Biophysical Studies of Nucleic Acid - Small Molecule Recognition

The presented work is a collection of studies performed to examine small molecule recognition of nucleic acids. Recognition of nucleic acids was examined using two types of small molecules: aminoglycosides and Hoechst dyes. Chapter One focuses on established experimental techniques (i.e. absorbance, isothermal titration calorimetry, flow cytometry, cell microscopy, etc.) in small molecule recognition of nucleic acids. The emphasis of studies in the group has been on two focus areas. (i) Thermodynamic characterization of mixed A-form and B-form DNA or RNA conformation with aminoglycosides, particularly neomycin-class. (ii) Characterization of new Hoechst-morpholine (HT-MO) derivatives’ interactions with nucleic acids. Focus area (i) is delved in Chapter Two, establishing a thermodynamic profile for neomycin-class aminoglycosides binding to the mixed conformation of 5’-GGGGCCCC-3’ (dG4C4) in various conditions. The studies demonstrated that the environmental condition (pH and salt concentration) plays a pivotal role in the thermodynamics of the model system. dG4C4 studies demonstrated several key points (i.e. aminoglycoside binding is sensitive to pH and salt conditions, intrinsic enthalpies are key to differential affinities of the aminoglycosides, etc) that will be discussed, including circular dichroism (CD) studies indicating that aminoglycoside binding induces a conformational shift toward A-form dG4C4. Chapter Three is an expansion of the aminoglycoside-G4C4 recognition, utilizing 5’-rGrGrGrGrCrCrCrC-3’ (rG4C4). The rG4C4 studies revealed that the buffer, pH, and aminoglycoside utilized all impact the thermodynamic profiles similarly to dG4C4. Chapter Three also compared the DNA and RNA sequences, dG4C4 and rG4C4. In all cases, the rG4C4-aminoglycoside interactions excelled comparatively to dG4C4. All experiments with dG4C4 and rG4C4 point to the neomycin-class aminoglycosides having a higher affinity for RNA, while DNA provides a flexible conformation in which the environment influences the conformation. Chapter Four demonstrates a shift to focus area (ii), which looks at new Hoechst derivatives’ interaction with nucleic acids. Previous group members investigated derivatives with modifications of the phenolic side of Hoechst 33258 with aliphatic linkers. More recent work examined a library of morpholine-Hoechst (HT-MO) derivatives that have the n-methyl piperazine group replaced with morpholine. Characterizing the novel HT-MO library interactions with 5’-CGCA3T3GCG-3’ and 5’-dA30•dT30-3’ demonstrated that HT-MO 6003 exhibited fluorescence and cellular uptake similar to commercially available Hoechst dyes, and the other HT-MO derivatives were significantly weaker.