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  • Puromycin br In the case of AT G Fig


    In the case of AT11 G4 (Fig. 4A), C8 was the most stabilizing ligand ( Tm = 24.0 °C) while C3 stabilized the least ( Tm = 13.5 °C). As re-ported previously for this set of acridine orange derivatives, the in-crease in the alkylamide chain enhances the Puromycin thermal stabilization in-duced by the ligands (Carvalho et al., 2018). Similarly, C8 promoted a stabilization of over 30 °C for AT11-B0 G4 (absolute value not de-termined due to lack of convergence in data fitting) while C3 and C5 equally stabilized the G4 structure by ca. 13.7 °C (Fig. 4B).
    Following CD studies, the apparent equilibrium dissociation con-stants (KD) for the complexes were determined by fluorimetric titrations (Fig. 5). The ligands produce a broad band centered at 568 nm after excitation at 498 nm. Upon adding pre-folded AT11 G4 to the ligands solution, an enhancement in the ligands fluorescence was observed (Fig. 5, top). Up to 10-fold fluorescence enhancement was observed which is indicative of strong association between the ligands and the 
    Regarding AT11-B0 G4, similar fluorescence enhancement was ob-served for all ligands (Fig. 5, bottom), with KD values of 1.1 × 10−6, 1.3 × 10−6 and 1.7 × 10−6 M for C3, C5 and C8, respectively (Table 2). Likewise, the complex formation is in the moderate-high affinity range which supports the ligand-induced stabilization observed in CD melting experiments. The Hill coefficients (n) suggest a cooperative binding mode. Overall, the ligands bind AT11 G4 and AT11-B0 G4 with similar affinities.
    One of the issues of using oligonucleotides as aptamers for drug delivery is serum degradation and susceptibility to nuclease (Carvalho et al., 2019). In order to evaluate whether the AT11 G4 and AT11-B0 G4 were stable in serum or if the ligand conjugation was able to protect from serum degradation due to G4 stabilization, free AT11 G4 and AT11-B0 G4 and their complexes were incubated in culture media supplemented with 10% or 50% fetal bovine serum (FBS). Taking into account that aptamers may take several days to internalize (Reyes-Reyes et al., 2010), this experiment was also performed at 72 h of in-cubation. As C8 promoted the highest thermal stabilization, the assay was only carried in the presence of this ligand. The results revealed by agarose gel electrophoresis are shown in Fig. 6. Free AT11 G4 seems to undergo some degradation after a 48 h incubation Puromycin as indicated by the progressive disappearance/decrease of the corresponding band (Fig. 6A). In the presence of C8 no apparent degradation can be ob-served suggesting that the complex is stable in 10% FBS (Fig. 6B). On
    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 stability 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