Study of the interaction of fluorescent dyes with several DNA structures

Abstract

This project studies the interaction of the fluorescent ligand R9 with various DNA structures, such as G-quadruplex, duplex, hairpin, and i-motif, using techniques like fluorescence spectroscopy, circular dichroism, UV-Vis spectroscopy, and high-performance liquid chromatography (HPLC). The main objective is to characterize the binding properties and structural effects that R9 induces in these DNA structures. The result shows that the R9 ligand binds to all the studied DNA structures, even though the strength of this interaction depends on the shape of each structure. In particular, the parallel G-quadruplex Pu22T14T23 stands out for its high affinity, which is evidenced by increased fluorescence, changes in UV-Vis absorption spectra, and a significant increase in thermal stability. Circular dichroism data suggest that, in G-quadruplex, R9 binds externally through π-π stacking between the nitrogenous bases and the ligand, as well as electrostatic interactions. In the case of the duplex H28, the ligand intercalates between the strands. Thermal denaturation measurements indicate that R9 enhances the stability of most DNA structures, raising their melting temperatures (Tm). This effect is clearly seen in the parallel G-quadruplex Pu22T14T23, which exceeds 95 ºC in the presence of R9. However, in hybrid structures like IDJ1 (which combines an i-motif and a hairpin), the effect of the ligand varies more, making some structural components less stable. Finally, the project included the set up of an HPLC system that had not been used for years. The restarting of the HPLC system confirmed the functionality of the equipment, although the separation of DNA structures was not optimal with the column used. In summary, this study demonstrates that the R9 ligand can stabilize non-canonical DNA structures, particularly G-quadruplex. This makes it a good candidate for studies on telomerase inhibition in cancer cells