Insights into the G-rich VEGF-binding aptamer V7t1: when two G-quadruplexes are better than one!

Abstract

The G-quadruplex-forming VEGF-binding aptamer V7t1 was previously found to be highly polymorphic in a K+-containing solution and, to restrict its conformational preferences to a unique, well-defined form, modified nucleotides (LNA and/or UNA) were inserted in its sequence. We here report an in-depth biophysical characterization of V7t1 in a Na+-rich medium, mimicking the extracellular environment in which VEGF targeting should occur, carried out combining several techniques to analyse the conformational behaviour of the aptamer and its binding to the protein. Our results demonstrate that, in the presence of high Na+ concentrations, V7t1 behaves in a very different way if subjected or not to annealing procedures, as evidenced by native gel electrophoresis, size exclusion chromatography and dynamic light scattering analysis. Indeed, not-annealed V7t1 forms both monomeric and dimeric G-quadruplexes, while the annealed oligonucleotide is a monomeric species. Remarkably, only the dimeric aptamer efficiently binds VEGF, showing higher affinity for the protein compared to the monomeric species. These findings provide new precious information for the development of improved V7t1 analogues, allowing more efficient binding to the cancer-related protein and the design of effective biosensors or theranostic devices based on VEGF targeting.

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

“…Compared to the Na + -rich medium, completely different was the behavior observed in both gels for V7t1 in TRIS/K + buffer (Figure 1b and Figure S4b), here analyzed for the first time. In addition, in these saline conditions, V7t1 showed the presence of two distinct bands corresponding to the monomeric and dimeric G4 structures found in HEPES/Na + buffer [17], but without detectable differences between the N.A. and A. samples (Figure 1b and Figure S4b, lanes 1 and 2).…”

“…Overall, no sensible difference in terms of aggregate In both gel assays, in the selected HEPES/Na + buffer, all the investigated V7t1 dimers showed a detectable difference if in N.A. or A. forms, as in the case of V7t1 [17]. In particular, bisV7t1T7 and bisV7t1HEG2 in the N.A.…”

“…All the designed linkers spanned similar lengths, in the range of 40-42 bonds, considered suitable to keep the extremities of two V7t1 sequences at a proper distance to allow their optimal folding [17].…”

“…The selected Na + -rich buffer mimes the blood salt composition and is indeed more appropriate for studying the structure-activity relationships of these aptamers in the extracellular media, although the stability of the G4 module is generally decreased in these saline conditions. In addition, the VEGF 165 protein was provided folded in this solution in which we had previously performed the key experiment demonstrating the preference of the protein for dimeric V7t1 [17].…”

A homodimeric aptamer variant generated from ligand-guided selection activates T-cell receptor cluster of differentiation three complex

“…In the last decade, increased interest has shown in truncating aptamers to variants that show enhanced activity (18,19). In fact, we and others have shown that the truncation and dimerization of aptamers resulted in the enhanced binding properties of molecules against cellular targets (18,20,21).…”