Fig. 2: Design and characterization of Y-shaped probes.
From: Spatial fluorescence barcode by transiently luminescent DNA beads
a Four-strand Y-shaped probe labeled with a Cy5 fluorophore and dual-BHQ3 quenchers, modified with a 3’ phosphonothioate group. Target recognition triggers TMSD, releasing the BHQ3 quencher-labeled Structure 2 and activating the Cy5 fluorescence of Structure 1. ExoIII digests the DNA target, allowing Structure 2 to rehybridize and fluorescence to reset autonomously. b Activation-reset cycling stability of single Y-shaped probe IV over six repeated activation-quenching cycles. Data are mean ± S.D. (n = 3 independent experiments), and the shaded areas represent error bars. The figure contains elements created by BioRender. c Orthogonality analysis among seven Y-shaped probes I-VII. The heatmap shows normalized AUC. d Sequential activation-reset cycling of seven Y-shaped probes I-VII. The probe mixture was tested across 14 cycles, with each probe triggered twice sequentially. The concentration of all DNA in the reaction system was 50 nM, the ExoIII concentration was 20 U mL⁻¹, the reaction temperature was 37 °C, and the buffer composition consisted of 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, and 1 mM DTT (pH 7). Data are mean ± S.D. (n = 3 independent experiments), and the shaded areas represent error bars. The figure contains elements created by BioRender. Source data can be found in the Source Data file.
