Biochemical Characterization and Optimization of RNA Aptamers Bound to Hoechst Dye Derivatives

Abstract

Ribonucleic acid (RNA) aptamers are single-stranded nucleic acids that are typically selected through a process known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX). These oligonucleotides can bind to small target molecules, including proteins and chemical toxins, with high specificity and affinity. They can be further modified with recognition elements, such as light-up fluorophore molecules, to become aptamer-based biosensors. In the year 2008, Sando et al. selected for an RNA aptamer that is capable of specific binding to a Hoechst dye derivative with bulky tert-Butyl (tBu) groups, which prevents the non-specific binding to DNA sequences. The group performed some preliminary biochemical characterization experiments; however little information is currently known about the specific molecular interactions between the optimized Aptamer II-mini3-4 sequence and the tBu Hoechst dye. Therefore, this research project aimed to develop a complete biochemical profile of the SELEX-selected 71-nucleotide (nt) Aptamer II sequence and the optimized 29-nt Aptamer II-mini3-4 sequence. In addition, new RNA aptamer variants were developed within this research project to not only gain a deeper understanding of the aptamer’s folded conformation and binding interactions with the tBu Hoechst dye, but to also optimize the aptamer system further.

In Chapter 2 fluorescence emission experiments were performed to determine if binding was present between the RNA aptamer variants and the derivative tBu Hoechst dyes. The fluorescence enhancement of each bound complex was also compared and evaluated to understand binding interactions within the aptamer systems. Moreover, fluorescence titration assays were conducted to estimate the binding affinities for between the interacting molecules.

In Chapter 3 native polyacrylamide gel (PAGE) experiments were conducted to determine the conformation of the tBu Hoechst-bound and unbound conformations of the RNA aptamer variants. In addition, isothermal titration calorimetry (ITC) experiments were employed as an alternative method to determine the dissociation constants of the studied aptamer systems. It was also used to obtain the thermodynamic and kinetic properties of each of the tBu Hoechst-binding RNA aptamers. The information gained within this research project was intended to lay the groundwork for future work including the structure elucidation of the Aptamer II-mini3-4 RNA sequence bound with the tBu Hoechst dye through nuclear magnetic resonance (NMR) spectroscopy techniques. It was also intended to aid in optimizing the aptamer system for its development as a building block for an aptamer-based biosensor that can be used for potential commercial applications.