Fig. 4.

Targeting and payload aptamers colocalize within endosomes. a–c AF488-anti-tail or 3WJdB (0.5 μM, green) were assembled with 3-fold molar excess of Cy5-labeled Waz and C10.36 (red), and colocalization of the two aptamer modules was assessed after 1 h-incubation in NALM6 cells. a Representative confocal microscopy images of fixed NALM6 cells show significant colocalization between targeting aptamers and AF488-anti-tail. b A reduction of colocalization between targeting and payload modules was found using 3WJdB as a consequence of reduced brightness and photostability of 3WJdB–DFHBI-1T compared with AF488 and Cy5, as well as a higher fluorescence background due to the unbound DFHBI-1T. c Strong colocalization between 3WJdB and either Waz or C10.36 was observed when AF488-labeled 3WJdB was used as imaging probe in place of 3WJdB–DFHBI-1T. d–f Cy3-labeled 3WJdB (green) was assembled with Waz aptamer, and colocalization with endocytic markers (red) was assessed after 1 h-incubation in NALM6 cells. d Representative confocal microscopy images of fixed and immunostained NALM6 cells show significant colocalization between Waz–3WJdB-Cy3 and Rab5 (early endosome marker). e A reduction of colocalization was found between Waz–3WJdB–Cy3 and Rab7 (late endosome marker). f NALM6 cells were co-incubated for 1 h with 0.5 μM AF488-labeled Tf and 0.5 μM Waz–3WJdB-Cy3 complex, then cells were fixed and imaged by confocal microscopy. A strong colocalization between Tf-AF488 and Waz–3WJdB–Cy3 was observed both in the cell periphery and perinuclear region of NALM6 cells. For all samples, Pearson’s correlation coefficient was used to estimate the extent of colocalization between targeting and payload modules of the aptamer platform or between Waz–3WJdB–Cy3 and endocytic markers (see also Supplementary Fig. 14). Images are representative of two independent experiments. Scale bars: 5 μm