RESUMO
Transient melting of the duplex-DNA (B-DNA) during DNA transactions allows repeated sequences to fold into non-B-DNA structures, including DNA junctions and G-quadruplexes. These noncanonical structures can act as impediments to DNA polymerase progression along the duplex, thereby triggering DNA damage and ultimately jeopardizing genomic stability. Their stabilization by ad hoc ligands is currently being explored as a putative anticancer strategy since it might represent an efficient way to inflict toxic DNA damage specifically to rapidly dividing cancer cells. The relevance of this strategy is only emerging for three-way DNA junctions (TWJs) and, to date, no molecule has been recognized as a reference TWJ ligand, featuring both high affinity and selectivity. Herein, we characterize such reference ligands through a combination of in vitro techniques comprising affinity and selectivity assays (competitive FRET-melting and TWJ Screen assays), functional tests (qPCR and Taq stop assays) and structural analyses (molecular dynamics and NMR investigations). We identify novel azacryptands TrisNP-amphi and TrisNP-ana as the most promising ligands, interacting with TWJs with high affinity and selectivity. These ligands represent new molecular tools to investigate the cellular roles of TWJs and explore how they can be exploited in innovative anticancer therapies.
RESUMO
Alternative DNA structures play critical roles in fundamental biological processes linked to human diseases. Thus, targeting and stabilizing these structures by specific ligands could affect the progression of cancer and other diseases. Here, we describe, using methods of molecular biophysics, the interactions of two oxidatively locked [Co2L3]6+ cylinders, rac-2 and meso-1, with diverse alternative DNA structures, such as junctions, G quadruplexes, and bulges. This study was motivated by earlier results demonstrating that both Co(III) cylinders exhibit potent and selective activity against cancer cells, accumulate in the nucleus of cancer cells, and prove to be efficient DNA binders. The results show that the bigger cylinder rac-2 stabilizes all DNA structures, while the smaller cylinder meso-1 stabilizes just the Y-shaped three-way junctions. Collectively, the results of this study suggest that the stabilization of alternative DNA structures by Co(III) cylinders investigated in this work might contribute to the mechanism of their biological activity.