Abstract
In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF165 binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.
Highlights
The Vascular Endothelial Growth Factor (VEGF) family comprises different signaling cytokine proteins involved in both vasculogenesis and angiogenesis processes [1,2,3,4,5]
In order to confirm the binding specificity of V7t1 and its covalent dimers for the VEGF165 protein, we investigated possible unspecific binding using as control bovine serum albumin (BSA)
A dimeric structure represents the simplest multivalent system and nucleic acid aptamers are especially suitable for the design of these constructs as they can be and ad hoc chemically modified
Summary
The Vascular Endothelial Growth Factor (VEGF) family comprises different signaling cytokine proteins involved in both vasculogenesis and angiogenesis processes [1,2,3,4,5]. The same research group prepared a targeted construct for the heparin binding site of the homodimeric VEGF protein made of two identical anti-VEGF DNA-based aptamers (VEa5, with the sequence 5 ATA CCA GTC TAT TCA ATT GGG CCC GTC CGT ATG GTG GGT GTG CTG GCC AGA TAG TAT GTG CAA TCA3 ) In this case the best results in terms of Kd values were obtained when no linker was introduced, while the presence of a linker containing from 10 to 20 T residues proved to be detrimental to the protein binding [19]. In preliminary in vitro investigations, their cytotoxic activity on human breast adenocarcinoma MCF-7 cells was evaluated
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