next, free biotin is added in excess. The fluorescence of the L0 nanoantenna does not change because its FAM moiety cannot reach any unoccupied biotin-binding sites⁵⁸, thus it remains unaffected by biotin binding. In contrast, the fluorescence of the L12 nanoantenna increases because its longer length allows its FAM moiety to weakly bind at an unoccupied biotin-binding site until it is ejected by the incoming higher affinity biotin molecule^55,56. (b) The L0 and L12 nanoantennas bind to SA. bAP then binds to the nanoantenna-SA platform. Next, free biotin is added in excess. The fluorescence of the L0 nanoantenna does not change upon addition of bAP because its FAM moiety cannot reach the biotin-binding sites where bAP binds and it also cannot reach the surface of bAP. Subsequent addition of free biotin also does not change its fluorescence for the same reason as without bAP and because most sites are full. In contrast, the fluorescence of the L12 nanoantenna increases upon addition of bAP because its longer length allows its FAM moiety to weakly bind at another biotin-binding site until it is ejected by the incoming high affinity biotin moiety of bAP, thereby allowing it to interact with bAP. Subsequent addition of free biotin does not substantially increase its fluorescence, indicative that the FAM moieties do not remain at the biotin-binding sites of SA. The spike observed upon addition of pNPP confirms that bAP has not been ejected. Although this experiment does not determine whether FAM is now interacting with bAP or with another location on SA, other parts of this paper demonstrate its interaction with bAP (for example, measurement of catalytic function). Conditions: 150 nM nanoantenna, 50 nM SA and 100 nM bAP in 1000 nM biotin in 200 mM Tris, 300 mM NaCl, 1 mM MgCl₂, pH 7.0, 37 °C.