Selective light-up of dimeric G-quadruplex forming aptamers for efficient VEGF165 detection

To develop efficient anticancer theranostic systems, we studied the interaction between a cyanine dye, analogue of thiazole orange (named CyOH), and two G-quadruplex-forming aptamers, V7t1 and 3R02, recognizing the Vascular Endothelial Growth Factor 165 (VEGF165) - an angiogenic protein overexpressed in cancer cells, responsible for the rapid growth and metastases of solid tumours. We demonstrated, by exploiting different biophysical techniques - i.e. gel electrophoresis, circular dichroism (CD), UV-vis and fluorescence spectroscopy that this cyanine interacted with both aptamers giving a marked fluorescence light-up only when bound to their dimeric forms. Interestingly, both oligonucleotides recognized VEGF165 with higher affinity when adopting dimeric G-quadruplexes, largely prevalent over their monomeric forms in pseudo-physiological conditions. Notably, the fluorescence light-up produced by the probe was maintained when the dimeric aptamer-CyOH complexes bound to the target protein. These complexes, tested on MCF-7 cancer cells using non-tumorigenic MCF-10A cells as control, were effectively internalized in cells and colocalized with a fluorescently-labelled anti-VEGF-A antibody, allowing both recognition and detection of the target. Our experiments showed that the studied systems are promising tools for anticancer theranostic strategies, combining the therapeutic potential of the G4-forming anti-VEGF aptamers with the diagnostic efficacy of the cyanine selective fluorescence light-up.

Automated summary

In order to develop efficient anticancer theranostic systems, the interaction between a cyanine dye (CyOH) and two G-quadruplex-forming aptamers (V7t1 and 3R02) was studied. These aptamers recognized the Vascular Endothelial Growth Factor 165 (VEGF165), which is overexpressed in cancer cells and responsible for the rapid growth and metastases of solid tumors. Biophysical techniques, such as gel electrophoresis, circular dichroism (CD), UV-vis spectroscopy and fluorescence spectroscopy, were used to demonstrate that the cyanine interacted with both aptamers, giving a fluorescence light-up when bound to their dimeric forms. The dimeric G-quadruplexes of the aptamers showed higher affinity for VEGF165 and the fluorescence light-up was maintained when the dimeric aptamer-CyOH complexes bound to the target protein. These complexes, tested on MCF-7 cancer cells with non-tumorigenic MCF-10A cells as control, were effectively internalized in cells and colocalized with a fluorescently-labeled anti-VEGF-A antibody, enabling recognition and detection of the target. The studied systems are promising tools for anticancer theranostic strategies, combining the therapeutic potential of the G4-forming anti-VEGF aptamers with the diagnostic efficacy of the cyanine selective fluorescence light-up.