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  • br Fig Saturation binding plots obtained by fluorescence titrations with

    2022-05-23


    Fig. 7. Saturation binding plots obtained by fluorescence titrations with nucleolin and fitted to Michaelis-Menten model for free Cy5-AT11 G4 and Cy5-AT11-B0 G4 (A and B, respectively), or for Cy5-AT11 and Cy5-AT11-B0 in the presence of ligand C8 (C and D, respectively).
    (B) AT11-B0 G4-RBD1,2. G4 structures are depicted in green with loop thymines responsible for binding highlighted in orange while RBD1,2 is represented in blue ribbon (except for atoms participating in H-bonding). H-bonds are shown as cyan lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    the other hand, free AT11-B0 (Fig. 6C) is resistant to serum nucleases, probably because of its higher thermal GSK'872 when compared to AT11 G4 and the lack of bulge in the G4 structure (Do et al., 2017). In the presence of C8 the stability was not affected (Fig. 6D). The results shown in Fig. 6E suggest that in 50% FBS free AT11 G4 is readily de-graded as seen from the absence of any band below 500 pb. In contrast, in the presence of ligand C8, the band corresponding to the AT11 G4-C8 complex was observed (Fig. 6F). These results suggest that the ligand significantly increases the serum stability of the aptamer up to 72 h. Free AT11-B0 G4 showed to be resistant to serum nucleases in 50% FBS (Fig. 6G) and the presence of ligand C8 did not affect this stability (Fig. 6H).
    As both AT11 G4 and AT11-B0 G4 sequences are AS1411 deriva-tives, the affinity of these two G4 structures towards nucleolin was 
    assessed by fluorimetric titrations using Cy5-labelled sequences. The saturation binding curves shown in Fig. 7A and B suggest fast saturation at low protein concentration which is indicative of high affinity. Indeed, the fitting of the data yielded KD values of 9.1 × 10−12 and 9.5 × 10−12 M for Cy5-AT11 G4 and Cy5-AT11-B0 G4, respectively, which are in the same range of values reported for other aptamers (Zhou and Rossi, 2017). To ascertain whether ligand binding would decrease the affinity of the G4 sequences towards nucleolin, a similar experiment was conducted with AT11-C8 and AT11-B0-C8 complexes. As seen in Fig. 7C and D, fast saturation was also attained in the pre-sence of the ligand, yielding KD values of 5.2 × 10−12 and 3.3 × 10−12 M for Cy5-AT11 G4 C8 and Cy5-AT11-B0 G4 C8, respectively. The KD values for the complexes are in the same picomolar range as the free G4 structures, suggesting that the presence of the ligand does not
    J. Figueiredo, et al.
    Fig. 9. Confocal fluorescence images of HeLa cells incubated with (A) Cy5-AT11-C8 and (B) Cy5-AT11-B0-C8 after 2 h at 37 °C. Nucleolin is stained with AlexaFluor 546®, C8 emits green fluorescence, Cy5-AT11 and Cy5-AT11-B0 are shown in red. Scale bar: 25 µm. Brightness was adjusted for all images equally. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Fig. 10. Confocal microscopy images of HeLa cells incubated with (A) Cy5-AT11 G4-C8 and (B) Cy5-AT11-B0 G4-C8 for 3 days. Cell nuclei are stained with Hoechst 33,342 (blue), C8 emits green fluorescence and Cy5-AT11 G4 or Cy5-AT11-B0 G4 are shown in red. Overlapping of the two stains, observed as yellow/orange regions, can be seen in the merged images of the cells. Scale bar: 25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Fig. 11. Confocal microscopy images of NHDF cells incubated for 3 days with
    (A) Cy5-AT11 G4-C8 and (B) Cy5-AT11-B0 G4-C8. Cell nuclei are stained with Hoechst 33,342 (blue), C8 emits green fluorescence and Cy5-AT11 G4 or -AT11-B0 G4 are shown in red. The combination of the two stains, observed as yellow/ orange regions, can be seen in the merged images of the cells. Scale bar: 25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)