New findings on the anti-HIV activity of d(TGGGAG) aptamers

Introduction: Recently, we reported the synthesis and characterization of a new mini-library of tetramolecular G quadruplexes based on the d(TGGGAG) sequence, which was previously shown to target the HIV-1 glycoprotein gp120. Oligodeoxynucleotides (ODNs) carrying aryl groups at the 5’-end through a phosphodiester bond were endowed with prominent anti-HIV activity. Biophysical studies on these aptamers demonstrated that there is no relationship between thermal stability of G4 structures and their anti-HIV activity. To identify the properties that make these tetramolecular G-quadruplexes good anti-HIV aptamers, we studied by ESI-MS the stoichiometry and the self-assembly kinetics of G-quadruplex structures based on the most active 5’-end modified d(TGGGAG) aptamers [5]. ESI-MS results showed that the conjugation of d(TGGGAG) at the 5’-end does not necessarily increase the folding rate of the G-quadruplex structure. Unexpectedly, the folding kinetics of the inactive G4 (unmodified sequence) was similar to that of the 5’-end modified sequences. In addition to the slow G4-kinetics revealed by ESI-MS studies, PAGE experiments on the modified d(TGGGAG) highlighted the strong effect of ODN concentration (single stranded) on the G4-folding kinetics. Results and Discussion: In order to clarify the role of G-quadruplex structures in the anti-HIV activity on the basis of the above kinetic studies, we investigated the anti-HIV activity of ODNs at different concentrations of G-quadruplex structure. All ODNs, as well as the unmodified sequence, were evaluated against a variety of HIV-1 viruses and resistant strains. SPR experiments were also carried out to evaluate their binding to the HIV envelope gp120 and gp41 proteins. Almost all compounds showed a good anti-HIV activity, suggesting the absence of a straight correlation between the amount of available G-quadruplex and anti-HIV activity of ODNs. Surprisingly, we found a strong anti-HIV activity of the unmodified sequence d(TGGGAG), in contrast to what has been extensively reported by Hotoda and others, who showed a lack of anti-HIV activity of the d(TGGGAG) sequence without 5’-end modifications. Also, by SPR experiments no straight correlation between gp120 binding and anti HIV activity was observed. Conclusions: The anti-HIV activity results highlight the absence of a straight correlation between the formation of G-quadruplex structures and the antiviral potential of ODNs, suggesting instead that the G4 is not the specie providing anti-HIV activity. In addition, anti-HIV data do not strictly correlate with the SPR results, suggesting that gp120 and gp41 is not the primary target of these aptamers