Fig. 1: miRNA detection scheme.
From: Quantification of purified endogenous miRNAs with high sensitivity and specificity
a Design of the target RNA containing let-7a sequence on the 5′-side, U15 linker on the 3′-side, and biotin at the 3′-end. b Design of DNA probes targeting let-7a. let-7a_PS, let-7a_PM, and let-7a_PT targeting the seed, mid, and tail regions of let-7a, respectively, are labeled with different fluorophores as depicted. c Scheme to accelerate miRNA detection. DNA probes are loaded in TtAgo, and their biding to surface-immobilized target miRNAs is monitored using multicolor single-molecule fluorescence imaging. d Single-molecule spots of 100 pM let-7a. e Single-molecule spots without target let-7a. f Single-molecule spots of 100 pM let-7a in the presence of ten times excess unlabeled DNA probes. g Representative fluorescence intensity time traces that show binding of bare DNA probes (top) and TtAgo-loaded DNA probes (bottom) on surface-immobilized target let-7a miRNAs. h Comparison of the binding rate constants (kon) of bare DNA probes (red) and TtAgo-loaded DNA probes (black). i Comparison of dissociation rate constants (koff) of bare DNA probes (red) and TtAgo-loaded DNA probes (black). To obtain the each data point of h and i, three or more independent experiments were conducted and at least 300 molecules in total were analyzed. Data are mean ± SEM. To obtain kon and koff of Ago-loaded DNA probes, 10 min imaging time was used. To obtain kon and koff of bare DNA probes, 1 h imaging time was used. Source data are available in the Source data file.
