Extended Data Fig. 6: Effect of FAM connection and isomer on SA binding, bAP binding, and pNPP hydrolysis.

(a) In most of this study until this point, we used a L12 ssDNA nanoantenna with 5′ thymine 6-carboxyfluorescein (5′ T 6-FAM). Here, however, we also tested other FAM connections on the same DNA sequence: 5′ 6-carboxyfluorescein (5′ 6-FAM), 5′ 5-carboxyfluorescein (5′ 5-FAM) and 3′ 5-carboxyfluorescein (3′ 5-FAM). (b) Shown are the quenching of fluorescence upon SA binding, the increase of fluorescence upon bAP binding, and the transient fluorescence spike during pNPP hydrolysis. Despite the similar fluorescence emission of 5-FAM and 6-FAM when conjugated to DNA, the various FAM nanoantennas display different trends for protein binding and pNPP hydrolysis. These differences are likely due to how the chemical connection subtly affects FAM-bAP interaction. All experiments were performed with n=1 biologically independent enzyme samples examined over 3 independent experiments. Data are presented as mean values ± SEM. Conditions: 15 nM nanoantenna, 5 nM SA, 10 nM homemade bAP, 30 μM pNPP, pH 8.0, 100 mM Tris, 10 mM NaCl, 30 °C. PMT voltage = 800 V.