Morphological effects of G-quadruplex stabilization using small molecule in Zebrafish

Zebrafish (Danio rerio) embryos are transparent and offer the advantage of studying early developmental changes due to ex-utero development making it an appropriate model to study gene expression changes as a result of molecular targeting. Zebrafish embryos were injected with a previously reported G-quadruplex selective ligand and the phenotypic changes were recorded. We have observed marked discrepancies in development of inter segmental vessels as a result. In silico analysis examined that putative G-quadruplex motif occur in the upstream promoter region of Cdh5 (N-Cadherin) gene. Real Time PCR based investigation, indicated that in Zebrafish, CDH-2 (ZN-cad) was significantly down-regulated in the ligand treated embryos. Biophysical characterization of the interaction of the ligand with the G-quadruplex motif found in this promoter yielded strong binding and stabilization of the G-quadruplex with this ligand. Hence, we report for the first time about the phenotypic impact of G-quadruplex targeting with a ligand in a vertebrate organism. This study has unveiled not only G-quadruplex targeting in non-human animal species, it has also unveiled that G-quadruplexes can provide a ready handle to understand the phenotypic effects of targeting certain important genes involved in differentiation and developmental processes in a living eukaryotic organism.