Synthesis and Binding of Flavone-Based Ammonium Salts to Triplex DNA

Abstract

Triplex DNA is formed when a third strand, the triplex-forming oligonucleotide (TFO), sequence specifically binds to a duplex. TFOs can be used for anti-gene therapy by targeting a certain sequence of duplex DNA and subsequently inhibiting the downstream transcription and translation of a specified protein. However, triplex formation is limited in both thermodynamic and kinetic aspects because of the charge repulsion of a third negatively charged strand.Recently, it was found that a flavonoid, quercetin, and its derivatives were able to stabilize triplex DNA without having an effect on duplex DNA. In this project, four flavone-based ammonium salt derivatives containing a positive charge were synthesized. The positively charged derivatives could counteract the negatively charged backbone repulsion and contribute to stabilizing the triplex. Additionally, these compounds have the added benefit of being more water soluble than their non-charged counterparts. These derivatives were evaluated with UV thermal denaturation studies to determine their effects on an intramolecular triplex DNA (15GCT). After observing triplex stabilization via UV thermal denaturation, the thermodynamics of DNA-ligand binding were further quantified using an isothermal titration calorimeter (ITC). The ITC is an instrument that measures the heat change within a sample cell as a ligand is titrated in. If binding occurs, there is a recorded heat change relative to a reference cell, and a variety of thermodynamic values, such as the binding constant (Kd), can be calculated. The results from UV thermal denaturation and ITC are compared to evaluate the binding and stabilization of these ammonium salt compounds on triplex DNA