Extended Data Fig. 5: Effect of various chemical modifiers near FAM on cDNA binding, SA binding, bAP binding, and pNPP hydrolysis.

Here, we investigated whether various chemical modifications near the dye (‘modifiers’) could affect the fluorescence signal of FAM by changing its interaction with bAP. (a) We used the L12 ssDNA FAM nanoantenna (5′ T 6-FAM) with a complementary strand containing the modifier located at the 3′-end. (b) For comparison, 1) is the cDNA without a modifier; 2) is the cDNA with phosphate; 3) is the cDNA with a hydrophobic C₁₆ alkane chain; 4) is the cDNA with a modifier that contains a disulfide that would normally be cleaved before use to provide thiol functionality; 5) is a cDNA with the cleaved thiol. (c) Example kinetic signatures and (d) summary of all results. In short, the SA and bAP binding steps display different intensities with each modifier, but nevertheless they are qualitatively similar in all cases (that is, signal up or signal down). The exception to this is the C₁₆ alkane chain, which results in fluorescence quenching when bAP binds. In all cases, the spike intensity during pNPP hydrolysis was similar. 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, 75 nM cDNA, 5 nM SA, 10 nM homemade bAP, 25 μM pNPP, pH 8.0, 100 mM Tris, 10 mM NaCl, 37 °C.