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    • br Results br Discussion In this study six new

    2021-10-11


    Results
    Discussion In this study, six new T30695 variants were synthesized with the acyclic R or S glycerol linker instead of lomitapide synthesis unit at 4, 8 or 12 T residue. Based on our previous studies on TBA-GQ, this type of modification should provoke local changes in GQ conformations in the final T30695-GQ variants, that without affecting the structural stabilities could still potentially influence biological activities of aptamers. Our CD, 1H NMR, EMSA and HRMS studies showed that the new synthesized T30695 variants are able to fold in dimeric GQ structures that resemble that of T30695 [12]. Based on the number of ammonium ions incorporated into the GQ structure, the ability to form dimers for all T30695-GQ variants was confirmed by HRMS results carried out in the presence of ammonium acetate ions. In addition, in presence of K+ or Na+ ions, all T30695-GQ variants also had a band with the same electrophoretic mobility as T30695-GQ, which in turn presented a lower electrophoretic mobility than 5′-TT-T30695-GQ, that is known to form monomolecular GQ [13,20]. CD melting experiments also showed that the Tm values were always much higher in the presence of K+ ions than Na+ ions and that significant differences between the Tm values of T30695-r4, −r8, or -r12 and the corresponding T30695-s4, or -s8, or -s12 GQs were noticeable mainly in the presence of 70 mM of Na+. The thermal stability of all described ONs was also explored using a buffer containing 140.7 mM of sodium (140Na buffer), that corresponds to the total concentration of monovalent ions in PBS (138 mM of Na+ and 2,7 mM of K+) (Figs. 3, 11 and 12S). As expected, using 140Na buffer all the G-quadruplex melting temperatures become both higher than that measured in 70Na buffer and broadly similar to that measured in PBS. In addition, some of the differences between Tm values of T30695-r and –s G-quadruplexes were still observed but strongly reduced (Fig. 11S). The overall of these phenomena state clearly that both the type and the concentration of cations in the solution contribute to the stability of G-quadruplexes. The trends of the Tm values of the T30695-r and-s GQ variants in different buffered conditions could be related to slight differences between Na+ and K+ GQ cores [[45], [46], [47], [48], [49]]. Indeed, X-Ray and NMR [[45], [46], [47], [48], [49]] studies on GQs showed that potassium ions due to their relatively large radii reside between the G-quartet planes, coordinating the eight O atoms of G bases of two successive G-quartets. Conversely, the small Na+ ions can occupy a range of different positions inside the GQ core, including the G-quartet in-plane site. As a consequence of the ion mobility, the compactness of the GQ core is smaller in the presence of Na+ than K+. Therefore the constrains related to the G-quartet distances and/or to the requisite co-planarity in each G-quartet could decrease with Na+ ions, thus allowing to similar stability of T30695-GQ and its r variants. HIV-1 IN complex recruits LEDGF/p75 during the integration of viral cDNA into specific regions of the DNA of the host cell [28,41,50,51]. In vitro experiments showed that, like viral DNA, also LEDGF/p75 promotes HIV-1 IN tetramerization [35,46]. However, in vitro FRET experiments showed that the resulting (HIV-1 IN)4-viral DNA and LEDGF/p75-(HIV-1 IN)4 tetramers have distinct conformations [42]. Furthermore, it appeared that the addition of LEDGF/p75 on preformed IN–viral DNA complex lets unaffected the IN tetramer conformations [50,51]. In view of the biochemical analyses of T30695 interaction with IN, that indicated the same binding site on IN tetramers for viral DNA and T30695 [7,8,16,18], in our LEDGF/p75 dependent integration assay, the aptamers were pre-incubated 1 h with HIV-1 IN, and LEDGF/p75 was successively added together with the ONs mimicking viral and host DNA. Despite this, the addition of LEDGF/p75 in the assay adversely affects the ability of T30695 and its variants to inhibit HIV-1 IN, as showed by the increasing of all the IC50 values. Moreover, the extend of this phenomenon depended on the position and the R or S chirality of glycerol T loop, at least partially, regardless the stability of T30695-GQ variants. Based on their molecular model results, Sgobba et al. suggested that T30695 binds to an IN tetramer, principally through its phosphate groups. In the final complex, the flexibility of the catalytic loops of the IN tetramer is reduced and the enzyme is held in catalytically inactive conformations [16]. Conversely, previous reported data showed that the binding of LEDGF/p75 to preformed viral DNA-(IN)4 complex, stabilizing catalytically active conformations of the HIV-1 IN tetramers, favors the products of viral DNA integration into the target DNA [51]. Therefore, due to the IN tetramer allostery, T30695 and LEDGF-p75, appeared to act in an opposite manner on the integration activity of the enzyme. In this context, our data suggest that the binding of LEDGF/p75 could, at least partially, allosterically restore the active conformations of the catalytic site in (HIV-1 IN)4 aptamer-GQ complexes, undermining the stability of the interactions between aptamer-GQ and the HIV-1 IN.